Giorgia Benzi

Alain Camasses

Yoshimura Atsunori

Yuki Katou

Katsuhiko Shirahige

Simonetta Piatti
email: simonetta.piatti@crbm.cnrs.fr

Al

M Kupiec

A common molecular mechanism underlies the role of Mps1 in chromosome biorientation and the spindle assembly checkpoint

Keywords: chromosome biorientation, error correction, Mps1, Spc105, spindle assembly checkpoint Subject Category Cell Cycle Mitosis, Chromosome biorientation, Spindle assembly checkpoint, Kinetochore, Mps1, Aurora B

The Mps1 kinase corrects improper kinetochore-microtubule attachments, thereby ensuring chromosome biorientation. Yet, its critical phosphorylation targets in this process remain largely elusive. Mps1 also controls the spindle assembly checkpoint (SAC), which halts chromosome segregation until biorientation is attained. Its role in SAC activation is antagonised by the PP1 phosphatase and involves phosphorylation of the kinetochore scaffold Knl1/Spc105, which in turn recruits the Bub1 kinase to promote assembly of SAC effector complexes. A crucial question is whether error correction and SAC activation are part of a single or separable pathways. Here, we isolate and characterise a new yeast mutant, mps1-3, that is severely defective in chromosome biorientation and SAC signalling. Through an unbiased screen for extragenic suppressors, we found that mutations lowering PP1 levels at Spc105 or forced association of Bub1 with Spc105 reinstate both chromosome biorientation and SAC signalling in mps1-3 cells. Our data argue that a common mechanism based on Knl1/Spc105 phosphorylation is critical for Mps1 function in error correction and SAC signalling, thus supporting the idea that a single sensory apparatus simultaneously elicits both pathways.

INDEX OF TABLES

 Table 1 :
RESUME Introduction

La ségrégation des chromosomes est un processus extrêmement délicat qui doit se dérouler de façon très précise pendant la mitose. En effet, si des erreurs surviennent pendant la séparation du matériel génétique, des cellules avec un nombre anormal de chromosomes (aneuploïdie) peuvent être générées et aboutir à plusieurs types de maladies. Par exemple, des défauts de ségrégation pendant la méiose causent de pathologies telles que le syndrome de Down (trisomie 21), de Edwards (trisomie 18) ou de Klinefelter (XXY). En revanche, les aneuploïdies engendrées au cours de la mitose sont plutôt liées au déclenchement ou au développement de cancer, en causant un niveau d'expression différent d'oncogènes ou oncosuppresseurs.

Pour que la ségrégation des chromosomes ait lieu sans failles, plusieurs mécanismes clés doivent se dérouler de façon exacte : les organelles qui forment les microtubules (spindle pole bodies chez la levure et centrosomes chez les autres eucaryotes) doivent se dupliquer une seule fois pendant la transition G1/S ; en phase S l'ADN doit être répliqué de manière fidèle et, en prophase, condensé en chromosomes mitotiques. Pendant ce temps, les chromatides soeurs sont jointes par des molécules de cohésine, jusqu'à l'anaphase. Pendant la prométaphase, les chromatides capturent les microtubules du fuseau mitotique par des structures protéiques présentes au niveau des centromères et appelées kinétochores. D'abord, les kinétochores se lient aux surfaces latérales des microtubules et seulement après aux extrémités des microtubules, en formant des liaisons dites « end-on ». De façon importante, les chromatides soeurs doivent se lier aux microtubules émanés des pôles opposés du fuseau, donnant lieu à une tension au niveau des kinétochores. Ce processus est appelé bi-orientation. Si les kinétochores ne se lient pas correctement aux microtubules, un mécanisme de surveillance appelé Spindle Assembly Checkpoint (SAC) arrête les cellules en métaphase pour permettre la correction des erreurs. Le processus de correction des erreurs détache les kinétochores mal orientés des microtubules et requiert la kinase Aurora B. Une fois que tous les chromosomes sont bi-orientés, le SAC s'éteint, le fuseau mitotique s'allonge ce qui permet aux deux chromatides de migrer vers les deux pôles de la cellule. Enfin, en télophase les chromatides atteignent les pôles de la cellule qui se divise par le processus de cytokinèse, générant ainsi deux cellules filles.

Mps1 est une kinase conservée qui joue des rôles essentiels pendant la mitose. Elle est impliquée dans l'activation du SAC à travers la phosphorylation de la protéine du kinétochore KNL1/Spc105, qui est requise pour le recrutement au kinétochore de la kinase Bub1. L'activation du SAC implique la formation d'un complexe appelé MCC (Mitotic Checkpoint Complex), qui inhibe l'ubiquitine-ligase APC/C et empêche la dégradation de ses cibles. Cela provoque l'arrêt des cellules en métaphase. De façon importante, la phosphorylation de KNL1/Spc105 est contrée par la phosphatase PP1, qui à son tour est nécessaire pour éteindre le SAC.

Mps1 est aussi impliquée directement dans la régulation de la bi-orientation des chromosomes. En particulier, Mps1 intervient dans la correction des fautes de liaison entre kinétochores et microtubules, de façon similaire à Aurora B. Cependant, ses cibles dans ce processus sont mal connues, surtout chez la levure. En outre, sa relation avec Aurora B est assez controversée, car il n'est pas encore clair si les deux kinases coopèrent ou si elles promeuvent la bi-orientation par des mécanismes différents.

Par conséquent, l'étude détaillée du rôle de Mps1 dans ce processus est d'importance fondamentale.

Résultats et discussion

Au cours de ma thèse, j'ai caractérisé un nouveau mutant thermosensible de MPS1, appelé mps1-3, qui à des températures restrictives est incapable de rectifier les liaisons incorrectes entre kinétochores et microtubules et montre des défauts très prononcés dans la ségrégation des chromosomes. De plus, ce mutant est aussi incapable d'activer le SAC et avance dans le cycle cellulaire malgré l'absence de tension au niveau des kinétochores, accumulant des taux élevés d'aneuploïdie. Par conséquent, il représente un outil précieux pour élucider le rôle de la kinase Mps1 dans la ségrégation des chromosomes. Au niveau du domaine kinase du mutant mps1-3 la serine 635 est mutée en phénylalanine. J'ai donc étudié si cette mutation provoque une diminution de l'activité kinase de la protéine Mps1-3, mais j'ai pu démontrer que la protéine Mps1-3 est, au contraire, hyperactive. De plus, la protéine ne se localise pas correctement aux kinétochores, suggérant que le phénotype du mutant mps1-3 est dû au manque de phosphorylation de cibles spécifiques aux kinétochores.

Pour obtenir plus d'informations sur le mécanisme par lequel Mps1 contrôle la ségrégation des chromosomes, j'ai effectué un crible génétique ayant pour but d'identifier des suppresseurs de la thermosensibilité du mutant mps1-3. Cette approche m'a permis d'isoler plusieurs suppresseurs capables de se diviser à des températures restrictives. Une caractérisation génétique détaillée m'a permis de définir la nature de ces suppresseurs, les classer comme intra-géniques ou extra-géniques et identifier les gènes mutés responsables de la suppression. En ce qui concerne les suppresseurs extra-géniques, j'ai trouvé des mutations dans le gène SPC105, codant pour la protéine du kinétochore Spc105/KNL1, ainsi que dans le gène GLC7, codant pour la sous-unité catalytique de la phosphatase PP1. De manière intéressante, les mutations dans ces deux gènes touchent les domaines d'interaction entre Spc105 et PP1, qui sont impliqués dans la localisation de PP1 aux kinétochores et son activité d'extinction du SAC. Cela suggère que la suppression de la thermosensibilité du mutant mps1-3 est liée à la réduction d'activité de la phosphatase PP1 aux kinetochores. Remarquablement, ces suppresseurs rétablissent à la fois l'activation du SAC et une correcte bi-orientation des chromosomes, suggérant que Mps1 régule les deux processus par le même mécanisme. Nous avons donc émis l'hypothèse que la phosphorylation de Spc105/KNL1 par Mps1, qui à son tour recrute Bub1 aux kinetochores, et qui est contre balancée par la phosphatase PP1, pourrait être critique pour le rôle de Mps1, à la fois, dans l'activation du SAC et dans la régulation de la bi-orientation des chromosomes. Confortant cette hypothèse, j'ai démontré que les niveaux de phosphorylation de Spc105, ainsi que la localisation de Bub1 aux kinétochores, sont fortement réduits chez le mutant mps1-3, tandis qu'ils sont correctement restaurés chez les suppresseurs. Si le mécanisme responsable des défauts mitotiques du mutant mps1-3 est l'absence de Bub1 aux kinétochores, le recrutement artificiel de Bub1 aux kinétochores pourrait supprimer les phénotypes du mutant mps1-3 à des températures restrictives. J'ai donc créé un système basé sur la forte affinité entre la GFP et un GFPbinding domain (GBD) pour lier artificiellement Bub1 à Spc105. Remarquablement cette liaison constitutive permet de restaurer une correcte ségrégation des chromosomes, ainsi qu'une activation partielle du SAC chez le mutant mps1-3.

En bref, avec cette étude j'ai pu démontrer que l'un de mécanismes fondamentaux par lesquels Mps1 contrôle la bi-orientation de chromosomes est la phosphorylation de Spc105, qui est également nécessaire pour la signalisation du SAC. Cette partie de ma thèse a été récemment publiée dans la revue EMBO Reports (Benzi et al., 2020).

Grâce au crible génétique, j'ai aussi identifié des suppresseurs qui portent des mutations dans le gène GRR1, codant pour l'une des protéines F-box du complexe ubiquitine-ligase SCF. Le mécanisme moléculaire par lequel ces mutations suppriment les phénotypes mitotiques du mutant mps1-3 n'a pas encore été élucidé, mais j'ai pu constater que la protéine Grr1 est exprimée à des niveaux moins élevés dans ces mutants que dans les souches sauvages. Ces données suggèrent que la protéine est déstabilisée et pourrait, par conséquent, diminuer l'activité du complexe SCF. De plus, j'ai pu constater que Grr1 est localisée au niveau des centromeres/kinétochores, bien que ces résultats soient encore préliminaires. Grr1 n'a jamais été trouvée aux kinétochores auparavant et cette observation, si validée, suggère un nouveau rôle du complexe SCF Grr1 dans le contrôle de la ségrégation des chromosomes. Pour l'instant, la relation entre Grr1 et Mps1 n'est pas claire et la signification de la suppression des défauts du mutant mps1-3 par les mutations grr1 est encore difficile à comprendre.

Cependant au cours de ma thèse j'ai essayé de mieux étudier ce phénomène en testant quelques hypothèses. En particulier, j'ai pu constater que Grr1 ne semble pas réguler ni la dégradation, ni la localisation de Mps1 aux kinétochores. Étant donné que PP1 contre balance Mps1 à la fois dans le SAC et dans la bi-orientation des chromosomes, nous avons supposé que Grr1 pourrait directement contrôler la dégradation d'un inhibiteur de PP1, qui, à son tour, rétablirait l'équilibre kinase/phosphatase en s'accumulant chez le mutant mps1-3. Cependant, les sous-unités régulatrices de PP1 que j'ai testées jusqu'à présent n'ont pas montré des niveaux plus élevés de protéines chez les mutants grr1, suggérant que Grr1 pourrait contrôler soit d'autres sous-unités de PP1, soit d'autres protéines non liées à la phosphatase PP1.

Le crible génétique m'as aussi permis d'identifier des suppresseurs intra-géniques, qui présentent une deuxième mutation dans MPS1. Ces mutations sont toutes dans le domaine catalytique de la kinase, ce qui nous a permis d'émettre l'hypothèse que le mécanisme derrière leur suppression pourrait être une baisse d'activité kinase par rapport à la protéine mutante Mps1-3. Puisque Mps1 contrôle son turnover aux kinétochores à travers son autophosphorylation, nous avons supposé que la localisation réduite de la protéine Mps1-3 aux kinétochores pourrait être due à l'augmentation de son activité kinase, qui diminuerait son temps de rétention aux kinétochores. J'ai donc testé l'activité kinase des suppresseurs, avec et sans la mutation mps1-3, et j'ai pu constater une diminution partielle de l'activité kinase de la plupart des protéines mutantes, surtout en ce qui concerne la phosphorylation d'un substrat externe plutôt que l'autophosphorylation. De plus, une analyse préliminaire par ChIPseq nous a permis de conclure que la localisation aux kinétochores de ces protéines mutantes est généralement rétablie. Malgré qu'il ait été montré que Mps1 interagit avec les kinétochores via son domaine N-terminale, mes données suggèrent que le domaine kinase dans la partie C-terminale contribue également à réguler la dynamique de la protéine aux kinétochores.

Conclusion

Dans l'ensemble, les données obtenues pendant ma thèse nous ont permis de mieux comprendre le mécanisme par lequel Mps1 contrôle la bi-orientation des chromosomes chez la levure. Nous avons démontré que le SAC ainsi que le processus de correction des erreurs de liaisons entre kinétochores et microtubules répondent à un même capteur qui implique Mps1 et déclenche les deux processus par la phosphorylation de Spc105. De plus, nos données préliminaires suggèrent un nouveau rôle du complexe SCF Grr1 comme possible partenaire de Mps1 dans la régulation de la bi-orientation des chromosomes. Plusieurs des mécanismes décrits ici sont conservés chez les eucaryotes et nos conclusions pourraient être valides pour d'autres organismes.

Mps1 est surexprimée dans plusieurs cancers et plusieurs inhibiteurs de Mps1 ont été développés pour le traitement du cancer, malheureusement sans succès jusqu'à présent. Une meilleure compréhension des mécanismes moléculaires utilisés par Mps1 pour contrôler la progression au cours de la mitose serait souhaitable pour envisager des nouvelles stratégies thérapeutiques.

Chromosome segregation is an essential process that must take place faithfully during mitosis. If chromosome segregation is unequal, daughter cells accumulate unbalanced numbers of chromosomes, called aneuploidies, which are associated to severe pathologies.

Mps1 is an essential, conserved kinase that plays different key roles during mitosis. In budding yeast it is implicated in the duplication of the microtubule organizing centers (called spindle pole bodies), and therefore for the formation of a bipolar spindle. Mps1 is also essential for the activation of the spindle assembly checkpoint (SAC), the surveillance mechanism that arrests the cells in metaphase until proper kinetochore-microtubule attachments are established. This is achieved through phosphorylation of the kinetochore protein Spc105/KNL1, which then recruits the checkpoint protein Bub1 to kinetochores. Spc105/KNL1 phosphorylation is reversed by the phosphatase PP1, which conversely displaces Bub1 from kinetochores and silences the checkpoint. Mps1 is also involved in chromosome bi-orientation, i.e. the process by which sister chromatids attach to microtubules emanating from opposite spindle poles. In particular, Mps1 contributes to error correction, which involves detachment of improper kinetochore-microtubule connections to improve their chance to attach properly. However, its role in this process is far from being fully understood. Indeed, particularly in budding yeast, the targets of Mps1 that are critical for chromosome bi-orientation are not known and relationships, if any, between Mps1 and the main player in the error correction process, i.e. the kinase Ipl1/Aurora B, are not understood. Therefore, deciphering the role of Mps1 in the control of chromosome bi-orientation through the identification of its critical substrates and a comprehension of its possible interplay with Ipl1/Aurora B are important goals that have been pursued during this thesis work.

For my studies I took advantage of a novel temperature sensitive mutant, mps1-3, which turned out to be specifically defective in chromosome segregation and SAC signaling, but capable to duplicate spindle pole bodies and form a bipolar spindle, unlike the majority of mps1 mutants characterized so far. This mutant bears a mutation in the kinase domain (Ser635Phe), but surprisingly the Mps1-3 protein displays increased kinase activity in vitro relative to the wild type protein. Conversely, the protein does not localize at kinetochores, suggesting that the mps1-3 mitotic defects stem from the lack of phosphorylation of critical kinetochore substrates.

To gain further insights into the mechanism by which Mps1 regulates chromosome bi-orientation, I performed a genetic screen for spontaneous suppressors of the temperature-sensitivity of the mps1-3 mutant. During my thesis I mainly focused on two classes of extragenic suppressors: one class comprised mutations in SPC105, which encodes for the kinetochore protein Spc105/KNL1, and the other included mutations in GLC7, that encodes for the catalytic subunit of the phosphatase PP1.

Remarkably, all suppressing mutations hit the Spc105-Glc7 interaction interface, suggesting that reduced levels of PP1 phosphatase activity at kinetochores underlie the suppression of the temperature-sensitivity of mps1-3 cells. Importantly, the suppressors restored both SAC signaling and proper chromosome bi-orientation, suggesting that the same molecular mechanism underlies the role of Mps1 in both processes. We have proposed that phosphorylation of Spc105/KNL1 by Mps1, which leads to Bub1 kinetochore recruitment and is antagonized by the phosphatase PP1, is not only required to trigger SAC signaling, but also to establish chromosome bi-orientation. Consistently, phosphorylation of Spc105 and Bub1 kinetochore localization, which were markedly affected in mps1-3 cells, were efficiently restored in the spc105 and GLC7 suppressors. Moreover, artificial recruitment of Bub1 on Spc105 suppressed the chromosome segregation defects of the mps1-3 mutant cells and partially restored SAC signaling.

Thus, a main conclusion of my thesis work is that SAC and error correction are triggered by a single sensory device involving Mps1 and antagonized by PP1.

Through the same genetic screen, I also found suppressors carrying mutations in an F-box protein of the SCF ubiquitin-ligase complex, Grr1, which I started characterizing. I could gain preliminary data indicating that Grr1 might localize at kinetochores, suggesting that the SCF Grr1 complex could be a novel player in the control of chromosome bi-orientation at kinetochores. The relationship between this ubiquitin-ligase and the kinase Mps1 will be further explored the future.

Finally, through the genetic screen above, I could also isolate intragenic suppressors, carrying a second mutation in the kinase domain of MPS1. Unlike the extragenic suppressors, the intragenic suppressors analyzed so far could restore Mps1 kinetochore localization. Since Mps1 regulates its own turnover at kinetochores by autophosphorylation, we postulated that the elevated kinase activity of the Mps1-3 mutant protein might be responsible for its displacement from kinetochores. In this view, the suppressors might re-establish kinetochore localization of Mps1-3 by decreasing its kinase activity. Surprisingly, although most of the suppressing mutations reduced the ability of Mps1-3 to phosphorylate an exogenous substrate (Spc105 or MBP), they did not significantly affect autophosphorylation, suggesting that Mps1-dependent phosphorylation of an as yet unknown kinetochore protein could influence Mps1 residence time.

INTRODUCTION 21

An overview of the mitotic cell cycle

Chromosome transmission is an extremely important process that needs to be finely tuned. Indeed, each cell must inherit a full set of chromosomes and, thus, a balanced amount of genetic information in order to be functional. Therefore, during the mitotic cell cycle replicated DNA must be equally partitioned between the two daughter cells through the delicate process of chromosome segregation.

The ensemble of processes that lead to separation of the replicated DNA and cytoplasm to make two new cells is called mitosis, from the Greek μίτος (mitos) that literally means "warp thread". This term has been coined by Walter Flemming in 1882, after visualization of chromatin that appeared as long threads (described in [START_REF] Sharp | An Introduction To Cytology[END_REF]. Mitosis occupies a relatively short time compared to the whole of the mitotic cell cycle that comprises also interphase, which is much longer compared to mitosis.

Interphase is further divided in G1, S and G2 phases. In G1 the cell grows in size and synthesizes mRNAs and proteins in preparation for subsequent steps leading to mitosis; during S phase DNA is replicated; finally, the G2 phase is the second growth and protein synthesis period in preparation for mitosis. Mitosis, in turn, includes four main stages: prophase (that can be divided into early prophase and prometaphase), metaphase, anaphase and telophase (see below). Mitosis is then followed by cytokinesis, which is the moment where the cytoplasm of the cell is finally divided in two, making the two new cells [START_REF] Cooper | The Eukaryotic Cell Cycle. The Cell: A Molecular Approach[END_REF].

Below is an overview of the essential processes that need to correctly take place in order to properly segregate chromosomes.

During the G1/S transition cells must duplicate once and only once the microtubule organizing centers (MTOCs), the structures responsible for microtubule nucleation. These assemblies (called spindle pole bodies or SPBs in budding yeast and centrosomes in other eukaryotes) nucleate microtubules that in mitosis get organized in a bipolar spindle, which in anaphase pulls chromosomes apart towards the two sides of the dividing cell (reviewed in [START_REF] Fu | The centrosome and its duplication cycle[END_REF][START_REF] Jaspersen | THE BUDDING YEAST SPINDLE POLE BODY: Structure, Duplication, and Function[END_REF]. In S phase (Synthesis) the DNA is accurately duplicated through DNA replication. In prophase the mitotic spindle starts forming and DNA is condensed from its interphasic organization into mitotic chromosomes. Condensin complexes are among the proteins that mediate this process (reviewed in [START_REF] Kalitsis | Condensin, master organizer of the genome[END_REF]. During this time the two replicated sister chromatids for each chromosome must be held together until segregation can take place, later in anaphase. Central to this process is another protein complex called cohesin (reviewed in [START_REF] Mehta | Cohesin: A guardian of genome integrity[END_REF]. During prometaphase, sister chromatids start to be attached to microtubules of the mitotic bipolar spindle through their kinetochores, protein assemblies built on centromeres. Kinetochores first take contact with the lateral surface of microtubules (lateral attachment), and then with microtubule tips (end-on attachments), generating more stable connections. Once all pairs of sister chromatids are attached in a bipolar fashion (i.e. to microtubules emanating from opposite sides of the spindle), they congress to the metaphase plate, the imaginary position equidistant from the spindle poles (reviewed in [START_REF] Tanaka | Kinetochore-microtubule interactions: steps towards bi-orientation[END_REF].

To achieve equal chromosome segregation, it is mandatory that sister chromatids get bipolarly attached (amphitelic attachment). If this does not occur, a surveillance mechanism called Spindle Assembly Checkpoint (SAC), halts the cell cycle in metaphase until bioriented attachments are generated (reviewed in Musacchio, 2015). During this temporary arrest, erroneous microtubulekinetochore attachments are corrected by destabilization of faulty connections and subsequent formation of new correct attachments. This process is mainly mediated by the Chromosomal Passenger Complex, whose catalytic subunit is the kinase Aurora B (reviewed in Krenn and Musacchio, 2015). Once these errors are corrected, the SAC is silenced, mainly thanks to the action of the protein phosphatase PP1, and anaphase ensues (reviewed in [START_REF] Corbett | Molecular Mechanisms of Spindle Assembly Checkpoint Activation and Silencing[END_REF]. During this stage, the bipolar spindle elongates pulling sister chromatids towards the poles of the cells. In telophase, then, the chromosomes reach the cell poles, the nuclear membrane reforms around the segregated chromosomes and shortly after the cytoplasm and the entire cell are divided during cytokinesis, generating the two separated daughter cells. Moreover, DNA starts to be decondensed again (Fig. 1, [START_REF] Bloom | Tension management in the kinetochore[END_REF]. Budding yeast mitosis is closed, meaning that the nuclear membrane never breaks down. Therefore chromosome segregation takes place inside the nucleus. During G1, yeast cells grow and start to duplicate the SPBs (spindle pole bodies, the microtubule organising centers); here first monopolar attachments take place. At the G1/S transition bud emergence occurs, SPBs duplicate and chromosomal DNA starts being replicated, leading to the temporary dissociation of kinetochores from microtubules. In G2 the spindle becomes bipolar and in metaphase sister chromatids bi-orient on the mitotic spindle, meaning that they attach to microtubules emanating from the two opposite spindle poles. During anaphase the spindle elongates and the two sets of sister chromatids segregate to spindle poles. Finally during telophase the nuclear membrane separates into two separated nuclei along with the two newformed cells, which separate through cytokinesis.

Consequences of chromosome missegregation

All the processes described above have to carefully take place in every cell cycle in order to segregate always one copy of each chromosome to each daughter cell and to avoid unbalanced chromosome numbers. Indeed, this phenomenon, called aneuploidy, can have serious consequences for the cell and the whole multicellular organism and are linked to many important diseases. Different pathologies arise from aneuploidies generated during meiosis or mitosis. Meiosis is the process that generates haploid gametes through two rounds of division: meiosis I and meiosis II. During meiosis I homologous chromosomes are segregated, whereas sister chromatids are segregated during meiosis II. Aneuploidies here are often caused by a phenomenon called "nondisjunction", where either homologous chromosomes or sister chromatids fail to separate or prematurely separate, thus leading to gametes with DNA content n+1 or n-1 [START_REF] Herman | Aneuploid[END_REF]. Therefore, these gametes will generate an entire organism carrying an extra or lacking a chromosome. Aneuploid zygotes only rarely complete embryonic development and are usually lethal. However, aneuploidy sometimes can be compatible with life and cause pathological disorders that have distinctive features depending on the chromosome involved: trisomy of chromosome 21 causes Down syndrome; trisomy of chromosome 13 causes Patau syndrome; trisomy of chromosome 18 causes Edwards syndrome; extra X chromosome/s in males causes Klinefelter syndrome; lack of one X chromosome in females causes Turner syndrome; an extra X chromosome in females cause Triple X syndrome; an extra Y chromosome in males causes Jacobs syndrome (for details see table 1).

One of the causes of nondisjunction is the early separation of sister chromatids that are normally held together until metaphase II by cohesin complexes. This phenomenon has been associated to the mother's age, as it becomes more frequent with increasing maternal age (reviewed in [START_REF] Kuliev | Meiotic and mitotic nondisjunction: lessons from preimplantation genetic diagnosis[END_REF].

Syndrome

Chromosome Symptoms The name of the syndrome, the chromosome involved and the main symptoms are indicated.

Errors during chromosome segregation can occur also during mitosis, generating a "mosaic", where only some cells in the body carry an unbalanced chromosome number. Aneuploidies in mitosis have been related to cancer. Indeed, they are highly prevalent in cancer cells and are thought to contribute to malignant transformation and tumor progression. Understanding the role of aneuploidy in cancer is not trivial, especially if one considers what is referred to as the "aneuploidy paradox" [START_REF] Sheltzer | The aneuploidy paradox: costs and benefits of an incorrect karyotype[END_REF]: indeed, aneuploidy is often detrimental for cells during the development of a tissue or an organism, but is well-tolerated in cancer cells. 90% of solid tumors present aneuploid cells, with about 25% of the genome being altered in copy number through structural or numerical chromosomal alterations (reviewed in Ben-David and Amon, 2020). A change in the number of chromosomes can modify the gene dosage of certain tumors suppressors or oncogenes that can help the tumor develop faster and/or in a more suitable environment.

Although for long time it has been debated whether aneuploidies could be the cause or the consequence of cancer progression, today some lines of evidence indicate that they are often the cause of cancer transformation. First, aneuploidies are found in primary tumors and specific chromosome aneuploidies correlate with distinct tumor phenotypes. Second, aneuploid tumor cell lines display an elevated rate of chromosome instability, indicating that aneuploidy is a dynamic chromosome mutational event associated with cell transformation. Third, many genes regulating chromosome segregation have been found mutated in cancer cells, suggesting that these mutations could underlie the aneuploidies that are found in cancer (reviewed in [START_REF] Sen | Aneuploidy and cancer[END_REF]. Therefore, aneuploidies during mitosis likely have a central role in cancer development and cells must avoid them by orchestrating a perfect chromosome segregation that always partitions the correct number of chromosomes between the two daughter cells. Understanding all the key mechanisms necessary for a proper chromosome segregation is therefore paramount for our complete comprehension of the processes leading to aneuploidy-related human diseases.

The following chapters of this introduction will describe in further details the critical steps that are required to properly segregate chromosomes during a mitotic cell cycle.

Building a bipolar spindle: spindle pole bodies and centrosomes

In order to be pulled to the two poles of the cells, chromosomes need to attach to spindle microtubules, which are generated by Microtubule Organizing Centers (MTOCs).

Budding yeast MTOCs are the Spindle Pole Bodies (SPBs) that nucleate both spindle and astral microtubules, which are responsible, respectively, for chromosome segregation and spindle positioning. One important feature of this organelle is that it is embedded in the nuclear envelope throughout the whole cell cycle. This is important to allow spindle formation and kinetochoremicrotubule attachments in an organism where mitosis is close, meaning that the nuclear envelope does not break down during mitosis (reviewed in [START_REF] Cavanaugh | Big Lessons from Little Yeast: Budding and Fission Yeast Centrosome Structure, Duplication, and Function[END_REF].

The structure of the SPB has been extensively studied by electron microscopy [START_REF] Adams | Localization of Core Spindle Pole Body (SPB) Components during SPB Duplication in Saccharomyces cerevisiae[END_REF][START_REF] Bullitt | The yeast spindle pole body is assembled around a central crystal of Spc42p[END_REF][START_REF] O'toole | High-voltage electron tomography of spindle pole bodies and early mitotic spindles in the yeast Saccharomyces cerevisiae[END_REF]. The SPB is a cylindrical organelle organized in three main plaques: the outer plaque is on the cytoplasmic side and is associated with cytoplasmic microtubules; the inner plaque is on the nucleoplasmic side and is associated with nuclear microtubules; the central plaque is responsible for the insertion of the SPB into the nuclear membrane.

One side of the central plaque is associated with a structure called "the half-bridge", which is the site for the assembly of the new SPB during SPB duplication. Moreover, there are two additional layers, called first and second intermediate layers (IL1 and IL2), which are situated between the outer and the central plaque (reviewed in [START_REF] Jaspersen | THE BUDDING YEAST SPINDLE POLE BODY: Structure, Duplication, and Function[END_REF].

The budding yeast SPB contains 18 different components, and almost all of them are encoded by essential genes (reviewed in [START_REF] Cavanaugh | Big Lessons from Little Yeast: Budding and Fission Yeast Centrosome Structure, Duplication, and Function[END_REF]. Spc42 forms the central core of the SPB, with about 1000 molecules assembling into trimers of dimers, generating a hexagonal lattice [START_REF] Bullitt | The yeast spindle pole body is assembled around a central crystal of Spc42p[END_REF][START_REF] Donaldson | Spc42p: a phosphorylated component of the S. cerevisiae spindle pole body (SPD) with an essential function during SPB duplication[END_REF]. Spc42 binds to Spc110 [START_REF] Adams | Localization of Core Spindle Pole Body (SPB) Components during SPB Duplication in Saccharomyces cerevisiae[END_REF], a protein that in turns binds to Spc98 [START_REF] Geissler | The spindle pole body component Spc98p interacts with the gamma-tubulin-like Tub4p of Saccharomyces cerevisiae at the sites of microtubule attachment[END_REF], a component of the γ-tubulin small complex (γ-TuSc), necessary for microtubule nucleation. This complex is composed of two molecules of the γ-tubulin protein Tub4 and two other proteins, Spc97 and Spc98, forming a Y-shape, with the N termini of Spc97 and Spc98 at the base of the Y, while the C terminal arms of each interact with one molecule of Tub4 (Fig. 3) (reviewed in [START_REF] Kilmartin | Lessons from yeast: the spindle pole body and the centrosome[END_REF]. Another SPB module is represented by the membrane proteins collectively called SPIN (SPB Insertion Network), which are Bbp1, Mps2, Nbp1 and Ndc1, almost all integral membrane proteins, except for Bbp1 (reviewed in [START_REF] Rüthnick | Duplication and Nuclear Envelope Insertion of the Yeast Microtubule Organizing Centre, the Spindle Pole Body[END_REF]. Ndc1, is a shared component between this SPB module and the Nuclear Pore Complex (NPC) that contributes to the SPB insertion into the nuclear envelope [START_REF] Araki | The Saccharomyces cerevisiae Spindle Pole Body (SPB) Component Nbp1p Is Required for SPB Membrane Insertion and Interacts with the Integral Membrane Proteins Ndc1p and Mps2p[END_REF].

The half bridge main component is the protein Sfi1, which forms a rigid filament that is connected to the nuclear membrane through the protein Kar1 [START_REF] Li | Structural role of Sfi1p-centrin filaments in budding yeast spindle pole body duplication[END_REF] (Fig. 4). The first step towards SPB duplication is the elongation of the half-bridge into an entire bridge. This depends on the anti-parallel addition of Sfi1 molecules to already existing molecules of Sfi1 on the half-bridge [START_REF] Kilmartin | Sfi1p has conserved centrin-binding sites and an essential function in budding yeast spindle pole body duplication[END_REF][START_REF] Li | Structural role of Sfi1p-centrin filaments in budding yeast spindle pole body duplication[END_REF] . At the beginning of the G1 phase, at the distal end of the bridge, a structure called satellite, carrying an ellipsoidal shape, is formed and is considered the SPB-precursor. This structure consists of components of the core SPB and membrane proteins of the SPB Insertion Network [START_REF] O'toole | High-voltage electron tomography of spindle pole bodies and early mitotic spindles in the yeast Saccharomyces cerevisiae[END_REF]. At the end of G1, when cells enter S phase, the satellite grows in size, becoming the so-called duplication plaque (DP). The last step is then the insertion of the new SPB into the nuclear membrane that requires the formation of a nuclear envelope pore, which is a curved part of the membrane where the inner nuclear membrane and the outer nuclear membrane are contiguous. Nuclear pore complexes could facilitate membrane incorporation of the new SPB [START_REF] Rüthnick | Characterization of spindle pole body duplication reveals a regulatory role for nuclear pore complexes[END_REF]. The insertion process is accompanied by a change in the orientation of the growing duplication plaque: the Spc42 lattice changes from a vertical to a parallel orientation relative to the nuclear envelope [START_REF] Rüthnick | Characterization of spindle pole body duplication reveals a regulatory role for nuclear pore complexes[END_REF]. Moreover, the insertion enables nuclear components of the SPB to localize to the new forming SPB. Finally, Spc110 and the γ-tubulin complex localize to the new-formed SPB, thus rendering it mature to nucleate microtubules [START_REF] Rüthnick | Characterization of spindle pole body duplication reveals a regulatory role for nuclear pore complexes[END_REF] (Fig. 5).

At this point, in order to form a bipolar spindle, the duplicated SPBs need to separate, inheriting both halves of the bridge in order to be competent for the next duplication cycle. Therefore, the bridge is cleaved in its center, by separating two molecules of Sfi1 [START_REF] Avena | Licensing of Yeast Centrosome Duplication Requires Phosphoregulation of Sfi1[END_REF][START_REF] Elserafy | Molecular mechanisms that restrict yeast centrosome duplication to one event per cell cycle[END_REF].

One important feature of the SPB duplication is that it is conservative, meaning that the newly formed SPB has always completely new SPB components, while the old SPB remains intact. In addition, the old SPB is preferentially segregated to the daughter cell (the bud), whereas the newly formed SPB usually stays into the mother cell after duplication (Pereira et al., 2001). Mps1 is an essential kinase that promotes SPBs duplication. It is a serine/threonine kinase that has been described as dual-specificity kinase because it can also phosphorylate tyrosines in vitro [START_REF] Lauzé | Yeast spindle pole body duplication gene MPS1 encodes an essential dual specificity protein kinase[END_REF]. Its name (MonoPolar Spindle 1) describes how it has been identified: mps1 temperaturesensitive mutants were showing monopolar spindles and disability to duplicate SPBs at restrictive temperatures (Winey et al., 1991). Indeed, Mps1 plays several roles in SPBs duplication (Schutz and Winey, 1998). First, phosphorylation of Spc42 by Mps1 is required for its assembly into a lattice and for the formation of the satellite (Castillo et al., 2002). Second, Mps1 phosphorylation of Spc29 is essential to promote the stability of the protein in vivo [START_REF] Holinger | Budding yeast centrosome duplication requires stabilization of Spc29 via Mps1-mediated phosphorylation[END_REF]. Moreover, Spc29

phosphorylation by Mps1 has been shown to be important for the recruitment in G1/S of the Mps2-Bbp1 complex on the newly formed SPB, in order to facilitate its insertion into the nuclear envelope (Araki et al., 2010). Third, Cdc31 is also phosphorylated by Mps1 and this seems to be important to control the cell cycle-specific physical association of Cdc31 with the protein Kar1 (Araki et al., 2010).

Mps1 phosphorylation of other SPB components have been also associated to functions unrelated to SPB duplication: Spc98 phosphorylation may influence microtubule attachment to the inner plaque of the SPB after the nucleation step [START_REF] Pereira | Spc98p Directs the Yeast γ-Tubulin Complex into the Nucleus and Is Subject to Cell Cycle-dependent Phosphorylation on the Nuclear Side of the Spindle Pole Body[END_REF]. Moreover, Spc110 phosphorylation by Mps1 is important for Spc110 function in mitosis during spindle formation [START_REF] Friedman | Yeast Mps1p Phosphorylates the Spindle Pole Component Spc110p in the N-terminal Domain[END_REF].

The microtubule organizing centers for spindle assembly in higher eukaryotes are called centrosomes.

Similar to SPBs, they duplicate once in the cell cycle in order to generate a bipolar mitotic spindle.

However, the structure of the centrosome in quite different from the yeast SPB. Indeed, it is composed of two orthogonally placed centrioles surrounded by the pericentriolar material -or matrix (PCM).

Centrioles are conserved organelles that are also inherent components of cilia. In mammalian cells they comprise nine short microtubule triplets organized in a symmetric cylinder called "the cartwheel" (reviewed in Wang et al., 2014). The PCM was originally described as an amorphous electron-dense mass of material [START_REF] Robbins | The centriole cycle in synchronized HeLa cells[END_REF]). However, using super-resolution microscopy, an organized structure with separable spatial domains has been more recently identified [START_REF] Fu | Structured illumination of the interface between centriole and peri-centriolar material[END_REF][START_REF] Lawo | Subdiffraction imaging of centrosomes reveals higher-order organizational features of pericentriolar material[END_REF]. Moreover, this structure contains proteins responsible for microtubule nucleation [START_REF] Gould | The pericentriolar material in Chinese hamster ovary cells nucleates microtubule formation[END_REF]. Indeed, the γ-tubulin ring complex (γ-TuRC) is embedded inside the PCM.

A single cartwheel is composed of a central ring (hub) from which nine filaments (spokes) emanate.

Each spoke connects to one tubulin monomer of the triplet microtubule through a structure called the pinhead. Usually there are multiple cartwheels in the centriole, and their number can vary between different organisms and stages of centriole maturation. For example, cartwheels are present in the centriole during interphase but disappear during mitosis in humans (reviewed in [START_REF] Hirono | Cartwheel assembly[END_REF]. The molecular structure of the cartwheel consists of a coiled-coil-mediated homodimer of the protein SAS-6/Bld-12. The interaction between homodimers mediated by adjacent N terminal domains results in further oligomerization of SAS-6 homodimers [START_REF] Kitagawa | Structural Basis of the 9-Fold Symmetry of Centrioles[END_REF]. Additionally, Cep135/Bld-10 contributes to form the structure [START_REF] Hiraki | Bld10p constitutes the cartwheel-spoke tip and stabilizes the 9-fold symmetry of the centriole[END_REF]. For what concerns the microtubule triplets, they consist of three different tubulins: γ/δ/ε. These triplets elongate and reach a mature length, which is determined by the incorporation of CP110, CEP97, Centrin-2 and hPOC (Fig. 6) [START_REF] Azimzadeh | hPOC5 is a centrin-binding protein required for assembly of full-length centrioles[END_REF][START_REF] Spektor | Cep97 and CP110 suppress a cilia assembly program[END_REF]. centriole, called the procentriole, which then elongates during G2 until reaching a similar size to the mother centriole. Before mitosis the daughter centriole starts accumulating PCM, becoming able to nucleate microtubules in order to form a full spindle (reviewed in [START_REF] Fu | The centrosome and its duplication cycle[END_REF]. The connection between the parental centrioles is called G1-G2 tether (GGT) and provides a loose link between the proximal ends of these centrioles. It is therefore established in G1 and disassembles in G2, when the two centrosomes separate in preparation for spindle assembly. The second type of centrioles connection is called S-M linker (SML), which forms during S phase and connects the forming procentriole to the parental adjacent centriole. This linker is tighter compared to the GGT and persists until the mother and daughter centrioles are disengaged during mitosis, when they are separated to get ready to start a new centrosome duplication cycle (reviewed in [START_REF] Nigg | The centrosome cycle: Centriole biogenesis, duplication and inherent asymmetries[END_REF]. The main components of the SML linker are rootletin, Nap1 and LRRC45 [START_REF] Yang | Rootletin interacts with C-Nap1 and may function as a physical linker between the pair of centrioles/basal bodies in cells[END_REF]. The latter, when phosphorylated by the kinase Nek2, gets displaced from the centrosomes leading to disruption of the centrosomal linker [START_REF] He | LRRC45 Is a Centrosome Linker Component Required for Centrosome Cohesion[END_REF].

During prophase, the centrosomes will generate a bipolar spindle that will pull themselves away separating them at the two opposite poles of the dividing cell (Fig. 7). At the beginning of G1, cells contain a single centrosome with two perpendicular centrioles. During G1 the link between the centrioles (purple bar) is dissolved (centriole disengagement) and centrioles are only connected by a fibrous structure (purple strands). In S phase, the duplicating centrosomes assemble two new centrioles perpendicular to the two existing ones. Then, the daughter centriole finally acquires the same features of the mother centriole and the fibrous linker between daughter and mother centrioles is cleaved. Due to the link between the old and the newly formed centriole for each couple (purple bars), the two centrosomes are now engaged and prevented from further replication. In late G2, the two centrosomes mature by recruiting additional PCM components (grey shadow) in order to form the mitotic spindle. The centrosomes then separate and move to the opposite sides of the nucleus. This is simultaneous with nuclear envelope breakdown (NEBD). From Barr and Gergely, 2007. Briefly, the key cell cycle kinases involved in centrosome duplication are: Polo-like kinase 4 (Plk4), which is essential for the assembly of the procentriole in G1/S [START_REF] Bettencourt-Dias | SAK/PLK4 is required for centriole duplication and flagella development[END_REF][START_REF] Habedanck | The Polo kinase Plk4 functions in centriole duplication[END_REF]; Polo-like kinase 1 (Plk1), which triggers the centriole disengagement during mitosis [START_REF] Lončarek | Centriole Reduplication during Prolonged Interphase Requires Procentriole Maturation Governed by Plk1[END_REF][START_REF] Tsou | Polo kinase and separase regulate the mitotic licensing of centriole duplication in human cells[END_REF]; Cdk2, that was shown to be important for centriole separation and centrosome duplication to begin [START_REF] Hinchcliffe | Requirement of Cdk2-cyclin E activity for repeated centrosome reproduction in Xenopus egg extracts[END_REF][START_REF] Matsumoto | Cyclin-dependent kinase 2 (Cdk2) is required for centrosome duplication in mammalian cells[END_REF][START_REF] Meraldi | Centrosome duplication in mammalian somatic cells requires E2F and Cdk2-cyclin A[END_REF][START_REF] Tokuyama | Specific phosphorylation of nucleophosmin on Thr(199) by cyclin-dependent kinase 2-cyclin E and its role in centrosome duplication[END_REF]. However, it has been then shown that Cdk2 depleted mouse fibroblasts have only a minor defect in centrosomes duplication and proliferate well [START_REF] Duensing | Cyclin-dependent kinase 2 is dispensable for normal centrosome duplication but required for oncogene-induced centrosome overduplication[END_REF].

Finally, the kinase Aurora A seems to be important for centrosome maturation and spindle microtubule growth [START_REF] Magnaghi-Jaulin | Aurora A Protein Kinase: To the Centrosome and Beyond[END_REF].

In contrast to its well established role in SPB duplication in budding yeast, the kinase Mps1 has no confirmed role in the duplication of centrosomes in higher eukaryotes. In human and mouse, Mps1 was initially shown to promote centrosome duplication [START_REF] Fisk | Human Mps1 protein kinase is required for centrosome duplication and normal mitotic progression[END_REF][START_REF] Fisk | The mouse Mps1p-like kinase regulates centrosome duplication[END_REF][START_REF] Kasbek | Preventing the Degradation of Mps1 at Centrosomes Is Sufficient to Cause Centrosome Reduplication in Human Cells[END_REF]. Indeed, the kinase appears to be important for the formation of centriolar assembly through SAS-6 phosphorylation [START_REF] Pike | Centriole assembly and the role of Mps1: defensible or dispensable?[END_REF]. Moreover, Mps1 phosphorylation on the protein Centrin 2 has been shown to stimulate the centriole assembly pathway [START_REF] Yang | Mps1 Phosphorylation Sites Regulate the Function of Centrin 2 in Centriole Assembly[END_REF]. However, contradictory data exist in the literature and the role of Mps1 in centrosome duplication has not been confirmed by others. For instance, chemical inhibition of Mps1 does not seem to have consequences on centrosome duplication (Kwiatkowski et al., 2010;Santaguida et al., 2010;Stucke et al., 2002). Therefore, Mps1 role in this process, if any, needs further investigation.

Building a bipolar spindle: power on with motor proteins

A mitotic spindle is composed of interpolar microtubules, which keep the SPBs/centrosomes in contact and push them apart during anaphase, and kinetochore microtubules, which connect the kinetochores to the SPB/centrosome (reviewed in [START_REF] Fraschini | Factors that Control Mitotic Spindle Dynamics[END_REF].

The first step towards the formation of a bipolar spindle is microtubule nucleation. The best model to describe the involvement of the γ-tubulin ring complex in microtubule nucleation is called "template model" (Fig. 8): adjacent γ-tubulin subunits interact laterally with each other forming a ring, by making shoulder-to-shoulder contacts [START_REF] Zheng | Nucleation of microtubule assembly by a gammatubulin-containing ring complex[END_REF]. This ring has therefore microtubule-likesymmetry, indicating that γ-tubulin complexes serve as microtubule templates. Indeed, it acts as a scaffold for α/β tubulin dimers, speeding up the assembly of the ring that forms the growing microtubule [START_REF] Kollman | Microtubule nucleating γTuSC assembles structures with 13-fold microtubule-like symmetry[END_REF]. Moreover, the γ-tubulin complex also acts as a cap for the microtubule minus-end while the microtubule continues growth from its plus-end. This provides stability and protection to the microtubule minus-end from enzymes that could lead to its depolymerization, and at the same time inhibits minus-end growth [START_REF] Wiese | A new function for the gamma-tubulin ring complex as a microtubule minus-end cap[END_REF]. During prophase, centrosomes/SPBs separate towards the two opposite poles and a bipolar spindle is formed. This phenomenon is mainly ensured by motor proteins of different classes. The main players in this process are proteins of the kinesin-5 family, which are essential for the formation of a bipolar spindle in many organisms, such as yeast, mammals and fly [START_REF] Blangy | Phosphorylation by p34cdc2 regulates spindle association of human Eg5, a kinesin-related motor essential for bipolar spindle formation in vivo[END_REF][START_REF] Enos | Mutation of a gene that encodes a kinesin-like protein blocks nuclear division in A. nidulans[END_REF][START_REF] Goshima | The roles of microtubule-based motor proteins in mitosis: comprehensive RNAi analysis in the Drosophila S2 cell line[END_REF][START_REF] Hagan | Novel potential mitotic motor protein encoded by the fission yeast cut7+ gene[END_REF][START_REF] Hoyt | Two Saccharomyces cerevisiae kinesin-related gene products required for mitotic spindle assembly[END_REF]. Kinesin-5 family members function as bipolar homotetramers, with pairs of motor heads at opposite ends of an elongated molecule [START_REF] Scholey | Structural basis for the assembly of the mitotic motor Kinesin-5 into bipolar tetramers[END_REF]. They use microtubules simultaneously as cargo and track, as they bind and walk on two anti-parallel microtubules at the same time, thereby sliding them apart [START_REF] Kapitein | The bipolar mitotic kinesin Eg5 moves on both microtubules that it crosslinks[END_REF]. Eg5 is the main kinesin-5 in mammals, whereas in budding yeast Cin8 and Kip1 are the main kinesin-5 members involved in this process. Interestingly, these yeast proteins can move both toward microtubule plus-or minus-ends using the same motility mechanism depending on the motor concentration [START_REF] Fridman | Kinesin-5 Kip1 is a bi-directional motor that stabilizes microtubules and tracks their plus-ends in vivo[END_REF][START_REF] Thiede | Regulation of bi-directional movement of single kinesin-5 Cin8 molecules[END_REF]. In yeast, the pushing force generated by Cin8 and Kip1 is counteracted by Kar3, a minus-end directed motor member of the kinesin-14 family [START_REF] Saunders | Kinesin-related proteins required for structural integrity of the mitotic spindle[END_REF].

MAP proteins are also important in budding yeast for spindle assembly. For example, the CLASP family protein Stu1, a non-motor protein that forms homodimers, binds to interpolar microtubules and provides an outward force on spindle poles, resulting essential for SPB separation and integrity of the mitotic spindle [START_REF] Yin | Stu1p is physically associated with betatubulin and is required for structural integrity of the mitotic spindle[END_REF].

Although kinesins-5 are essential for spindle elongation, they do not seem to be the only force that generates the spindle and separates centrosomes. Indeed, other motor proteins are important for this process. One of them is the minus-end directed motor dynein. Although the precise role of dynein in this mechanism is still under debate, strong evidence confirmed that dynein is required for this process [START_REF] Eshel | Cytoplasmic dynein is required for normal nuclear segregation in yeast[END_REF][START_REF] Vaisberg | Cytoplasmic dynein plays a role in mammalian mitotic spindle formation[END_REF]. Moreover, dynein plays a role in spindle positioning by pulling on microtubules (reviewed in di [START_REF] Di Pietro | Regulation of mitotic spindle orientation: an integrated view[END_REF].

Motor proteins of the kinesin-12 family have also been implicated in bipolar spindle formation, but do not appear essential for this process, since they rather seem to act redundantly with kinesin-5 [START_REF] Tanenbaum | Kif15 cooperates with eg5 to promote bipolar spindle assembly[END_REF][START_REF] Vanneste | The role of Hklp2 in the stabilization and maintenance of spindle bipolarity[END_REF].

Other proteins that have been implicated in spindle formation are chromokinesins. These are kinesins that bind to chromosome arms and walk along microtubules growing from the centrosomes, generating the force that pushes chromosomes away from spindle poles (reviewed in [START_REF] Tanenbaum | Mechanisms of Centrosome Separation and Bipolar Spindle Assembly[END_REF]. However, at the same time, microtubule used as tracks by chromokinesins experience a force that pushes spindle poles apart, suggesting that these kinesins can also promote centrosomes separation together with other kinesins [START_REF] Dumont | Force and length in the mitotic spindle[END_REF].

Finally, it has been proposed that in Drosophila microtubule pushing forces may contribute to centrosome separation [START_REF] Cytrynbaum | A force balance model of early spindle pole separation in Drosophila embryos[END_REF]. Indeed, while growing from one centrosome, microtubules will encounter the other centrosome and can exert a pushing force on it, especially early during centrosome separation when centrosomes are close to each other.

Shaping mitotic chromosomes: the role of condensin complex

In order to guarantee a correct chromosome segregation, chromatin needs to be converted from loosely organised interphase DNA, to highly compacted, individual chromosomes. To achieve this, chromatin fibers require to be extensively looped. The main responsible for this DNA compaction/condensation is the multiproteic complex condensin [START_REF] Hirano | Condensins, chromosome condensation protein complexes containing XCAP-C, XCAP-E and a Xenopus homolog of the Drosophila Barren protein[END_REF]. The majority of eukaryotes contain two type of condensin complexes, condensin I and II, while fungi lack condensin II. Each complex contains two subunits of the SMC (Structural Maintenance of Chromosomes) protein family, SMC2 and SMC4. These subunits contain an ATP-binding cassette (ABC) ATPase domain (head domain) at one end and a hinge domain on the other end, separated by an anti-parallel coiled-coil of about 50nm [START_REF] Anderson | Condensin and cohesin display different arm conformations with characteristic hinge angles[END_REF]. The two subunits associate through their hinge domain and form a V-shape molecule. The head domains get in contact interposing two molecules of ATP between them and lose their contact upon ATP hydrolysis [START_REF] Lammens | Structural biochemistry of ATP-driven dimerization and DNAstimulated activation of SMC ATPases[END_REF].

The two SMC subunits are common to the two condensin complexes, whereas the SMC binding partners, members of the kleisin protein family, change between the two types of condensin. Kleisins bind to the SMC2 head domain through their N-termini and to the SMC4 head through their Ctermini. This creates a closed ring-like structure [START_REF] Onn | Reconstitution and subunit geometry of human condensin complexes[END_REF]. The kleisin subunit in condensin I is CAP-H whereas in condensin II is CAP-H2. In addition, these subunits contact other condensin subunits through their HEAT repeat-containing domain, which are CAP-D2 and CAP-G in condensin I and CAP-D3 and CAP-G2 in condensin II (Fig. 9) [START_REF] Ono | Differential contributions of condensin I and condensin II to mitotic chromosome architecture in vertebrate cells[END_REF][START_REF] Yeong | Identification of a subunit of a novel Kleisin-beta/SMC complex as a potential substrate of protein phosphatase 2A[END_REF]. The eukaryotic condensin complexes (I and II) share the same SMC proteins core subunits (SMC2/4). In contrast, they have distinct sets of non-SMC regulatory subunits, which comprise a kleisin-subunit (CAP-H or CAP-H2) and two HEAT repeats containing proteins (CAP-D2/G and CAP-D3/G2). From Hirano, 2016.

The two condensin complexes in mammals play slightly different roles and show different localization in space and time. Indeed, condensin I is in the cytoplasm during interphase and is brought to chromosomes only after nuclear envelope breakdown (NEBD), during prometaphase.

Condensin II, in contrast, localizes in the nucleus from interphase to mitosis and participates to the first steps of DNA condensation. After NEBD, the two condensins together support chromosome condensation in the nucleus to assure a correct chromosome segregation. Finally, at the end of mitosis, condensin I is exported out of the nucleus and is re-localized in the cytoplasm until prophase of the next cell cycle [START_REF] Ono | Spatial and Temporal Regulation of Condensins I and II in Mitotic Chromosome Assembly in Human Cells[END_REF][START_REF] Ono | Differential contributions of condensin I and condensin II to mitotic chromosome architecture in vertebrate cells[END_REF]. In addition, condensin I is found in the outer part of chromosome axes, whereas condensin II is enriched in the inner part of axes [START_REF] Ono | Differential contributions of condensin I and condensin II to mitotic chromosome architecture in vertebrate cells[END_REF].

Moreover, condensin I is more mobile than condensin II and interacts with chromosomes in a more dynamic manner (Gerlich et al., 2006a).

Contrary to what happens in higher eukaryotes, condensin in S. cerevisiae localizes in the nucleus during the whole cell cycle [START_REF] Freeman | The condensin complex governs chromosome condensation and mitotic transmission of rDNA[END_REF]. This could be due to the fact that in budding yeast cell cycle S phase and M phase partially overlap, since the spindle starts to form at the end of S phase and there is no clear G2 phase.

The two main enzymatic activities of condensin are an ATPase-dependent positive supercoiling of closed dsDNA (double-stranded DNA) in presence of the enzyme topoisomerase II during replication and transcription, and ATP-independent DNA reannealing activity that reduces unwound DNA fragments generated after transcription has taken place (reviewed in [START_REF] Kalitsis | Condensin, master organizer of the genome[END_REF]. The interaction of condensins with DNA depends on ATP binding: indeed, ATP binding leads to closure of the condensin molecule to form a ring, whereas ATP hydrolysis helps to disrupt the SMC/kleisin interface, thus opening the DNA gate to start topological genome re-organization [START_REF] Woo | Structural Studies of a Bacterial Condensin Complex Reveal ATP-Dependent Disruption of Intersubunit Interactions[END_REF].

The most accredited model to explain the mechanism of DNA compaction by condensin structures is the "loop extrusion model", which suggests that a single condensin molecule can bind at two chromatin points and can slide the DNA to generate a loop that becomes progressively larger (Fig. 10; [START_REF] Ganji | Real-time imaging of DNA loop extrusion by condensin[END_REF][START_REF] Kim | DNA-loop extruding condensin complexes can traverse one another[END_REF]. More recently, condensin functioning has been better described with the use of Hi-C analysis: during prophase, an array of loops would be formed, that during prophase acquires a helical arrangement, with consecutive loops emanating from a central spiral-staircase condensin scaffold [START_REF] Gibcus | A pathway for mitotic chromosome formation[END_REF]. One strand of DNA is anchored by the kleisin and HEAT-repeated subunits (in yellow) of the condensin complex, which extrudes a loop of DNA. From [START_REF] Ganji | Real-time imaging of DNA loop extrusion by condensin[END_REF] As already mentioned, the two condensin complexes play different roles during mitotic chromosome compaction in vertebrates: condensin II participates to early DNA compaction, starting with chiral looping and/or loop extrusion. Then, higher-order assembly leads to linear organization and axial shortening of chromosomes [START_REF] Hirano | Condensin-Based Chromosome Organization from Bacteria to Vertebrates[END_REF]. This step could also involve HEAT-mediated condensincondensin interactions [START_REF] Kinoshita | Balancing acts of two HEAT subunits of condensin I support dynamic assembly of chromosome axes[END_REF]. Then, when condensin I is imported in the nucleus, it contributes to the compaction of chromatin causing lateral compaction possibly through its supercoiling activity [START_REF] Kimura | ATP-dependent positive supercoiling of DNA by 13S condensin: a biochemical implication for chromosome condensation[END_REF]. Indeed, when condensin II is removed, there is a loss of axial compaction with chromosomes resulting longer and less rigid [START_REF] Abe | The initial phase of chromosome condensation requires Cdk1-mediated phosphorylation of the CAP-D3 subunit of condensin II[END_REF][START_REF] Green | Contrasting roles of condensin I and condensin II in mitotic chromosome formation[END_REF][START_REF] Ono | Differential contributions of condensin I and condensin II to mitotic chromosome architecture in vertebrate cells[END_REF]; in contrast, when condensin I is removed, lateral condensation is altered and mitotic chromosomes result shorter and larger [START_REF] Green | Contrasting roles of condensin I and condensin II in mitotic chromosome formation[END_REF][START_REF] Ono | Condensin II initiates sister chromatid resolution during S phase[END_REF](Fig. 11). In the first case it is possible to notice shorter and larger chromosomes, whereas in the second case they are longer and less rigid. From [START_REF] Kalitsis | Condensin, master organizer of the genome[END_REF] Condensin is not only important for chromatin compaction during mitosis, but has also many other important roles during interphase. For instance, chromatin fibers can fold into interaction domains, called topologically associated domains (TADs), which mediate the interactions between enhancers and promoters for gene expression. Condensin has recently emerged as a regulator of TADs organization [START_REF] Crane | Condensin-driven remodelling of X chromosome topology during dosage compensation[END_REF][START_REF] Li | Widespread rearrangement of 3D chromatin organization underlies polycomb-mediated stress-induced silencing[END_REF][START_REF] Van Bortle | Insulator function and topological domain border strength scale with architectural protein occupancy[END_REF]. In addition, condensin plays a role in the compaction and segregation of rDNA (ribosomal DNA) [START_REF] Paul | Condensin Depletion Causes Genome Decompaction Without Altering the Level of Global Gene Expression in Saccharomyces cerevisiae[END_REF], which is one of the most difficult regions to segregate due to high level of repetition, active transcription and late replication [START_REF] D'ambrosio | Condensin-dependent rDNA decatenation introduces a temporal pattern to chromosome segregation[END_REF][START_REF] Freeman | The condensin complex governs chromosome condensation and mitotic transmission of rDNA[END_REF]. Moreover, condensin is enriched at the tRNA genes in many organisms [START_REF] D'ambrosio | Condensin-dependent rDNA decatenation introduces a temporal pattern to chromosome segregation[END_REF][START_REF] Iwasaki | Interaction between TBP and Condensin Drives the Organization and Faithful Segregation of Mitotic Chromosomes[END_REF][START_REF] Kranz | Genome-wide analysis of condensin binding in Caenorhabditis elegans[END_REF][START_REF] Van Bortle | Insulator function and topological domain border strength scale with architectural protein occupancy[END_REF][START_REF] Yuen | Condensin II is anchored by TFIIIC and H3K4me3 in the mammalian genome and supports the expression of active dense gene clusters[END_REF]. In budding yeast, for example, it is required to cluster tRNA genes in trans, i.e. between different chromosomes, in order to support tight regulation of tRNA expression [START_REF] Haeusler | Clustering of yeast tRNA genes is mediated by specific association of condensin with tRNA gene transcription complexes[END_REF][START_REF] Iwasaki | Centromeric localization of dispersed Pol III genes in fission yeast[END_REF][START_REF] Paul | Condensin Depletion Causes Genome Decompaction Without Altering the Level of Global Gene Expression in Saccharomyces cerevisiae[END_REF].

Finally, condensin has a role in transcription. For example, in Drosophila melanogaster, where homologous chromosomes remain paired during interphase, enhancers can act in trans, between different chromosomes, to regulate promoters [START_REF] Mellert | Transvection is common throughout the Drosophila genome[END_REF]. Condensin antagonizes this process (called transvection), possibly by creating separated chromosomes through the loop extrusion action, like in mitosis [START_REF] Hartl | Chromosome alignment and transvection are antagonized by condensin II[END_REF][START_REF] Smith | Maintenance of interphase chromosome compaction and homolog pairing in Drosophila is regulated by the condensin cap-h2 and its partner Mrg15[END_REF].

Importantly, centromeric condensin has a role in the establishment of a good centromere conformation, which leads to proper sister chromatid geometry that allows chromosome biorientation [START_REF] Yong-Gonzalez | Condensin function at centromere chromatin facilitates proper kinetochore tension and ensures correct mitotic segregation of sister chromatids[END_REF]. In budding yeast the Shugoshin protein Sgo1, which has an established role in pericentric cohesion protection and recruitment of the CPC complex (further described in the next chapters), has also an important role in the recruitment of condensin at centromeres, thus providing flexibility to the pericentric region and facilitating chromosome biorientation (Peplowska et al., 2014;Verzijlbergen et al., 2014).

Different proteins, mostly kinases and phosphatases, are known to regulate condensin loading on chromosomes. AKAP95 (A Kinase-Anchoring-Protein) localizes to the nuclear matrix in interphase and binds to chromatin during mitosis, where it targets condensin I after NEBD [START_REF] Eide | Distinct but overlapping domains of AKAP95 are implicated in chromosome condensation and condensin targeting[END_REF].

Moreover, pRb (Retinoblastoma) is necessary for CAP-D3 localization on mitotic chromosomes in Drosophila [START_REF] Longworth | RBF1 promotes chromatin condensation through a conserved interaction with the Condensin II protein dCAP-D3[END_REF]. In interphase condensin I is phosphorylated by CK2 (casein kinase 2), which suppresses its supercoiling activity [START_REF] Takemoto | Negative regulation of condensin I by CK2-mediated phosphorylation[END_REF], and by CDK1 in mitosis, which instead increases its DNA supercoiling activity [START_REF] Abe | The initial phase of chromosome condensation requires Cdk1-mediated phosphorylation of the CAP-D3 subunit of condensin II[END_REF]. Additionally, Aurora B kinase can also phosphorylate condensin I and this is necessary for its loading on chromosomes [START_REF] Lipp | Aurora B controls the association of condensin I but not condensin II with mitotic chromosomes[END_REF][START_REF] Takemoto | Analysis of the role of Aurora B on the chromosomal targeting of condensin I[END_REF]. Finally, Mps1 phosphorylates the condensin II subunit CAP-H2 and this event is required for proper loading of condensin II on chromatin [START_REF] Kagami | Mps1 phosphorylation of condensin II controls chromosome condensation at the onset of mitosis[END_REF].

Dephosphorylation by phosphatases is also important. Indeed, PP2A interacts with and dephosphorylates the condensin II subunit CAP-D3, facilitating condensin II chromosomal recruitment [START_REF] Yeong | Identification of a subunit of a novel Kleisin-beta/SMC complex as a potential substrate of protein phosphatase 2A[END_REF].

Holding chromatids together: the cohesion complex

From the beginning of DNA replication in S phase until the establishment of chromosome biorientation, sister chromatids need to be held together by cohesion. This is essential in order to avoid precocious chromosome segregation that can lead to unequal numbers of chromosomes in daughter cells. Chromatid cohesion is exerted by a multiprotein complex called cohesin. Cohesin complexes are composed of four core subunits that are conserved in all eukaryotes. In budding yeast cohesin subunits are Smc1, Smc3, Scc1 and Scc3 during mitosis, whereas in meiosis Scc1 is replaced with Rec8 [START_REF] Haering | Structure and stability of cohesin's Smc1-kleisin interaction[END_REF][START_REF] Haering | Molecular Architecture of SMC Proteins and the Yeast Cohesin Complex[END_REF]. In vertebrates the kleisin Scc1 is called Rad21 (also referred to as!"!kleisin). In addition, two paralogs of Scc3 exist, named SA1 and SA2. Cohesins can contain any of the two proteins, but not the two at the same time [START_REF] Losada | Identification and characterization of SA/Scc3p subunits in the Xenopus and human cohesin complexes[END_REF]. Moreover, SA2-containing cohesin is threefold more abundant than SA1-containing cohesin, and it seems that only the former is responsible for sister chromatids cohesion, while the latter is involved in gene regulation and replication of telomeres (Remeseiro et al., 2012b(Remeseiro et al., , 2012a)). Smc1 and Smc3 are members of the SMC (Structural Maintenance of Chromosomes) protein family and are ATPases (reviewed in [START_REF] Mehta | Cohesin: A guardian of genome integrity[END_REF]. As already described for the condensin subunits SMC2/4, they carry a hinge domain on one end and an ATPase head domain on the other end, separated by two anti-parallel coiled coils. The interactions between the hinge domains allow the formation of V-shaped heterodimers of SMC proteins [START_REF] Hirano | Hinge-mediated dimerization of SMC protein is essential for its dynamic interaction with DNA[END_REF][START_REF] Melby | The symmetrical structure of structural maintenance of chromosomes (SMC) and MukB proteins: long, antiparallel coiled coils, folded at a flexible hinge[END_REF]. Moreover, the two proteins interact on the other end through their ATPase domains with the help of Scc1/Rad21 [START_REF] Schleiffer | Kleisins: a superfamily of bacterial and eukaryotic SMC protein partners[END_REF]. ATP binding is important for the binding of Scc1/Rad21 to the Smc1-Smc3 heterodimer. The fourth subunit, Scc3/SA2, is associated to Scc1/Rad21 and contains HEAT repeats domains, which are responsible for protein-protein interactions [START_REF] Neuwald | HEAT repeats associated with condensins, cohesins, and other complexes involved in chromosome-related functions[END_REF]. The four cohesin core subunits, which are all essential proteins, therefore form a closed ring structure with an internal diameter of 40nm [START_REF] Haering | Molecular Architecture of SMC Proteins and the Yeast Cohesin Complex[END_REF].

Besides the core subunits, additional proteins associate to the ring structure. Pds5 is a large HEAT repeat-containing protein, highly conserved and essential in budding yeast. In vertebrates there are two homologs of this protein, Pds5A and Pds5B (reviewed in [START_REF] Mehta | Cohesin: A guardian of genome integrity[END_REF]. Pds5 is both a positive and negative regulator of cohesion depending on the protein it binds to (soronin or Wapl1, further described below). Another binding partner is Wapl1 (wings apart-like protein 1). The Cterminal domain of this protein, which also consists of HEAT repeats, is important for cohesin binding, whereas the N-terminal domain is necessary for the binding to Pds5. Wapl1 is a negative regulator of cohesion that opens the cohesin ring at the interface Smc3-Scc1 allowing exit of DNA from the ring [START_REF] Chatterjee | Structural insights into the regulation of cohesion establishment by Wpl1[END_REF][START_REF] Huis In 't Veld | Characterization of a DNA exit gate in the human cohesin ring[END_REF][START_REF] Ouyang | Structure of the human cohesin inhibitor Wapl[END_REF]. The third protein and regulator is Sororin, which is required for cohesion establishment and maintenance [START_REF] Rankin | Sororin, a substrate of the anaphase-promoting complex, is required for sister chromatid cohesion in vertebrates[END_REF][START_REF] Schmitz | Sororin is required for stable binding of cohesin to chromatin and for sister chromatid cohesion in interphase[END_REF]. Sororin homologs have been found in metazoans, but not in fungi (Fig. 12). The cohesin ring is formed by two rod-shaped SMC proteins, Smc1 (in red) and Smc3 (in light blue), that hold a dimerization domain on one end and an ATPase domain on the other. The interaction between the dimerization domains creates a V-structure. The ring is closed by an αkleisin subunit (in yellow), which binds to Smc3 and Smc1 and also, with its central domain, to Scc3/SA and Pds5. Pds5 in turn binds to Wapl1. Adapted from Nasmyth, 2011.

Cohesion loading takes place after chromosome segregation and before the beginning of DNA replication of the next cell cycle, in interphase. This mechanism is mediated by the cohesin-loading complex Scc2-Scc4 (Nipb1 and Mau2 in humans) [START_REF] Ciosk | Cohesin's binding to chromosomes depends on a separate complex consisting of Scc2 and Scc4 proteins[END_REF][START_REF] Watrin | Human Scc4 is required for cohesin binding to chromatin, sister-chromatid cohesion, and mitotic progression[END_REF] and requires ATP hydrolysis [START_REF] Hu | ATP Hydrolysis Is Required for Relocating Cohesin from Sites Occupied by Its Scc2/4 Loading Complex[END_REF]. Indeed, Scc2/Nipb1 stimulates the ATPase activity of Smc1-Smc3, allowing their hinge domain to be opened and DNA to enter [START_REF] Hu | ATP Hydrolysis Is Required for Relocating Cohesin from Sites Occupied by Its Scc2/4 Loading Complex[END_REF]. In addition, this complex is essential to load cohesin at the right chromosomal places: indeed, it interacts with the mediator transcriptional complex in mammalian cells and the RSC chromatin-remodeling complex in budding yeast in order to load cohesin at promoter, nucleosomal free, DNA regions [START_REF] Huang | A Role for the RSC chromatin remodeler in regulating cohesion of sister chromatid arms[END_REF][START_REF] Kagey | Mediator and cohesin connect gene expression and chromatin architecture[END_REF][START_REF] Muñoz | A Role for Chromatin Remodeling in Cohesin Loading onto Chromosomes[END_REF]. The cohesin ring is at the same time opened by the protein Wapl1 bound to Pds5, in order to allow its movement across the chromatin after being loaded. Instead of being kept in the chromosomal sites where it has been loaded, cohesin is repositioned onto sites of convergent transcription in yeast and at CCCTC-binding factor (CTCF) sites in mammals. CTCF has a zing finger domain, and cohesin recruitment to these sites depends on the presence of CTCF [START_REF] Lee | Specific regulation of CENP-E and kinetochores during meiosis I/meiosis II transition in pig oocytes[END_REF][START_REF] Merkenschlager | CTCF and cohesin: linking gene regulatory elements with their targets[END_REF][START_REF] Rubio | CTCF physically links cohesin to chromatin[END_REF][START_REF] Wendt | Cohesin mediates transcriptional insulation by CCCTC-binding factor[END_REF]. The mechanism by which cohesin is translocated to these sites is still unknown.

However, one possibility is that cohesin rings are pushed along chromosomes by the transcriptional machinery [START_REF] Lee | Specific regulation of CENP-E and kinetochores during meiosis I/meiosis II transition in pig oocytes[END_REF][START_REF] Busslinger | Cohesin is positioned in mammalian genomes by transcription, CTCF and Wapl[END_REF][START_REF] Davidson | Rapid movement and transcriptional re-localization of human cohesin on DNA[END_REF].

Cohesin loading on the correct chromosome sites is not sufficient to have sister chromatid cohesion. Indeed, cohesion establishment between sister chromatids takes place later, during S phase, and requires acetylation of two Lysines of Smc3/SA2 (K105 and K106) and interaction of cohesin with Sororin [START_REF] Chan | Pds5 promotes and protects cohesin acetylation[END_REF][START_REF] Zhang | Acetylation of Smc3 by Eco1 Is Required for S Phase Sister Chromatid Cohesion in Both Human and Yeast[END_REF]. Smc3/SA2 acetylation depends on two cohesin acetyltransferase (CoATs), Esco1 and Esco2 in vertebrates [START_REF] Zhang | Acetylation of Smc3 by Eco1 Is Required for S Phase Sister Chromatid Cohesion in Both Human and Yeast[END_REF]. While Esco2 seems to have the most important role in cohesion establishment, Esco1 is more involved in gene expression regulation [START_REF] Alomer | Esco1 and Esco2 regulate distinct cohesin functions during cell cycle progression[END_REF]. These CoATs seem to be recruited through the interaction with DNA replication factors like PCNA, the processivity factor of the DNA Pol δ [START_REF] Moldovan | PCNA controls establishment of sister chromatid cohesion during S phase[END_REF]. Acetylation of Smc3/SA2 is thought to be important to stabilize the ring and to avoid its opening. In addition, the protein Sororin, which has a conformation similar to Wapl1, binds to Pds5 and represses Wapl1 binding and its ring-releasing activity [START_REF] Nishiyama | Sororin mediates sister chromatid cohesion by antagonizing Wapl[END_REF].

In budding yeast, there is only one CoAT, Eco1, that is required for Smc3 acetylation and whose recruitment depends on Pds5 [START_REF] Chan | Pds5 promotes and protects cohesin acetylation[END_REF]. Moreover, there is no Sororin homolog in yeast, suggesting that cohesin acetylation could be sufficient to counteract the ring-releasing action of Wapl1.

How cohesin complexes hold together the two sister chromatids is still not clear. Different models have been proposed (Fig. 13; reviewed in [START_REF] Mehta | Cohesin: A guardian of genome integrity[END_REF]: the one ring model, also called "embrace model", is the most popular and envisions that one single cohesin ring entraps the two sister chromatid fibers; the two rings model, called also "the handcuff model" envisions that each cohesin ring embraces one chromatin fiber and one molecule of Scc3/SA2 bridges the two cohesin rings; the multimeric bracelet model proposes that Scc1/Rad21 connects Smc heterodimers to form multimeric filaments that entrap chromatin inside this oligomeric bracelet; finally, the fourth model, called the rod shaped model, describes a rod shaped structure of cohesion, based on transmitted electron microscopy images of minichromosomes [START_REF] Surcel | Cohesin interaction with centromeric minichromosomes shows a multi-complex rod-shaped structure[END_REF]. C: bracelet model: cohesin rings oligomerize in a hand-to-hand fashion to hold the sister chromatids together. D: rod model: cohesin rings associate with one another by the SMC coiled coils to make a rod-like structure. From [START_REF] Mehta | Cohesin: A guardian of genome integrity[END_REF] Most of the sister chromatid cohesion is maintained throughout DNA replication, until prophase in vertebrate cells and budding yeast meiosis, and until anaphase in budding yeast mitosis. Indeed, when vertebrate cells enter prophase, most of the cohesin on chromosome arms is removed, whereas it is maintained at centromeres until anaphase. Not only cohesin, but also its associated proteins are removed from chromosomes, like Wapl1, Pds5, Sororin, Scc2/Scc4 (reviewed in [START_REF] Zheng | Regulation of sister chromatid cohesion during the mitotic cell cycle[END_REF]. Cohesin dissociation depends on different factors, mainly mitotic kinases: Plk1 phosphorylation of SA1-SA2 decreases binding of cohesin to chromatin [START_REF] Sumara | The dissociation of cohesin from chromosomes in prophase is regulated by Polo-like kinase[END_REF]; Plk1 and Aurora B phosphorylation of Sororin causes its displacement from Pds5, which therefore can be bound again by Wapl1 in order to destabilize the ring [START_REF] Nishiyama | Aurora B and Cdk1 mediate Wapl activation and release of acetylated cohesin from chromosomes by phosphorylating Sororin[END_REF].

Removing the majority of cohesin during prophase has an important role in the cohesin cycle, since it allows the cohesin to become soluble and ready for the next cell cycle. In this way, it can be reloaded on chromatin during interphase. Additionally, cohesin removal during prophase I of meiosis is necessary for the resolution of recombination intermediates (called chiasmata; [START_REF] Buonomo | Disjunction of Homologous Chromosomes in Meiosis I Depends on Proteolytic Cleavage of the Meiotic Cohesin Rec8 by Separin[END_REF][START_REF] Kitajima | Shugoshin collaborates with protein phosphatase 2A to protect cohesin[END_REF][START_REF] Kudo | Resolution of Chiasmata in Oocytes Requires Separase-Mediated Proteolysis[END_REF].

Importantly, centromeric cohesin is protected from the prophase pathway and from separasemediated cleavage until anaphase, when cohesin will be completely disrupted. The cohesin protector belongs to the protein family Shugoshin (which in Japanese literally means "guardian spirit") [START_REF] Watanabe | Shugoshin: guardian spirit at the centromere[END_REF], which comprises Sgo1 and Sgo2 in vertebrates and Sgo1 in budding yeast. Sgo1 is mainly involved in cohesion protection at centromeric regions and chromosome biorientation in mitosis, whereas Sgo2 is the meiotic ortholog of Sgo1 that protects meiotic cohesion. During meiosis I, sister chromatids mono-orient by attaching to the same spindle pole, whereas homologous chromosomes bind to opposite sides of the spindle in order to be segregated. Only once homolog biorientation takes place, separase becomes active (reviewed in [START_REF] Marston | Shugoshins: Tension-Sensitive Pericentromeric Adaptors Safeguarding Chromosome Segregation[END_REF]. However, separase only acts on chromosomes arms. Indeed, until biorientation of sister chromatids on the meiosis II spindle, centromeric cohesin is protected by Shugoshin from the action of separase. The target of separase in meiosis is the protein Rec8, which is cleaved thanks to its phosphorylation [START_REF] Brar | Rec8 phosphorylation and recombination promote the step-wise loss of cohesins in meiosis[END_REF][START_REF] Clyne | Polo-like kinase Cdc5 promotes chiasmata formation and cosegregation of sister centromeres at meiosis I[END_REF][START_REF] Katis | Rec8 phosphorylation by casein kinase 1 and Cdc7-Dbf4 kinase regulates cohesin cleavage by separase during meiosis[END_REF][START_REF] Lee | Role of Polo-like Kinase CDC5 in Programming Meiosis I Chromosome Segregation[END_REF]. At pericentromeres meiotic Shugoshin counteracts Rec8 phosphorylation by recruiting the phosphatase PP2A associated to the B' regulatory subunit, which dephosphorylates Rec8, thereby preventing its cleavage in meiosis I [START_REF] Ishiguro | Shugoshin-PP2A counteracts casein-kinase-1dependent cleavage of Rec8 by separase[END_REF][START_REF] Kitajima | Shugoshin collaborates with protein phosphatase 2A to protect cohesin[END_REF][START_REF] Riedel | Protein phosphatase 2A protects centromeric sister chromatid cohesion during meiosis I[END_REF][START_REF] Xu | Structure and function of the PP2A-shugoshin interaction[END_REF]. This happens for both yeast and vertebrates meiosis, whereas in mitosis this pathway has been found only in vertebrates. In vertebrates, mitotic Sgo1 protects pericentromeric cohesin from the prophase pathway, therefore allowing cohesion removal only on chromosomes arms [START_REF] Kitajima | Shugoshin collaborates with protein phosphatase 2A to protect cohesin[END_REF][START_REF] Riedel | Protein phosphatase 2A protects centromeric sister chromatid cohesion during meiosis I[END_REF][START_REF] Tang | PP2A Is Required for Centromeric Localization of Sgo1 and Proper Chromosome Segregation[END_REF]. Here, Sgo1 phosphorylation by CDK enables Sgo1 binding to cohesin, recruitment of the PP2A phosphatase and protection of cohesion [START_REF] Liu | Phosphorylation-enabled binding of Sgo1-PP2A to cohesin protects sororin and centromeric cohesion during mitosis[END_REF]. Different activities of Sgo1-PP2A allow protection: most importantly, Sgo1-PP2A prevents cohesin dissociation through dephosphorylation of SA2 and Scc1 [START_REF] Hauf | Dissociation of cohesin from chromosome arms and loss of arm cohesion during early mitosis depends on phosphorylation of SA2[END_REF][START_REF] Kitajima | Shugoshin collaborates with protein phosphatase 2A to protect cohesin[END_REF][START_REF] Mcguinness | Shugoshin prevents dissociation of cohesin from centromeres during mitosis in vertebrate cells[END_REF]; moreover, it is implicated in sororin maintenance at centromeres [START_REF] Nishiyama | Sororin mediates sister chromatid cohesion by antagonizing Wapl[END_REF], it counteracts Aurora B and CDK, maintaining sororin in its dephosphorylated state and preserving its association with Pds5; finally, it prevents cohesin removal by Wapl1, precluding its binding to cohesin through interaction with the same interface [START_REF] Dreier | Regulation of sororin by Cdk1-mediated phosphorylation[END_REF][START_REF] Liu | Phosphorylation-enabled binding of Sgo1-PP2A to cohesin protects sororin and centromeric cohesion during mitosis[END_REF][START_REF] Nishiyama | Sororin mediates sister chromatid cohesion by antagonizing Wapl[END_REF][START_REF] Hara | Structure of cohesin subcomplex pinpoints direct shugoshin-Wapl antagonism in centromeric cohesion[END_REF].

Sgo1 pericentromeric localization depends on phosphorylation of histone H2A by the mitotic kinase Bub1 (Fernius and Hardwick, 2005;Kawashima et al., 2010;[START_REF] Perera | Sgo1 establishes the centromeric cohesion protection mechanism in G2 before subsequent Bub1-dependent recruitment in mitosis[END_REF]. Moreover, Sgo1 interaction with cohesin requires its phosphorylation by the kinase Cdk1 [START_REF] Liu | Phosphorylation-enabled binding of Sgo1-PP2A to cohesin protects sororin and centromeric cohesion during mitosis[END_REF]. The kinase Mps1 seems to be important for Sgo1 centromeric localization in budding yeast (Benzi et al., 2020;Storchová et al., 2011) and in vertebrate cells for centromeric localization of both Sgo2 in meiosis and Sgo1 in mitosis [START_REF] El Yakoubi | Mps1 kinase-dependent Sgo2 centromere localisation mediates cohesin protection in mouse oocyte meiosis I[END_REF]van der Waal et al., 2012). However, at least during yeast mitosis, where a prophase pathway and centromeric cohesin protection might not exist, this localization has another meaning and could be rather related to the function of Sgo1 in the maintenance at centromeres of the Chromosomal Passenger Complex (CPC, Kawashima et al., 2007;Peplowska et al., 2014;Vanoosthuyse et al., 2007;Verzijlbergen et al., 2014). Moreover, Aurora B kinase and Sgo1 seem to have a reciprocal relationship being each other important for their centromeric localization: in Drosophila, for example, Shugoshin phosphorylation by Aurora B promotes its association with chromosomes [START_REF] Resnick | INCENP and Aurora B promote meiotic sister chromatid cohesion through localization of the Shugoshin MEI-S332 in Drosophila[END_REF]. In addition, in human cells Sgo2 phosphorylation by Aurora B allows its binding to the phosphatase PP2A [START_REF] Tanno | Phosphorylation of mammalian Sgo2 by Aurora B recruits PP2A and MCAK to centromeres[END_REF].

A recent study has uncovered a new role for meiotic Sgo2, which acts as a pseudo-substrate and inhibitor of separase in mitosis through binding to the spindle checkpoint protein Mad2 [START_REF] Hellmuth | Securin-independent regulation of separase by checkpoint-induced shugoshin-MAD2[END_REF]. Upon checkpoint silencing and APC/C re-activation, separase is liberated from Sgo2/Mad2, being Sgo2 substrate of the APC [START_REF] Karamysheva | Multiple Anaphase-promoting Complex/Cyclosome Degrons Mediate the Degradation of Human Sgo1[END_REF], thus adding an additional function to shugoshins in order to protect pericentromeric cohesion.

In the face of the prophase pathway, cohesion on chromosome arms is never completely dissolved during mitosis, but there are always few molecules of cohesin that are kept until their complete disruption in anaphase. This is probably due to the fact that few molecules of Sgo1 are also present and bind to cohesin on chromosome arms [START_REF] Giménez-Abián | Regulation of Sister Chromatid Cohesion between Chromosome Arms[END_REF].

Once sister chromatids are bi-oriented, i.e. attached to microtubules emanating from opposite spindle poles, the Spindle Assembly Checkpoint (further described in the next chapters) is silenced and the APC/C is re-activated, thereby leading to ubiquitylation and degradation of its substrates Securin and Cyclin B. Securin is bound until anaphase to Separase and inhibits its cleaving action [START_REF] Ciosk | An ESP1/PDS1 complex regulates loss of sister chromatid cohesion at the metaphase to anaphase transition in yeast[END_REF][START_REF] Waizenegger | Regulation of Human Separase by Securin Binding and Autocleavage[END_REF]. Moreover, in vertebrates Separase is also inhibited by phosphorylation by Cdk1 and physical interaction with Cyclin B [START_REF] Gorr | Mutual inhibition of separase and Cdk1 by two-step complex formation[END_REF][START_REF] Stemmann | Dual inhibition of sister chromatid separation at metaphase[END_REF], which is also interrupted with reactivation of the APC/C. Once Separase is again active, it cleaves the cohesin subunit Scc1/Rad21 at two sites, leading to destruction of the cohesin rings that were still present and to separation of sister chromatids [START_REF] Hauf | Cohesin cleavage by separase required for anaphase and cytokinesis in human cells[END_REF][START_REF] Uhlmann | Cleavage of cohesin by the CD clan protease separin triggers anaphase in yeast[END_REF].

Then, the resulting proteolytic fragments of Scc1/Rad21 are degraded through a ubiquitin-dependent pathway [START_REF] Rao | Degradation of a cohesin subunit by the N-end rule pathway is essential for chromosome stability[END_REF]. Scc1/SA2 cleavage by Separase is enhanced by phosphorylation by Polo Kinase in different organisms [START_REF] Alexandru | Phosphorylation of the cohesin subunit Scc1 by Polo/Cdc5 kinase regulates sister chromatid separation in yeast[END_REF][START_REF] Hauf | Dissociation of cohesin from chromosome arms and loss of arm cohesion during early mitosis depends on phosphorylation of SA2[END_REF][START_REF] Sumara | The dissociation of cohesin from chromosomes in prophase is regulated by Polo-like kinase[END_REF].

Importantly, as already mentioned, in vertebrates only very few molecules, that are kept at centromeres, are cleaved by this pathway (about 10%) (Gerlich et al., 2006b;[START_REF] Waizenegger | Two distinct pathways remove mammalian cohesin from chromosome arms in prophase and from centromeres in anaphase[END_REF]. However, this reaction remains essential for sister chromatid separation (Fig. 14). The Scc2/Scc4 complex promotes cohesin loading in telophase and early G1. During DNA replication, Esco1/2 and Sororin are required to establish chromosome cohesion. In prophase, cohesin is removed from the chromosome arms by the action of mitotic kinases and the Wapl1/Pds5 complex, whereas cohesin at centromeres is protected by the Sgo1/PP2A complex until anaphase. After sister chromatid bi-orientation and SAC silencing, Separase is activated and cleaves centromeric cohesin, enabling sister chromatid separation in anaphase. From [START_REF] Zheng | Regulation of sister chromatid cohesion during the mitotic cell cycle[END_REF].

An outlook on kinetochores

In order to be equally segregated, sister chromatids bind to microtubules coming from opposite spindle poles through complex protein structures called kinetochores that are assembled on centromeres. More than 50 proteins, mostly conserved across the eukaryotic kingdom, assemble to form a kinetochore, which can be divided into two parts: the inner and the outer kinetochore.

Proteins of the inner kinetochore interact with the centromeric chromatin, whereas proteins of the outer kinetochore directly contact microtubules (reviewed in [START_REF] Yamagishi | Kinetochore composition and its function: lessons from yeasts[END_REF].

The innermost part of the kinetochore is the histone H3 variant Centromeric Protein A (CENP-A, Cse4 in budding yeast), which binds to the other histones to form centromere-specific nucleosomes [START_REF] Earnshaw | Identification of a family of human centromere proteins using autoimmune sera from patients with scleroderma[END_REF][START_REF] Palmer | A 17-kD centromere protein (CENP-A) copurifies with nucleosome core particles and with histones[END_REF]. CENP-A interacts with proteins of the Constitutive Centromere Associated Network (CCAN), a complex of the inner kinetochore composed of different proteins including CENP-C and CENP-T [START_REF] Fukagawa | The Centromere: Chromatin Foundation for the Kinetochore Machinery[END_REF] (Fig. 15). CENP-T (Cnn1 in budding yeast) forms a tetramer with CENP-W, CENP-S and CENP-X that is proposed to be essential for the establishment of centromeric chromatin necessary to form an intact kinetochore [START_REF] Gascoigne | Induced Ectopic Kinetochore Assembly Bypasses the Requirement for CENP-A Nucleosomes[END_REF][START_REF] Nishino | CENP-T-W-S-X Forms a Unique Centromeric Chromatin Structure with a Histone-like Fold[END_REF]. Moreover, mammalian CENP-T directly interacts with the Ndc80 complex, a component of the outer kinetochore, through its Nterminus [START_REF] Nishino | CENP-T provides a structural platform for outer kinetochore assembly[END_REF]. The inner kinetochore protein CENP-C (Mif2 in budding yeast) acts as a structural hub for kinetochore assembly [START_REF] Przewloka | CENP-C Is a Structural Platform for Kinetochore Assembly[END_REF][START_REF] Screpanti | Direct Binding of Cenp-C to the Mis12 Complex Joins the Inner and Outer Kinetochore[END_REF]. In budding yeast its localization depends on CENP-A [START_REF] Tanaka | CENP-C Functions as a Scaffold for Effectors with Essential Kinetochore Functions in Mitosis and Meiosis[END_REF][START_REF] Westermann | Architecture of the budding yeast kinetochore reveals a conserved molecular core[END_REF].

The core of the outer kinetochore is the KMN network, which is composed of Knl1, the Mis12 complex and the Ndc80 complex (reviewed in Pesenti et al., 2016) (Fig. 15). The Ndc80 complex is composed of dimers of Hec1 (Ndc80 in budding yeast)-Nuf2 and of Spc24-Spc25. The complex is directly responsible for contacting microtubules. Indeed, the two proteins Ndc80 and Nuf2 bear calponin-homology (CH) domains that form the microtubule-binding surface [START_REF] Alushin | Multimodal microtubule binding by the Ndc80 kinetochore complex[END_REF](Alushin et al., , 2010;;Ciferri et al., 2008;[START_REF] Wei | The Ndc80/HEC1 complex is a contact point for kinetochoremicrotubule attachment[END_REF]. Moreover, the globular domain of the Spc24-Spc25 dimer connects the Ndc80 complex to the inner kinetochore through direct interaction with CENP-T [START_REF] Gascoigne | Induced Ectopic Kinetochore Assembly Bypasses the Requirement for CENP-A Nucleosomes[END_REF][START_REF] Malvezzi | A structural basis for kinetochore recruitment of the Ndc80 complex via two distinct centromere receptors[END_REF][START_REF] Nishino | CENP-T provides a structural platform for outer kinetochore assembly[END_REF][START_REF] Petrovic | The MIS12 complex is a protein interaction hub for outer kinetochore assembly[END_REF]. The Mis12 complex is composed of four subunits: Mis12 (Mtw1 in yeast), Mis13 (Dsn1 in yeast), Mis14 (Nsl1 in yeast) and Nnf1. This complex acts as a platform for the formation of the KMN network complex (Cheeseman et al., 2006) and connects other components of the outer kinetochore to the inner kinetochore through the interaction with CENP-C [START_REF] Screpanti | Direct Binding of Cenp-C to the Mis12 Complex Joins the Inner and Outer Kinetochore[END_REF] (Fig. 15). Knl1 (Spc105 in budding yeast) has a conserved microtubule-binding domain in its N-terminus (Cheeseman et al., 2006;[START_REF] Welburn | Aurora B phosphorylates spatially distinct targets to differentially regulate the kinetochoremicrotubule interface[END_REF] and the protein is important for both microtubule attachments and activation of the spindle assembly checkpoint (better described in the next chapters), being a kinetochore scaffold for many proteins involved in this cascade.

In budding yeast there is an additional complex, the Dam1 complex (DASH in fission yeast), which forms a ring around microtubules and has an essential role in coupling chromosome movement to microtubule depolymerisation (Asbury et al., 2006). Importantly, this complex is essential to retain the Ndc80 complex to the microtubule (Grishchuk et al., 2008;Lampert et al., 2010a;Schmidt et al., 2012a;Tien et al., 2010;Westermann et al., 2006). A direct homolog of this complex in humans has not been found, although the Ska complex has been proposed to be the functional counterpart of the Dam1 complex [START_REF] Jeyaprakash | Structural and functional organization of the Ska complex, a key component of the kinetochoremicrotubule interface[END_REF]Welburn et al., 2009). The kinetochore is assembled on the centromere to form an interface for the interaction with microtubules. Major components are the Constitutive Centromere Associated Network (CCAN) and the KMN (Knl1, Mis12 and the Ndc80 complex) network. The CCAN binds to chromatin through the CENP-A-containing nucleosomes. The KMNnetwork binds to the CCAN through interaction with CENP-C or CENP-T. Finally, the Ndc80 complex directly contacts microtubules. From Hara and Fukagawa, 2018.

First contact: establishing kinetochore-microtubule interactions

The first step towards bi-orientation is the contact between kinetochores and microtubules. This is possible because microtubules are highly dynamic and oscillate between periods of growth and shrinkage. Indeed, during polymerization, the tubulin dimers forming microtubules are in a GTPbound state. However, GTP can be hydrolysed to GDP, and GDP-tubulin at the tips of filaments is more subject to depolymerisation, leading to rapid shrinking of the microtubule filament. On the other end, GTP-bound tubulin can induce microtubule growth. This tug of war between growth and shrinkage is referred to as "dynamic instability of microtubules" [START_REF] Weisenberg | Microtubule Formation in vitro in Solutions Containing Low Calcium Concentrations[END_REF]. Microtubule dynamics increases considerably when cells enter mitosis compared to interphase. During its growing phase, a microtubule can move up to several micrometres, and its trajectory can continuously change.

This mechanism inspired the "search and capture" model [START_REF] Kirschner | Beyond self-assembly: From microtubules to morphogenesis[END_REF]: growing microtubules would search for a target to hit on their way, and once kinetochores are touched by microtubules, they can capture a microtubule suppressing its dynamics and resulting in the formation of a stable connection (reviewed in [START_REF] Heald | Thirty years of search and capture: The complex simplicity of mitotic spindle assembly[END_REF]. The first connection is in a first place between the kinetochores and the lateral surface of the microtubule, called microtubule lattice, that provides a larger contact surface (=lateral attachments). Lateral attachments are then converted into end-on attachments, further described below.

The search and capture model is not the only mechanism behind the efficiency of the kinetochoremicrotubule attachments, but other processes are involved. In cells that undergo an open mitosis, one important aspect that contributes to microtubule capture is the formation of a gradient of Ran-GTP around chromosomes over a range of 20 μm that directs spindle pole microtubules towards the kinetochores [START_REF] Caudron | Spatial Coordination of Spindle Assembly by Chromosome-Mediated Signaling Gradients[END_REF]. However, this does not take place in organisms with a closed mitosis, like budding yeast (Tanaka et al., 2005). Moreover, in yeast, fly and vertebrate cells microtubules are generated not only from spindle poles, but also from kinetochores (reviewed in [START_REF] Tanaka | Kinetochore-microtubule interactions: the means to the end[END_REF]. The Ran-GTP gradient is also essential for this process [START_REF] Torosantucci | Localized RanGTP Accumulation Promotes Microtubule Nucleation at Kinetochores in Somatic Mammalian Cells[END_REF]. These microtubules interact with the spindle pole-deriving microtubules, facilitating kinetochore loading on the lateral surface of the microtubule [START_REF] Khodjakov | Minus-end capture of preformed kinetochore fibers contributes to spindle morphogenesis[END_REF][START_REF] Kitamura | Kinetochores Generate Microtubules with Distal Plus Ends: Their Roles and Limited Lifetime in Mitosis[END_REF][START_REF] Maiato | Kinetochore-driven formation of kinetochore fibers contributes to spindle assembly during animal mitosis[END_REF]. Once kinetochores are loaded, in yeast kinetochore-derived microtubules seem to disappear [START_REF] Kitamura | Kinetochores Generate Microtubules with Distal Plus Ends: Their Roles and Limited Lifetime in Mitosis[END_REF], whereas in fly and vertebrates they contact the spindle pole and are stabilised [START_REF] Khodjakov | Minus-end capture of preformed kinetochore fibers contributes to spindle morphogenesis[END_REF][START_REF] Maiato | Kinetochore-driven formation of kinetochore fibers contributes to spindle assembly during animal mitosis[END_REF]. This could be due to a general difference in the polarity of these microtubules, which presents their plus ends distal to kinetochores in yeast and opposite in other species [START_REF] Khodjakov | Minus-end capture of preformed kinetochore fibers contributes to spindle morphogenesis[END_REF][START_REF] Kitamura | Kinetochores Generate Microtubules with Distal Plus Ends: Their Roles and Limited Lifetime in Mitosis[END_REF][START_REF] Maiato | Kinetochore-driven formation of kinetochore fibers contributes to spindle assembly during animal mitosis[END_REF].

Once kinetochores are laterally attached to microtubules (Fig. 15), they are transported towards the spindle poles along the microtubule filaments (lateral sliding). This is promoted by motor proteins, which is dynein in vertebrate cells [START_REF] King | Dynein is a transient kinetochore component whose binding is regulated by microtubule attachment, not tension[END_REF][START_REF] Yang | Kinetochore Dynein Is Required for Chromosome Motion and Congression Independent of the Spindle Checkpoint[END_REF] and Kar3 (a member of the kinesin-14 family) in yeast (Tanaka et al., 2005(Tanaka et al., , 2007a)). Then, the plus-end of the microtubule shrinks and gets in contact with the kinetochore, leading to end-on attachment. Here kinetochores are pulled poleward as microtubules shrink (end-on pulling) (Fig. 16). These attachments are more stable: indeed, detachment never happens, whereas it does happen in about 5% of lateral attachments (Tanaka et al., 2007b).

In budding yeast, the Dam1 complex has turned out to be an important regulator of kinetochoremicrotubule attachments. Indeed, it is essential for end-on attachments by interacting and recruiting Ndc80 to the plus-ends and by stabilising its binding to microtubules (Lampert et al., 2010[START_REF] Lampert | Molecular requirements for the formation of a kinetochore-microtubule interface by Dam1 and Ndc80 complexes[END_REF].

Moreover, it converts the microtubule depolymerising forces, deriving from the curling of protofilaments that depolymerise, to pulling forces that move chromosomes towards the spindle pole (Westermann et al., 2006). This is possible because the complex forms a ring around the microtubules, which has also the possibility to slide along the microtubule filament [START_REF] Miranda | The yeast DASH complex forms closed rings on microtubules[END_REF][START_REF] Westermann | Formation of a dynamic kinetochore-microtubule interface through assembly of the Dam1 ring complex[END_REF]. As mentioned before, no homologs of the Dam1 complex seem to exist in higher eukaryotes. However, the Spindle and Kinetochore Associated (SKA) complex plays essential roles in stabilising kinetochore-microtubule attachments in human cells [START_REF] Gaitanos | Stable kinetochoremicrotubule interactions depend on the Ska complex and its new component Ska3/C13Orf3[END_REF]Welburn et al., 2009). Indeed, it sustains the microtubule binding of Ndc80, being functionally similar to the Dam1 complex (Schmidt et al., 2012;Welburn et al., 2009).

Once kinetochores are transported along the microtubules towards the spindle poles, one sister kinetochore has to become attached to microtubules emanating from one pole, while the other sister has to be attached to those emanating from the opposite pole. This extremely important process is referred to as sister chromatids bi-orientation, also known as amphitelic attachment. One mechanism that allows chromosome bi-orientation is the back-to-back orientation of sister chromatids [START_REF] Nicklas | CHROMOSOME MICROMANIPULATION[END_REF]: if one of the sister kinetochores attach to microtubules and faces one spindle pole, the other kinetochore will face the opposite pole and will normally be captured only from microtubules emanated from this pole (Östergren, 1951). This is possible because cohesion forces counterbalance the pulling forces and support inflexibility of sister kinetochore geometry [START_REF] Tanaka | Identification of cohesin association sites at centromeres and along chromosome arms[END_REF][START_REF] Tanaka | Chromosome bi-orientation on the mitotic spindle[END_REF].

During meiosis I the situation is quite different. Indeed, sister chromatids segregate to the same spindle pole, a process referred to as "mono-orientation". Here, the kinetochore geometry of sister chromatids is side-by-side, rather than back-to-back and this facilitates their orientation to the same pole (Fig. 17;[START_REF] Goldstein | Kinetochore structure and its role in chromosome orientation during the first meiotic division in male D. melanogaster[END_REF][START_REF] Lee | Specific regulation of CENP-E and kinetochores during meiosis I/meiosis II transition in pig oocytes[END_REF][START_REF] Parra | Involvement of the cohesin Rad21 and SCP3 in monopolar attachment of sister kinetochores during mouse meiosis I[END_REF]. Many factors influence sister chromatids mono-orientation in meiosis and they differ from organism to organism. First, kinetochores seem to be very closely associated [START_REF] Goldstein | Kinetochore structure and its role in chromosome orientation during the first meiotic division in male D. melanogaster[END_REF]. Indeed, it has been shown that sister kinetochores are cross-linked through MIS12 and NDC80 [START_REF] Li | Fused sister kinetochores initiate the reductional division in meiosis I[END_REF]. In budding yeast the multiproteic complex Monopolin is crucial for clamping the two sister kinetochores, which create a single microtubule-binding unit [START_REF] Petronczki | Monopolar attachment of sister kinetochores at meiosis I requires casein kinase 1[END_REF][START_REF] Rabitsch | Kinetochore recruitment of two nucleolar proteins is required for homolog segregation in meiosis I[END_REF][START_REF] Tóth | Functional genomics identifies monopolin: a kinetochore protein required for segregation of homologs during meiosis i[END_REF]. Monopolin associates with kinetochores from late phophase I until the end of meiosis I [START_REF] Tóth | Functional genomics identifies monopolin: a kinetochore protein required for segregation of homologs during meiosis i[END_REF]. Mono-orientation in meiosis I also depends on the temporal regulation of kinetochoremicrotubule attachments, since Monopolin cannot bind to kinetochores that are already associated with microtubules [START_REF] Miller | Meiosis I chromosome segregation is established through regulation of microtubule-kinetochore interactions[END_REF]. In fission yeast, instead, a fundamental role in chromatids mono-orientation is played by modifications in centromeric cohesion: indeed, the meiosis-specific cohesin subunit Rec8 is enriched at the central core of centromeres and this is important to favour the side-by-side geometry of sister kinetochores [START_REF] Sakuno | Kinetochore geometry defined by cohesion within the centromere[END_REF][START_REF] Watanabe | Cohesin Rec8 is required for reductional chromosome segregation at meiosis[END_REF]. The meiosis-specific factor Moa1 promotes cohesion at the core centromere [START_REF] Yokobayashi | The kinetochore protein Moa1 enables cohesion-mediated monopolar attachment at meiosis I[END_REF]. When Rec8 disappears from centromeres, in metaphase II, sister kinetochore biorientation for meiosis II is allowed. Rec8 has been shown to play a similar role in many organisms, including mice [START_REF] Parra | Involvement of the cohesin Rad21 and SCP3 in monopolar attachment of sister kinetochores during mouse meiosis I[END_REF]. In contrast, in budding yeast Rec8 does not seem to be required for sister kinetochore mono-orientation [START_REF] Monje-Casas | Kinetochore orientation during meiosis is controlled by Aurora B and the monopolin complex[END_REF][START_REF] Tóth | Functional genomics identifies monopolin: a kinetochore protein required for segregation of homologs during meiosis i[END_REF]. This can be due to the fact that since here a single microtubule attaches to one kinetochore (or to a pair of sister kinetochores in meiosis I) [START_REF] Winey | Three-dimensional ultrastructure of Saccharomyces cerevisiae meiotic spindles[END_REF], sister kinetochore fusion is probably a much more efficient mechanism to allow mono-orientation in this organism. Sometimes sister chromatids' geometry is not sufficient for amphitelic attachments. Indeed, there can be different kinds of incorrect kinetochore-microtubule attachments: monotelic attachments, where only one kinetochore is attached, syntelic attachments, where both sister kinetochores are attached from microtubules coming from the same pole, or merotelic attachments where a single kinetochore is attached from microtubules emanating from the two spindle poles (Fig. 18). Monotelic attachment: one of the sister kinetochores attaches to microtubules, whereas the other does not. Syntelic attachment: both sister kinetochores attach to microtubules emanating from the same spindle pole. Amphitelic attachment: sister kinetochores attach to microtubules emanating from opposite spindle poles. Merotelic attachment: one sister kinetochore attaches simultaneously to microtubules extending from both spindle poles. From Tanaka et al., 2005. One particular feature that leads to improper attachments concerns fungi undergoing a close mitosis. Indeed, in organisms undergoing an open mitosis spindle microtubules can interact with kinetochores only after the nuclear envelope is broken down at the beginning of mitosis. Conversely, in other organisms like budding yeast that undergo close mitosis and where nuclear microtubules are produced throughout the cell cycle [START_REF] Winey | The spindle cycle in budding yeast[END_REF], kinetochores can constantly interact with microtubules. In particular, in budding yeast the first contact takes place during the G1 phase of the cell cycle, when kinetochores stay attached to microtubules in the vicinity of the SPB [START_REF] Guacci | Centromere position in budding yeast: evidence for anaphase A[END_REF][START_REF] Jin | Centromere clustering is a major determinant of yeast interphase nuclear organization[END_REF]Tanaka et al., 2002). During DNA replication in S phase, kinetochores disassemble from centromeres and detach from microtubules [START_REF] Kitamura | Kinetochore-microtubule interaction during S phase in Saccharomyces cerevisiae[END_REF]. Then, a new SPB is generated close to the old one and afterwards the kinetochores reassemble on both replicated centromeres. At this point the old SPB is more mature than the new one, and therefore generates more microtubules (Tanaka et al., 2005), thus possibly generating incorrect attachments. Only after the new SPB matures and starts to separate from the old one, there will be the same chance for kinetochores to capture microtubules coming from both spindle poles.

Halt, no access! The Spindle Assembly Checkpoint

When errors in kinetochore-microtubule attachment occur, it is crucial for cells to protect themselves, avoiding to progress in the cell cycle and missegregate chromosomes.

During the last century, from the 1950's, it has been shown that spindle poisons not only destroy the spindle structure, but also arrest cells in mitosis; moreover, in cells where the chromosomes cannot attach to microtubules upon inactivation of kinetochore proteins, anaphase is delayed by several hours. It has then been discovered that a set of proteins is necessary for arresting cells in mitosis in presence of this kind of problems (reviewed in [START_REF] Joglekar | A Cell Biological Perspective on Past, Present and Future Investigations of the Spindle Assembly Checkpoint[END_REF]. Indeed, we know now that if errors in kinetochore-microtubule attachments occur, an important and conserved surveillance mechanism called Spindle Assembly Checkpoint (SAC, also referred to as the Mitotic checkpoint or spindle checkpoint) prevents the metaphase to anaphase transition until these errors are corrected and all chromosomes are stably attached to the spindle in a bipolar fashion.

SAC is a signalling cascade that includes a network of proteins that responds to even one single improperly attached kinetochore. Altogether, SAC proteins delay the metaphase-anaphase transition through the inactivation of the E3 ubiquitin ligase Anaphase Promoting Complex/Cyclosome (APC/C). The APC, in turn, triggers sister chromatid separation and exit from mitosis through ubiquitylation and subsequent degradation of several substrates, including Securin, the inhibitor of Separase, to allow disruption of cohesion complexes around sister chromatids, and cyclin B, whose degradation leads to mitotic exit (Fig. 19).

SAC catalyses the formation of an inhibitory complex, called the Mitotic Checkpoint Complex (MCC), which is composed of four proteins: Cdc20, Mad2, BubR1/Mad3 and Bub3 (reviewed in Musacchio, 2015).

The APC/C recognises different kinds of motifs through its co-factor Cdc20, which has binding pockets on its surface to interact with degrons of the substrate: D Box (Destruction Box) motifs, containing the RxxLxxxxN consensus sequence; KEN Box motifs, containing the KENxxxN consensus sequence; ABBA motifs, containing the

[ILVF]x[ILMVP][FHY]x[DE] consensus
sequence [START_REF] Morgan | The Cell Cycle: Principles of Control[END_REF].

Recent models envision different levels of inhibition of the MCC complex on the APC: first, the MCC would act as a pseudo-substrate inhibitor of the APC/C (through one of the KEN motifs of BubR1) [START_REF] Burton | Mad3p, a pseudosubstrate inhibitor of APCCdc20 in the spindle assembly checkpoint[END_REF][START_REF] Chao | Structure of the mitotic checkpoint complex[END_REF][START_REF] Lara-Gonzalez | BubR1 blocks substrate recruitment to the APC/C in a KEN-box-dependent manner[END_REF][START_REF] Malureanu | BubR1 N terminus acts as a soluble inhibitor of cyclin B degradation by APC/C(Cdc20) in interphase[END_REF]. Second, MCC would bind to APC Cdc20 (through the second KEN motif of BubR1) and directly inhibit the APC itself [START_REF] Hein | Stable MCC binding to the APC/C is required for a functional spindle assembly checkpoint[END_REF][START_REF] Izawa | The mitotic checkpoint complex binds a second CDC20 to inhibit active APC/C[END_REF]. The first, apical kinase that is recruited at kinetochores for SAC activation is Mps1.

As described in the previous chapters, Mps1 was initially characterised for its role in SPB duplication in yeast. However, it has been later found, again in budding yeast, to be part of the SAC. Consistently, cells carrying the temperature-sensitive mps1-1 mutant, although generating a monopolar spindle, were not able to arrest in mitosis, independently from their defect in SPBs duplication (Weiss and Winey, 1996).

The catalytic activity of the Mps1 kinase domain is mostly inhibited by its N-terminal extension (NTE) that keeps the kinase domain hidden until a conformational change occurs. Mps1 gets activated in mitosis thanks to its trans-autophosphorylation on several residues, due to dimerization that allows the conformational change (Combes et al., 2018).

Mps1 kinetochore localisation depends on its interaction with the NDC80 complex through two different interaction modules: the NTE and the TPR domain (N-terminal tetratricopeptide repeat domain) interact with the calponin homology (CH) domain of the NDC80 subunit HEC1 (Hiruma et al., 2015), close to the HEC1-microtubule interface; the MR (Middle Region) interacts with the CH domain of NUF2, partner of HEC1 [START_REF] Ji | Kinetochore attachment sensed by competitive Mps1 and microtubule binding to Ndc80[END_REF] (Fig. 20). This latter interaction at the beginning of mitosis involves an inactive form of the kinase Mps1, which presents a low kinetochore turnover (Dou et al., 2015). Indeed, kinase-dead mutants of Mps1 or chemically inhibited Mps1 exhibit higher levels of kinetochore localisation, suggesting that Mps1 kinase activity promotes its own kinetochore release (Hewitt et al., 2010;Jelluma et al., 2010;Kwiatkowski et al., 2010;Sliedrecht et al., 2010). The long residence at kinetochores stimulates transautophosphorylation of Mps1 molecules that have dimerized (Hewitt et al., 2010), driving rapid protein activation (Jelluma et al., 2008;[START_REF] Kang | Autophosphorylation-dependent activation of human Mps1 is required for the spindle checkpoint[END_REF][START_REF] Mattison | Mps1 Activation Loop Autophosphorylation Enhances Kinase Activity[END_REF][START_REF] Wang | Structural and mechanistic insights into Mps1 kinase activation[END_REF]. Now, one of the two interaction sites with the NDC80 complex is lost (MR-NUF2 interaction, since NUF2 is able to interact only with the inactive kinase) and Mps1 kinetochore turnover increases (Dou et al., 2015)(Fig. 21).

Another factor that has been shown to be essential for Mps1 kinetochore localisation is ARHGEF17, a Rho family GTPase exchange factor protein, which seems to act independently of its Rho GEF catalytic activity. Indeed, phosphorylated ARHGEF17 forms a complex with inactive Mps1 and localises it at kinetochores, where Mps1 then phosphorylates ARHGEF17 to induce its own release from kinetochores [START_REF] Isokane | ARHGEF17 is an essential spindle assembly checkpoint factor that targets Mps1 to kinetochores[END_REF].

The Aurora B kinase, whose major role is in the correction of faulty kinetochore-microtubule attachments (for details, see next paragraph), promotes Mps1 recruitment to unattached kinetochores at the onset of mitosis (Nijenhuis et al., 2013;Saurin et al., 2011;Zhu et al., 2013). One of the Aurora B targets is HEC1, the direct receptor of Mps1 at kinetochores, which led to the hypothesis that Aurora B might induce Mps1 localisation by phosphorylating the N-terminal tail of HEC1. In this view, upon bipolar attachments and tension establishment, when Aurora B kinase and its substrates are spatially separated (model further described below), Ndc80 would no longer be phosphorylated and this could extinguish Mps1 kinetochore localisation and SAC signalling (Fig. 21; Dou et al.

2019).

Importantly, Aurora B could be required for the maintenance of checkpoint signalling also by inducing the kinetochore recruitment of other checkpoint components (Bub1, Bub3, Mad1, Mad2; Vigneron et al., 2004). However, in mammalian cells it has been recently shown that Aurora B activity alone is insufficient for effective Mps1 kinetochore recruitment, and that an essential role is played by the kinase Cdk1-cyclin B1 through phosphorylation of Mps1 S281 in the kinetochore-binding domain. This phosphorylation is essential for checkpoint signalling and is counteracted by the phosphatase PP2A-B55 for SAC silencing (Hayward et al., 2019a). In the absence of Aurora B activity (a), the TPR domain inhibits the NTE (A) and HEC1 interacts with NUF2, blocking the MR-NUF2 interaction (B). When Aurora B is active (b), the NTE is released from the TPR inhibition. ARHGEF17 interacts with inactive Mps1 (D) and localises it to kinetochores where it binds to the NDC80 complex via the NTE and MR regions (E). The phosphorylation of the HEC1 tail by Aurora B prevents the binding to NUF2 and allows the MR to bind there. Binding of inactive Mps1 to the NDC80 with both subunits causes tight binding and long kinetochore residence time. The slow turnover allows Mps1 autoactivation, which will change conformation and will bind only HEC1 (F). Preventing MR binding to NUF2 lowers the affinity of Mps1 for the NDC80 complex, causing a higher turnover rate at kinetochores. From Pachis and Kops, 2018.

The first kinetochore event that is necessary for SAC activation is the phosphorylation by Mps1 of the kinetochore protein KNL1/Spc105 on its MELT repeats (consensus amino acid sequence "Met-Glu-Leu-Thr"). This phosphorylation event during prophase is essential for the kinetochore recruitment of Bub1 and Bub3, the first proteins of the checkpoint cascade that form a dimer. Bub3 is indeed able to read the phosphorylated amino acids of the MELT repeats (Primorac et al., 2013), which are up to 19 in human KNL1 and 6 in yeast Spc105. Each phosphorylated MELT repeat can bind one molecule of Bub3-Bub1 (London et al., 2012;Primorac et al., 2013;Shepperd et al., 2012;Yamagishi et al., 2012). Bub1, then, provides a binding interface for BubR1, Cdc20 and the Mad1-Mad2 complex [START_REF] Di Fiore | The ABBA motif binds APC/C activators and is shared by APC/C substrates and regulators[END_REF]Jia et al., 2016). However, the budding yeast BubR1 homolog, Mad3, has not been found enriched at kinetochores when the spindle checkpoint is activated (Gillett et al., 2004, Ruggiero et al., 2020). Despite being a kinase, Bub1 kinase activity is not required for SAC signaling (Fernius and Hardwick, 2005;[START_REF] Mcguinness | Regulation of APC/C Activity in Oocytes by a Bub1-Dependent Spindle Assembly Checkpoint[END_REF][START_REF] Sharp-Baker | Spindle Checkpoint Protein Bub1 Is Required for Kinetochore Localization of Mad1, Mad2, Bub3, and Cenp-E, Independently of Its Kinase Activity[END_REF]. Furthermore, in yeast Bub1 is localized at kinetochores already during S phase (Benzi et al., 2020;Gillett et al., 2004), and this is due to an early phosphorylation of Spc105 by Mps1 (London et al., 2012). In addition, the metazoan KNL1 protein contains two sequences known as KI motifs that bind directly to Bub1 and BubR1 independently of Mps1 [START_REF] Kiyomitsu | Protein Interaction Domain Mapping of Human Kinetochore Protein Blinkin Reveals a Consensus Motif for Binding of Spindle Assembly Checkpoint Proteins Bub1 and BubR1[END_REF][START_REF] Krenn | KI motifs of human Knl1 enhance assembly of comprehensive spindle checkpoint complexes around MELT repeats[END_REF][START_REF] Krenn | Structural analysis reveals features of the spindle checkpoint kinase Bub1-kinetochore subunit Knl1 interaction[END_REF].

Mps1 promotes MCC activation also through phosphorylation of Bub1 (London and Biggins, 2014) and Mad1: phosphorylated Bub1 binds to the Mad1-Mad2 complex, while phosphorylated Mad1 directly interacts with Cdc20 and this is essential for APC/C inhibition (Ji et al., 2018). Indeed, mutations in Mps1 phosphorylation sites on Mad1 or Bub1 abolish SAC signaling in human cells (Ji et al., 2018;London and Biggins, 2014). In addition, Mps1 activity early in mitosis is required for the recruitment of the RZZ complex, a kinetochore complex composed of four subunits (Rod, Zwilch, Zw10 and Zwint-1), which in turn recruits at kinetochores Mad1-C-Mad2 (Fig. 22) (Santaguida et al., 2010). One important formulation that has helped understanding how the MCC is generated is the "Mad2 template model". In fact, Mad2 exists in two different conformers: O-Mad2 (Open conformation), which is mostly in the cytoplasm and represents the majority of Mad2; C-Mad2 (Closed conformation, with two β-sheets moving across the face of the protein), which is the conformation adopted when the protein is bound to Mad1 or Cdc20. Mad1 and C-Mad2 form a tetramer at kinetochores, and because Mad2 can dimerize, this represents a receptor for O-Mad2. Once bound to the tetramer, O-Mad2 is converted into C-Mad2 bound to Cdc20. Indeed, Mad1 and Cdc20 share a Mad2-binding motif and C-Mad2 adopts the same conformation in both complexes. The C-Mad2-Cdc20 complex dissociates from the Mad1-C-Mad2 complex and in the cytosol continues to act as a receptor for O-Mad2 in order to convert it into C-Mad2. Therefore, the inhibitory signal is amplified because the C-Mad2-Cdc20 complex converts more and more molecules of O-Mad2 into Cdc20bound C-Mad2 [START_REF] De Antoni | The Mad1/Mad2 Complex as a Template for Mad2 Activation in the Spindle Assembly Checkpoint[END_REF]. This is a fundamental prerequisite for MCC formation and APC inhibition (Fig. 23). Mad2 is also important for positioning the KEN-box degron of Mad3, which is the N-terminal domain through which Mad3 binds to Cdc20 [START_REF] Chao | Structure of the mitotic checkpoint complex[END_REF]. This is critical to promote the interaction between BubR1/Mad3 and Cdc20. Indeed, BubR1/Mad3 seems to be the essential inhibitor of the interaction between the APC/C and its substrates, since Mad3 has been shown to compete with Cdc20 for its substrates binding in a KEN-dependent manner and occupy its KEN-box binding site [START_REF] Burton | Mad3p, a pseudosubstrate inhibitor of APCCdc20 in the spindle assembly checkpoint[END_REF][START_REF] Chang | Molecular architecture and mechanism of the anaphase-promoting complex[END_REF][START_REF] Sczaniecka | The spindle checkpoint functions of Mad3 and Mad2 depend on a Mad3 KEN box-mediated interaction with Cdc20-anaphase-promoting complex (APC/C)[END_REF]. Therefore, Mad3 acts as a pseudosubstrate for what concerns KEN-box containing substrates [START_REF] Burton | Mad3p, a pseudosubstrate inhibitor of APCCdc20 in the spindle assembly checkpoint[END_REF]. the INCENP ortholog is Sli15, Survivin is Bir1, Borealin is Nbl1, and Aurora B is Ipl1 (Fig. 24).

Better under tension: the error correction mechanism

When sister chromatids are not properly bi-oriented, Aurora B destabilises improper kinetochoremicrotubule attachments until proper, under tension, connections are generated [START_REF] Cimini | Aurora kinase promotes turnover of kinetochore microtubules to reduce chromosome segregation errors[END_REF][START_REF] Knowlton | Aurora B is enriched at merotelic attachment sites, where it regulates MCAK[END_REF][START_REF] Lampson | Correcting improper chromosomespindle attachments during cell division[END_REF][START_REF] Muñoz-Barrera | Increased Aurora B activity causes continuous disruption of kinetochore-microtubule attachments and spindle instability[END_REF].

The CPC complex acts from the centromeres, where it is localised during mitosis, until anaphase. The centromeric localization relies on the Survivin subunit, which binds to histone H3 phosphorylated by the kinase Haspin during mitosis [START_REF] Vader | Survivin mediates targeting of the chromosomal passenger complex to the centromere and midbody[END_REF].

Another important event for CPC centromeric localization is the phosphorylation of histone H2A by the kinase Bub1, which in turn recruits Sgo2 that interacts with the subunit of the CPC Borealin (Fischböck-Halwachs et al., 2019;Kawashima et al., 2007Kawashima et al., , 2010)). Moreover, the interaction between Borealin and Sgo2 requires phosphorylation of Borealin by the kinase Cdk1 (Tsukahara et al., 2010) (Fig. 24).

Fission yeast Bir1, the homologue of Survivin, seems to mediate interactions between CPC and histones H3 and H2A (Tsukahara et al., 2010;Yamagishi et al., 2010). However, it has been recently published that this pathway, although important for CPC centromeric recruitment, does not impact on kinetochore recruitment or activity of kinetochore-associated Aurora B kinase (see below).

Instead, Bub1 seems important for the kinetochore recruitment of Aurora B independently from its kinase activity and histone phosphorylation [START_REF] Broad | Aurora B kinase is recruited to multiple discrete kinetochore and centromere regions in human cells[END_REF].

Figure 24 : The Chromosomal Passenger Complex and its recruitment to centromeres. Scheme depicting the structure of the CPC complex and the molecular details behind the centromeric recruitment of the complex, in which the activity of several kinases and histone modifications play a major role. Through these mechanisms, the CPC is recruited to the inner centromere between the two sister kinetochores, where it will perform its functions. From Vader and Lens, 2010. The first model that has been proposed for Aurora B functioning at centromeres to phosphorylate kinetochore substrates is a tension-related model (Tanaka et al., 2002). When, during prometaphase, improper kinetochore-microtubule attachments lack tension, kinetochores are closer to the centromeres and can be phosphorylated by the kinase Aurora B. Once errors have been corrected and new connections under tension are generated, kinetochores are more distant from the kinase at centromeres and therefore substrates are no longer accessible. During anaphase, then, when sister chromatid cohesion is disrupted and tension is released, removing the CPC complex from the centromeres, through relocalization to the spindle, is essential in order to avoid the kinase Aurora B to keep destabilising the attachments (Fig. 25) (Tanaka et al., 2002).

Figure 25 : The tension-related model. When kinetochores are not under tension, Ipl1/Aurora B at centromeres is closer to its substrates and able to phosphorylate them. Once kinetochores are under tension, they are stretched away from centromeres and Ipl1/Aurora B is farther away from its kinetochore substrates and unable to keep phosphorylating them. From Tanaka et al., 2002. Recent work in budding yeast challenged this model: mutants in Sli15 lacking the N-terminal part of the protein (sli15ΔN), which is required to bring Ipl1 to centromeres, are viable and capable of supporting proper chromosome segregation. Moreover, these mutants, as well as sli15 mutants in which the Cdk1 phosphorylation sites required to prevent premature localization of Ipl1 to the spindle are abolished, restore viability in the absence of Bir1, the homologue of Survivin that brings the CPC to centromeres (Campbell and Desai, 2013). These results therefore challenged the requirement of the kinase Aurora B/Ipl1 at centromeres for its activity. Rather, it was proposed that local concentration of Aurora B/Ipl1, either at centromeres or on microtubules, prompts its activation and subsequent phosphorylation of kinetochore targets (Campbell and Desai, 2013). There could be different explanations for these observations. One is that Ipl1 could localize at kinetochores, besides at centromeres, in order to phosphorylate its substrates for the error correction pathway. Consistent with this hypothesis, recent data showed that in budding yeast Sli15 interacts with the C-terminal part of the Ctf19 subunit of the COMA complex (Fischböck-Halwachs et al., 2019;García-Rodríguez et al., 2019). Importantly, synthetic lethality between deletion of the Ctf19 or Mcm21 subunit of the COMA complex and the sli15ΔN allele was rescued by artificial tethering of Sli15ΔN to the COMA complex. This also restored proper chromosome segregation (Fischböck-Halwachs et al., 2019).

Thus, targeting Sli15 to the COMA complex is sufficient to position Ipl1 close to its kinetochore substrates and allows its function in the correction of kinetochore-microtubule attachments (Fischböck-Halwachs et al., 2019;García-Rodríguez et al., 2019;[START_REF] Makrantoni | Phosphorylation of Sli15 by Ipl1 is important for proper CPC localization and chromosome stability in Saccharomyces cerevisiae[END_REF]. However, this model does not explain how Aurora B/Ipl1 would phosphorylate its substrates only upon lack of tension.

Another model that has been proposed for Aurora B is called the "dog leash" (Fig. 26): this model takes into account the fact that the CPC localized at centromeres can reach kinetochores, where the substrates are, thanks to the INCENP extension. Indeed, INCENP, by bridging the two CPC modules (i.e. the localization module and the kinase module), would act as a leash from the localization module at centromeres to the kinase and would allow Aurora B, at the end of the leash, to phosphorylate its substrates only within the limits defined by the length of the linker. When kinetochores lack tension, the substrates on the outer kinetochore are closer and reachable by Aurora B. When the kinetochores stretch due to bipolar microtubule interactions, the substrates become unreachable and Aurora B stops detaching kinetochore-microtubule attachments (Krenn and Musacchio, 2015). A. The idea behind the dog leash model is that the localisation model of the CPC (the owner) is tethered at the base of centromeres. INCENP acts as a "dog leash" allowing "the dog" (Aurora B) to phosphorylates substrates only in the limit defined by the length of the leash. B-C. Application of the leash model. B. Under low tension Aurora B can reach the kinetochore and phosphorylate its substrates. C. Under high tension substrates have crossed the limit defined by the leash and can no longer be phosphorylated by the kinase. From Krenn and Musacchio, 2015.

One of the main substrates phosphorylated by Aurora B/Ipl1 is the subunit of the NDC80 complex HEC1/Ndc80. Aurora B phosphorylates the N-terminal part of the protein on multiple residues. These phosphorylations are progressive, and their degree is maximal when tension is low. As a consequence, the more phosphorylation sites are phosphorylated, the more Ndc80 loses affinity for microtubules (Alushin et al., 2010;Cheeseman et al., 2006;Ciferri et al., 2008;DeLuca et al., 2006;Zaytsev et al., 2015Zaytsev et al., , 2014)).

Another substrate of the kinase is the complex Dam1 in budding yeast and its functional homolog SKA complex. Both these complexes are essential to retain the Ndc80 complex at depolymerising microtubule tips (Asbury et al., 2006;Grishchuk et al., 2008;Lampert et al., 2010;Schmidt et al., 2012;Tien et al., 2010;Welburn et al., 2009;Westermann et al., 2006). Aurora B-dependent phosphorylation of these complexes negatively regulates this association, prompting again dissociation of Ndc80 from microtubules (Abad et al., 2014;Chan et al., 2012;Kalantzaki et al., 2015;Lampert et al., 2010b;[START_REF] Maure | The Ndc80 loop region facilitates formation of kinetochore attachment to the dynamic microtubule plus end[END_REF]Schmidt et al., 2012;Tien et al., 2010). Aurora B-dependent phosphorylations have been shown to be antagonised by both the phosphatases PP1 and PP2A. For what concerns the phosphatase PP1, in yeast it has been shown that truncated forms of GLC7, the catalytic subunit of PP1, suppress ipl1 mutations, suggesting that the phosphatase acts opposing the kinase in vivo [START_REF] Francisco | Type 1 Protein Phosphatase Acts in Opposition to Ipll Protein Kinase in Regulating Yeast Chromosome Segregation[END_REF]. In mammalian cells it has been shown that phosphorylation of a kinetochore-targeted sensor, which is phosphorylated by Aurora B, is increased in cells where PP1 is not localised at kinetochores in metaphase, suggesting that PP1 at kinetochores is important to dephosphorylate Aurora B kinetochore substrates (Liu et al., 2010).

On the other hand, in mammalian cells generation of stable kinetochore-microtubule attachments depends on PP2A-B56 and depletion of PP2A increases phosphorylation of Aurora B substrates [START_REF] Foley | Formation of stable attachments between kinetochores and microtubules depends on the B56-PP2A phosphatase[END_REF]. However, which substrates are dephosphorylated by PP1 or PP2A in this context has not been elucidated. Moreover, the relationships between Aurora B kinase and the phosphatases is complicated by the presence of feedback loops. For example, interaction between PP1 and the protein scaffold Repo-man is responsible for dephosphorylation of histone-H3, which is phosphorylated by Haspin in order to localise the CPC [START_REF] Qian | PP1/Repo-man dephosphorylates mitotic histone H3 at T3 and regulates chromosomal aurora B targeting[END_REF]. Aurora B, by phosphorylating directly Repo-man, inhibits its chromatin recruitment, thus preventing the dephosphorylation of histone-H3 [START_REF] Qian | Aurora B defines its own chromosomal targeting by opposing the recruitment of the phosphatase scaffold Repo-Man[END_REF]. Another example of complex regulation is the phosphorylation by Aurora B of the RVxF binding domain to the phosphatase PP1 on the kinetochore protein KNL1 (Liu et al., 2010;Rosenberg et al., 2011). By inhibiting PP1 kinetochore recruitment, the kinase therefore prevents SAC silencing (details described in in the next paragraph).

The Mps1 kinase has been involved in the regulation of kinetochore-microtubule attachments, and, in particular, of the error-correction mechanism (Jones et al., 2005;Maure et al., 2007;Santaguida et al., 2010). However, the role of Mps1 in this pathway is ill-defined. For example, the relationships between Mps1 and Aurora B are far from being fully understood. In mammalian cells Mps1 acts upstream of Aurora B, being important for its kinetochore recruitment through phosphorylation of the CPC complex subunit Borealin (Jelluma et al., 2008). On the other hand, Aurora B seems to operate upstream of Mps1, by guiding the kinetochore localization of SAC proteins, including Mps1 (Santaguida et al., 2011;Saurin et al., 2011;Vigneron et al., 2004). In contrast, in budding yeast the two kinases seem to work independently (Maure et al., 2007;Storchová et al., 2011). Therefore, the precise connections between the two kinases in the error-correction pathway need to be further characterised.

The substrates of Mps1 in chromosome bi-orientation also need to be identified. In mammalian cells Mps1 phosphorylates the SKA complex to destabilise tensionless kinetochore-microtubule attachments (Maciejowski et al., 2017). In contrast, in budding yeast phosphorylation by Mps1 of the functional homolog of the SKA complex, the Dam1 complex (specifically of the Dam1 protein), is not required for chromosome bi-orientation (Kalantzaki et al., 2015;Shimogawa et al., 2006).

In budding yeast Mps1 also phosphorylates Ndc80. However, this phosphorylation seems to be important only for SAC signalling, and mutation of these phosphorylation sites to alanine or aspartate does not have any effect on chromosome segregation or kinetochore-microtubule attachments (Kemmler et al., 2009).

The only Shugoshin protein in yeast, Sgo1, has been involved in the regulation of chromosome segregation through centromere maintenance of the CPC (Kawashima et al., 2007;Tsukahara et al., 2010;Verzijlbergen et al., 2014) and could be a target of Mps1 in this process (Storchová et al., 2011).

One interesting mechanism that has been proposed lately in Drosophila envisions that Mps1 could indirectly regulate chromosome segregation by controlling the nuclear import of Mad1 through phosphorylation of its nuclear pore complex scaffold Tpr/Megator: phosphorylation by Mps1 is essential for the release of the Mad1/Megator interaction, thus leading to kinetochore recruitment of Mad1. Abrogation of this pathway has consequences on both SAC activation and chromosome segregation [START_REF] Cunha-Silva | Mps1-mediated release of Mad1 from nuclear pores ensures the fidelity of chromosome segregation[END_REF]. This conclusion is in line with other studies. Indeed, a mitotic role for Mad1 other than SAC has been hypothesised in the past, since mad1 mutants in Drosophila were showing lagging chromatids caused at least partly by persistent merotelic linkages [START_REF] Emre | A mitotic role for Mad1 beyond the spindle checkpoint[END_REF]. In addition, in S. pombe Mad1 has a role in chromosome congression by recruiting at kinetochores the kinesin Cut7 (homolog of Eg5), which is implicated in microtubule sliding to establish spindle bipolarity [START_REF] Akera | Mad1 promotes chromosome congression by anchoring a kinesin motor to the kinetochore[END_REF]. However, in budding yeast, where Mps1 is essential for chromosome biorientation, Mad1 is completely dispensable for cell viability [START_REF] Li | Feedback control of mitosis in budding yeast[END_REF] and therefore other substrates must underlie the role of Mps1 in the error correction pathway.

So far, all other phosphorylations by Mps1 of kinetochore proteins have been related to SAC activation.

Green light: Spindle Assembly Checkpoint silencing

Once all tensionless kinetochore-microtubule attachments have been corrected and chromosome biorientation has been achieved, SAC can be silenced.

It has been proposed that SAC silencing is a two-step process: first, kinetochore attachment (occupancy) may decrease the capacity of SAC activation, by diminishing the localisation of key SAC proteins; then, after bipolar attachments, the tension at sister kinetochores could completely abolish SAC signalling by triggering the dephosphorylation events that will be discussed below (reviewed in Wang et al. 2014). Indeed, one key event that triggers checkpoint silencing is the delocalisation of Mps1 from the kinetochores. An appealing hypothesis envisions that Mps1 competes with microtubules for the binding to Ndc80: when microtubules are not attached, Mps1 strongly binds to its kinetochore receptor, while it gets dislodged when end-on attachments are established (Hiruma et al., 2015;[START_REF] Ji | Kinetochore attachment sensed by competitive Mps1 and microtubule binding to Ndc80[END_REF]. However, Mps1 can maintain SAC arrest also after being removed from kinetochores, suggesting that its removal from kinetochores is not sufficient for SAC silencing [START_REF] Maldonado | Constitutive Mad1 targeting to kinetochores uncouples checkpoint signalling from chromosome biorientation[END_REF]). An alternative, complementary hypothesis is that microtubules do not directly compete with Mps1 for binding to Ndc80, but rather microtubule binding would increase the distance between Mps1 and its crucial substrates for SAC signalling (Musacchio, 2015). This hypothesis is supported by the evidence that any perturbation to this distance re-activates SAC signalling [START_REF] Aravamudhan | The kinetochore encodes a mechanical switch to disrupt spindle assembly checkpoint signalling[END_REF].

In Drosophila the phosphatase PP1 has been shown to inactivate Mps1 by dephosphorylating its Tloop, which is normally essential for its catalytic activity. This dephosphorylation takes place at kinetochores and in the cytosol, and inactivation of both pools of Mps1 during metaphase is essential to ensure SAC silencing (Moura et al., 2017). Interestingly, in mammalian cells it has been recently demonstrated that the kinetochore pool of the phosphatase PP2A-B56 regulates the Mps1 T-loop phosphorylation, hence regulating its activation status (Hayward et al., 2019b). It is interesting to note, however, that in Drosophila the PP1 phosphatase action has mainly consequences on the SAC role of Mps1, whereas in mammalian cells the activity of PP2A-B56 mainly counteracts the role of Mps1 in the error correction mechanism (Hayward et al., 2019b;Moura et al., 2017). The reason for this difference is at the moment unclear.

Another regulatory process that switches off Mps1 is its degradation in anaphase mediated by the APC Cdc20 and APC Cdh1 ubiquitin ligase complexes [START_REF] Cui | Degradation of the Human Mitotic Checkpoint Kinase Mps1 Is Cell Cycle-regulated by APC-cCdc20 and APC-cCdh1 Ubiquitin Ligases[END_REF][START_REF] Palframan | Anaphase Inactivation of the Spindle Checkpoint[END_REF].

Besides Mps1, also the activity of the Aurora B kinase is suppressed when bi-orientation is established. Therefore, the decreased activity of these two kinases tips the balance between kinase/phosphatase activities in favour of phosphatases. First, the phosphatase PP2A, bound to its kinetochore scaffold BubR1, dephosphorylates the SILK/RVSF motif at the N-terminus of the KNL1 protein, which is normally phosphorylated by Aurora B, enabling the binding of the phosphatase PP1 [START_REF] Nijenhuis | Negative feedback at kinetochores underlies a responsive spindle checkpoint signal[END_REF] (Fig. 27). Binding of PP1 to this motif results in the dephosphorylation of the MELT repeats of KNL1, resulting in loss of kinetochore recruitment of the dimer Bub1/Bub3 and the other associated SAC components (Bokros et al., 2016;Liu et al., 2010;Meadows et al., 2011;Pinsky et al., 2009;Jessica S. Rosenberg et al., 2011;Vanoosthuyse and Hardwick, 2009). This phenomenon is highly conserved. Indeed, in budding yeast deletion of the Spc105/PP1 interaction motif is lethal, and it has been shown that this is due to lack of SAC silencing. Indeed, viability can be rescued by either deleting SAC components or fusing a wild type (but not a catalytically inactive) PP1 to Spc105, demonstrating that dephosphorylation of the kinetochore substrate Spc105 is essential for SAC silencing (Jessica S. Rosenberg et al., 2011). PP1 is associated with other PIPs (PP1 interacting proteins) that often carry an RVxF motif.

For example, in fission yeast the motor protein Klp5/6 (kinesin 8) is an additional PIP and its kinetochore recruitment of PP1 is necessary for SAC silencing in this model system (Meadows et al., 2011).

Another important kinetochore-targeting subunit of PP1 is Sds22. Sds22 and PP1 mutually depend on each other for their kinetochore localisation [START_REF] Posch | Sds22 regulates aurora B activity and microtubule-kinetochore interactions at mitosis[END_REF]. However, Sds22 does not carry the classical PP1-binding motif and its interaction with PP1 is mediated by another subunit, called Inhibitor-3, which instead harbours the motif [START_REF] Bollen | The extended PP1 toolkit: designed to create specificity[END_REF]. In budding yeast the Inhibitor-3 homolog (called Ypi1) acts as a positive regulator of nuclear PP1, since YPI1 depletion leads to a reduction of nuclear PP1 and loss of nuclear Sds22. Moreover, expression of a Ypi1 variant that binds more weakly PP1 activates the SAC and suppresses the temperature sensitivity of an ipl1 mutant [START_REF] Bharucha | Ypi1, a Positive Regulator of Nuclear Protein Phosphatase Type 1 Activity in Saccharomyces cerevisiae[END_REF]. Only in vertebrates, Repo-Man in complex with PP1 is important to dephosphorylate histone H3 during mitosis, determining a decreased localisation of the CPC [START_REF] Qian | PP1/Repo-man dephosphorylates mitotic histone H3 at T3 and regulates chromosomal aurora B targeting[END_REF]. Finally, in yeast, the PIP Fin1 seem to be essential to regulate PP1 activity for SAC silencing, since when it is mislocalised SAC is inappropriately silenced due to PP1 activity [START_REF] Akiyoshi | Quantitative proteomic analysis of purified yeast kinetochores identifies a PP1 regulatory subunit[END_REF].

One important feature of SAC silencing is the delocalisation of Mad1 and Mad2 from kinetochores, which is referred to as "stripping". This mechanism depends on the minus-end-directed motor protein Dynein, which localises at unattached kinetochores and dissociates upon kinetochore attachments [START_REF] King | Dynein is a transient kinetochore component whose binding is regulated by microtubule attachment, not tension[END_REF]. Dynein delocalises SAC components from kinetochores towards the spindle poles, along k-fibers (microtubules attached to kinetochores) [START_REF] Howell | Cytoplasmic dynein/dynactin drives kinetochore protein transport to the spindle poles and has a role in mitotic spindle checkpoint inactivation[END_REF][START_REF] Mische | Dynein light intermediate chain: an essential subunit that contributes to spindle checkpoint inactivation[END_REF]. The kinetochore localisation of Dynein depends on the RZZ complex (Rod/Zw10/Zwilch), which binds directly to NDC80 [START_REF] Cheerambathur | Crosstalk between microtubule attachment complexes ensures accurate chromosome segregation[END_REF][START_REF] Gama | Molecular mechanism of dynein recruitment to kinetochores by the Rod-Zw10-Zwilch complex and Spindly[END_REF]. Moreover, the protein Spindly mediates the interaction between Dynein and the RZZ complex [START_REF] Gassmann | A new mechanism controlling kinetochore-microtubule interactions revealed by comparison of two dynein-targeting components: SPDL-1 and the Rod/Zwilch/Zw10 complex[END_REF][START_REF] Griffis | Spindly, a novel protein essential for silencing the spindle assembly checkpoint, recruits dynein to the kinetochore[END_REF]. This mechanism, however, is not sufficient for SAC silencing. Moreover, it is not conserved, as no obvious homologs of Spindly or the RZZ complex are present, for instance, in budding yeast (Fig. 28).

SAC silencing also requires that existing MCC, soluble and APC/C-bound, is disassembled and/or degraded. In metazoans an important pathway for SAC silencing involves direct MCC disassembly by two proteins: p31 comet and TRIP13 (Thyroid receptor hormone interacting protein) (Fig. 28). p31 comet is a HORMA domain protein and it has been identified as a Mad2-binding protein [START_REF] Habu | Identification of a MAD2-binding protein, CMT2, and its role in mitosis[END_REF][START_REF] Xia | Conformation-specific binding of p31comet antagonizes the function of Mad2 in the spindle checkpoint[END_REF]. TRIP13 is an AAA + family ATPase. Together the two proteins specifically recognise soluble C-Mad2 and convert it into the O-Mad2 conformation, preventing its binding to the MCC complex [START_REF] Ye | TRIP13 is a protein-remodeling AAA+ ATPase that catalyzes MAD2 conformation switching[END_REF]. In addition, p31 comet structurally mimics the active conformation of Mad2, binds to the MCC and extracts it from the complex. Moreover, experimental evidence suggests that p31 comet and TRIP13 can also bind to the APC/C-MCC and extract C-Mad2 from the MCC, although less efficiently than MCC-C-Mad2 not bound to the APC/C [START_REF] Mansfeld | APC15 drives the turnover of MCC-CDC20 to make the spindle assembly checkpoint responsive to kinetochore attachment[END_REF][START_REF] Westhorpe | p31comet-mediated extraction of Mad2 from the MCC promotes efficient mitotic exit[END_REF]. Mad2 extraction would then destabilise the MCC, thus allowing APC/C reactivation (Fig. 28). A homolog of p31 comet , Tyc1 (Tiny yeast comet 1) has been recently found in yeast, but no link with SAC silencing has been found [START_REF] Schuyler | Peptide inhibitors of the anaphase promoting-complex that cause sensitivity to microtubule poison[END_REF].

Another pathway required for the disassembly of the MCC is the ubiquitylation and degradation of Cdc20, which is APC/C-dependent [START_REF] Goh | Cdc20 protein contains a destruction-box but, unlike Clb2, its proteolysisis not acutely dependent on the activity of anaphase-promoting complex[END_REF][START_REF] Prinz | The regulation of Cdc20 proteolysis reveals a role for the APC components Cdc23 and Cdc27 during S phase and early mitosis[END_REF][START_REF] Shirayama | The Polo-like kinase Cdc5p and the WD-repeat protein Cdc20p/fizzy are regulators and substrates of the anaphase promoting complex in Saccharomyces cerevisiae[END_REF]. Since Cdc20 is continuously synthesised, ubiquitylated and degraded, its turnover limits the amount of Cdc20 during the metaphase arrest. However, once the MCC starts to be disassembled and the Mad1-C-Mad2 complex is stripped away from the kinetochores, the new Cdc20, free from the MCCbinding, can represent an advantage for APC/C rapid reactivation. (Fig. 28).

Other proteins have been recently proposed to play a role in the MCC APC-bound disassembly: one example is the APC subunit Apc15, whose RNAi causes a phenotype similar to p31 comet RNAi (delay in anaphase onset) and increases Mad2-Cdc20 levels [START_REF] Mansfeld | APC15 drives the turnover of MCC-CDC20 to make the spindle assembly checkpoint responsive to kinetochore attachment[END_REF][START_REF] Uzunova | APC15 mediates CDC20 autoubiquitylation by APC/C(MCC) and disassembly of the mitotic checkpoint complex[END_REF]. Indeed, the protein causes ubiquitylation and degradation of Cdc20 [START_REF] Mansfeld | APC15 drives the turnover of MCC-CDC20 to make the spindle assembly checkpoint responsive to kinetochore attachment[END_REF][START_REF] Uzunova | APC15 mediates CDC20 autoubiquitylation by APC/C(MCC) and disassembly of the mitotic checkpoint complex[END_REF]. Moreover, its depletion has a synthetic effect with p31 comet depletion [START_REF] Mansfeld | APC15 drives the turnover of MCC-CDC20 to make the spindle assembly checkpoint responsive to kinetochore attachment[END_REF]. Its yeast homolog Mnd2 is also required for SAC silencing [START_REF] Foster | The APC/C subunit Mnd2/Apc15 promotes Cdc20 autoubiquitination and spindle assembly checkpoint inactivation[END_REF].

However, in stark contrast, in fission yeast Apc15 stabilizes MCC-APC/C complexes, thus ensuring the SAC arrest [START_REF] May | Fission Yeast Apc15 Stabilizes MCC-Cdc20-APC/C Complexes, Ensuring Efficient Cdc20 Ubiquitination and Checkpoint Arrest[END_REF]. Another important protein in this pathway is CUEDC2, which has been shown to bind to Cdc20 and promote its release from Mnd2, thereby acting as a negative regulator of SAC silencing [START_REF] Gao | Cdk1-phosphorylated CUEDC2 promotes spindle checkpoint inactivation and chromosomal instability[END_REF] (Fig. 28).

Introduction

Accurate chromosome segregation is a fundamental aspect of mitosis and secures the genetic stability of a cell lineage. During the mitotic cell cycle, sister chromatids that have been generated by chromosome replication in S phase must attach through their kinetochores to microtubules emanating from opposite spindle poles in M phase. This occurs through an error correction mechanism where kinetochores dynamically attach and detach from microtubules until bipolar arrangement of sister chromatids is finalised (reviewed in ref. [START_REF] Lampson | Mechanisms to avoid and correct erroneous kinetochore-microtubule attachments[END_REF]). Bipolar attachment, also referred to as amphitelic attachment, leads to tension across kinetochores, which arises from the balance of pulling forces exerted by microtubules and counteracting forces exerted by sister chromatid cohesion (reviewed in ref. [START_REF] Bloom | Tension management in the kinetochore[END_REF]). This tug of war leads also to chromosome congression to the metaphase plate.

Key to the error correction process is the evolutionary conserved CPC (chromosome passenger complex) that is composed by the Aurora B kinase and three additional centromere-targeting and activating subunits (INCENP, survivin and borealin). The CPC detects lack of kinetochore tension (e.g. in case of monotelic attachment where only one sister kinetochore is bound to microtubules or syntelic attachment where both sister kinetochores are attached to the same spindle pole) and favours amphitelic attachments by destabilising incorrect kinetochore-microtubule connections through phosphorylation of specific kinetochore substrates (reviewed in ref. [3]). The Mps1 kinase is also required for the error correction pathway in many organisms [START_REF] Jelluma | Mps1 phosphorylates borealin to control Aurora B activity and chromosome alignment[END_REF][START_REF] Jones | Chemical genetics reveals a role for Mps1 kinase in kinetochore attachment during mitosis[END_REF][START_REF] Maure | Mps1 kinase promotes sisterkinetochore bi-orientation by a tension-dependent mechanism[END_REF][7][START_REF] Storchová | Bub1, Sgo1, and Mps1 mediate a distinct pathway for chromosome biorientation in budding yeast[END_REF]. In human cells Mps1 acts in concert with Aurora B in this process [7,9,[START_REF] Van Der Waal | Mps1 promotes rapid centromere accumulation of Aurora B[END_REF]. Additionally, Mps1specific phosphorylation targets, like the kinetochore Ska complex, have been recently involved in the error correction mechanism [11]. Importantly, in budding yeast Mps1 seems to operate independently of Aurora B in chromosome biorientation and its critical targets are unknown [START_REF] Maure | Mps1 kinase promotes sisterkinetochore bi-orientation by a tension-dependent mechanism[END_REF][START_REF] Storchová | Bub1, Sgo1, and Mps1 mediate a distinct pathway for chromosome biorientation in budding yeast[END_REF][START_REF] Kalantzaki | Kinetochore-microtubule error correction is driven by differentially regulated interaction modes[END_REF]. Although the budding yeast functional orthologue of the SKA complex, the Dam1 complex, is phosphorylated by Mps1, mutations altering these phosphorylations have no impact on chromosome segregation [START_REF] Kalantzaki | Kinetochore-microtubule error correction is driven by differentially regulated interaction modes[END_REF]13].

The error correction process is intimately coupled to a conserved surveillance device, the spindle assembly checkpoint or SAC, that prevents sister chromatid separation and the onset of anaphase until all chromosomes are bioriented on spindle microtubules. SAC signalling fires at unattached kinetochores, where SAC proteins are recruited, and is propagated to the whole cell for inhibition of the E3 ubiquitin ligase anaphase-promoting complex (APC) bound to its activator Cdc20. A soluble protein complex called MCC (mitotic checkpoint complex) and composed of the SAC components Bub3, BubR1, Mad2 and Cdc20 is essential for APC/Cdc20 inhibition. This, in turn, prevents degradation of APC substrates, such as securin and cyclin B, thereby halting the cell cycle before the onset of anaphase (reviewed in ref. [START_REF] Musacchio | The molecular biology of spindle assembly checkpoint signaling dynamics[END_REF]).

The Mps1 kinase acts at the apex of the SAC signalling pathway, by phosphorylating the Knl1/Spc105 kinetochore scaffold on its MELT repeats, which in turn recruit the Bub3-Bub1 SAC complex [15][16][START_REF] Shepperd | Phosphodependent recruitment of Bub1 and Bub3 to Spc7/KNL1 by Mph1 kinase maintains the spindle checkpoint[END_REF][START_REF] Yamagishi | MPS1/Mph1 phosphorylates the kinetochore protein KNL1/Spc7 to recruit SAC components[END_REF]. Subsequent binding of the SAC factor Mad1 to Bub1, together with Mps1-mediated Mad1 phosphorylation, is a prerequisite for recruitment of Mad2 to kinetochores and MCC assembly [START_REF] Ji | A sequential multi-target Mps1 phosphorylation cascade promotes spindle checkpoint signaling[END_REF][START_REF] London | Mad1 kinetochore recruitment by Mps1mediated phosphorylation of Bub1 signals the spindle checkpoint[END_REF][START_REF] Hervas-Aguilar | Bub3-Bub1 binding to Spc7/KNL1 toggles the spindle checkpoint switch by licensing the interaction of Bub1 with Mad1-Mad2[END_REF][START_REF] Moyle | A Bub1-Mad1 interaction targets the Mad1-Mad2 complex to unattached kinetochores to initiate the spindle checkpoint[END_REF][23]. Consistently, phosphorylation of Knl1/Spc105 MELT repeats by Mps1 is essential for SAC signalling [15,[START_REF] Shepperd | Phosphodependent recruitment of Bub1 and Bub3 to Spc7/KNL1 by Mph1 kinase maintains the spindle checkpoint[END_REF][START_REF] Yamagishi | MPS1/Mph1 phosphorylates the kinetochore protein KNL1/Spc7 to recruit SAC components[END_REF]. The Knl1/Spc105 kinetochore protein has emerged as a key factor not only as a platform for SAC signalling, but also to silence the SAC once all pairs of sister chromatids are bipolarly attached. Indeed, Knl1/Spc105 harbours at its N-terminus an "RVSF" amino acid motif that binds to PP1, which extinguishes SAC signalling at least partly by dephosphorylating Knl1/Spc105 MELT repeats [START_REF] Bokros | Fin1-PP1 helps clear spindle assembly checkpoint protein Bub1 from kinetochores in anaphase[END_REF][START_REF] Liu | Regulated targeting of protein phosphatase 1 to the outer kinetochore by KNL1 opposes Aurora B kinase[END_REF][START_REF] Meadows | Spindle checkpoint silencing requires association of PP1 to both Spc7 and kinesin-8 motors[END_REF][START_REF] Pinsky | Protein phosphatase 1 regulates exit from the spindle checkpoint in budding yeast[END_REF][START_REF] Rosenberg | KNL1/Spc105 recruits PP1 to silence the spindle assembly checkpoint[END_REF][29]. Additionally, PP1 dephosphorylates the catalytic loop of Mps1, thus further silencing SAC [30].

Aurora B has also been implicated in SAC signalling. While an early model proposed that its role in the SAC would be indirect and linked to the generation of unattached kinetochores during the correction of tension-less kinetochore-microtubule attachments [31][32][33][34], increasing evidence supports a direct role of Aurora B in SAC activation from unattached kinetochores [9,[35][36][37][38][39]. For instance, Aurora B promotes kinetochore recruitment of several SAC factors, including Mps1, and weakens PP1 binding to Knl1 through phosphorylation of Knl1 RVSF motif, suggesting that it acts upstream of SAC signalling [7,9,[START_REF] Liu | Regulated targeting of protein phosphatase 1 to the outer kinetochore by KNL1 opposes Aurora B kinase[END_REF]31,40].

The involvement of Mps1 and Aurora B in both error correction and SAC signalling raises a crucial, yet unsolved, question: are these two pathways concomitantly elicited by a common upstream sensory device or, alternatively, they respond to distinct defects (i.e. lack of kinetochore tension and lack of kinetochore attachment)? Is it possible to activate SAC without engaging the error correction machinery and vice versa? If the molecular machineries detecting these defects were separable, it should be possible to generate hypo-or hyper-morphic mutants in the two apical kinases that selectively affect either pathway without perturbing the other.

Here, we report the characterisation of a novel temperaturesensitive mps1 mutant of budding yeast (named mps1-3) that is defective in chromosome biorientation and SAC signalling. An unbiased genetic screen for extragenic suppressors of the lethality of mps1-3 cells at high temperature revealed that mutations affecting the Spc105-PP1 interface restore proper chromosome biorientation as well as a proficient SAC response. Furthermore, artificial anchoring of Bub1 near the MELT repeats of Spc105 in mps1-3 cells results in a similar rescue. Altogether, our data indicate that Mps1 promotes both error correction and SAC signalling through a common molecular mechanism based on Spc105 phosphorylation at the MELT repeats and recruitment of the Bub1 kinase to kinetochores, thereby strengthening the idea that a single sensory apparatus engages both processes.

Results

The novel mps1-3 mutant is defective in chromosome segregation and proficient in SPB duplication

The targets of budding yeast Mps1 in the error correction pathway are unknown so far. Dissecting the function of Mps1 in kinetochore error correction is hampered by its involvement in the duplication of spindle pole bodies (SPBs) and the assembly of a bipolar spindle [41], the first step towards chromosome biorientation.

We isolated the mps1-3 mutant through a random mutagenesis screen for temperature-sensitive mps1 mutants (see Materials and Methods). Haploid mps1-3 cells grew slowly at 25-30°C and were unable to form colonies at temperatures above 32°C (Fig 1A). Sequencing of the mps1-3 allele revealed a single mutation replacing the conserved serine 635 in the kinase domain by phenylalanine (Fig 1B). To characterise the primary defects underlying the temperature sensitivity of mps1-3 cells, we synchronised in G1 mps1-3 cells, together with a wild-type control and the well-characterised mps1-1 mutant [41], and released cells at the restrictive temperature of 34°C. FACS analysis of DNA contents revealed the expected bimodal distribution of DNA contents in wild-type cells, while mps1-3 and mps1-1 cells showed abroad distribution of DNA contents after cytokinesis (90-105 min), indicative of massive chromosome missegregation (Fig 1C). However, while mps1-1 cells were unable to assemble bipolar spindles due to their failure to duplicate SPBs and to activate the SAC [41], mps1-3 cells proficiently formed bipolar spindles and elongated them during anaphase (Fig 1D and Appendix Fig S1A), suggesting that mps1-3 might be a separation of function mutant specifically defective in chromosome biorientation but proficient at SPB duplication.

To quantify the mps1-3 chromosome segregation defects, we used a genetic assay based on the ability of diploid cells to mate with a haploid tester strain upon events leading to loss of one of the two copies of the MAT locus, including loss of one entire chromosome III [42]. We used wild-type and homozygous mps1-1/mps1-1, mps1-3/mps1-3 diploid cells that were grown for a defined number of generations at 25°C. Already under these permissive conditions, the appearance of mating-competent cells was 1,000-to 2,000-fold higher in the mps1 mutants relative to the wild type (Fig 1E ), suggesting pronounced chromosome III loss even in conditions that allow cell proliferation.

To follow directly chromosome biorientation in mps1-3 haploid cells, we visualised by fluorescence microscopy the segregation of chromosome V tagged with the TetO/TetR system 1.4 kb from the centromere (CEN5-GFP) [43]. Cells were also expressing the SPB marker Spc97-mCherry and carried a cdc15-2 temperature-sensitive allele to arrest cells in telophase. G1-synchronised cells were released at the restrictive temperature of 34°C, and samples were collected at 120 and 150 min from the release. As expected, near the totality of wild-type cells bioriented and properly segregated the two sister chromatids of chromosome V at opposite SPBs, while almost all mps1-1 cells underwent CEN5 mono-orientation on the single unduplicated SPB (Fig 1F). Strikingly, the vast majority of mps1-3 mutant cells displayed duplicated SPBs that separated far apart, but at 120 0 the two chromatids had segregated correctly to opposite spindle poles in 42% of the cells, whereas in 41% of the cells the two chromatids had remained at the same spindle pole. Only 4% of the cells did not duplicate the SPBs, while 12% of the cells had duplicated SPBs that remained very close to each other in the mother cell, accompanied by lack of CEN5 segregation (Fig 1F). We conclude that the mps1-3 mutant, though capable to duplicate the SPBs and form bipolar spindles, is specifically defective in chromosome biorientation at 34°C. It is worth noting, however, that at higher temperatures (37°C) mps1-3 cells also failed SPB duplication and bipolar spindle assembly, suggesting that mps1-3 is a hypomorphic mutant.

Centromeric and pericentromeric cohesin is crucial for chromosome biorientation, but while centromeres transiently split in metaphase upon bipolar attachment, pericentromeric cohesion resists to pulling spindle forces [43]. We therefore asked if premature loss of pericentromeric cohesion could underlie the chromosome biorientation defect of mps1-3 cells. To answer this question, we tagged with the TetO/TetR system the pericentromere of chromosome V (TetO array at À13 kb from the centromere ref. [43]) and analysed its behaviour in metaphase-arrested cells by fluorescence microscopy. After a pre-synchronisation in G1, cells carrying the temperaturesensitive cdc16-1 allele (compromising APC/C activity at high temperatures and arresting cells in metaphase [44]) were released at 34°C. Under these conditions, sister pericentromeric regions of chromosome V did not disjoin in most mps1-3 cells, similar to wild-type and mps1-1 cells (Appendix Fig S1B). Thus, the chromosome biorientation defect of mps1-3 cells is not explained by a premature loss of pericentric cohesion.

Biorientation defects could be due to lack of kinetochore-microtubule attachments, or to a failure in the correction of faulty kinetochore-microtubule connections that are not under tension. To discriminate between these two processes, we imaged live cells with GFP-tagged CEN5 and expressing Tub1-mCherry to visualise microtubules. Cells were pre-incubated at 34°C for 1 h before filming at the same temperature. Under these conditions, while 100% of wildtype cells correctly bioriented the two sister CEN5 and segregated them to opposite spindle poles (n = 201, Figs 1G and3H), only 51.3% of mps1-3 mutant cells bioriented CEN5, whereas 48.7% of the cells did not (n = 179, Figs 1G and3H). During the whole movie, we did not detect lagging chromosomes or CEN5 signals off the spindle in mps1-3 cells, suggesting that kinetochore-microtubule attachment is not affected. Furthermore, in contrast to ipl1 mutant cells that missegregate chromosomes mostly towards the old SPB [45], we found no such bias in mps1-3 cells under the above conditions (Fig 3H). Interestingly, however, when we filmed mps1-3 after a G1 arrest and release at 34°C (i.e. during the first cell cycle at restrictive temperature, when the pre-existing SPB is likely fully functional), we found a strong missegregation bias towards the bud (Appendix Fig S1C). This bias tends to be lost in the subsequent cell cycles (Appendix Fig S1C). From these data, we conclude that mps1-3 cells are defective in the correction of monopolar attachments, like ipl1 mutants and in agreement with previous conclusions [START_REF] Maure | Mps1 kinase promotes sisterkinetochore bi-orientation by a tension-dependent mechanism[END_REF]. Additionally, they could alter the normal pattern of SPB asymmetric inheritance (see Discussion).

The mps1-3 mutant does not engage the spindle assembly checkpoint

To assess if mps1-3 cells can engage the SAC, pre-synchronised G1 cells were released at 34°C in presence of the microtubule-depolymerising drug nocodazole. FACS analysis of DNA contents showed that wild-type cells arrested as expected in metaphase with 2C DNA contents, indicative of proficient SAC signalling. Contrariwise, mps1-3 and mps1-1 cells kept progressing through the cell cycle accumulating 4C DNA contents, indicating that they cannot engage a SAC response (Fig 2A). This was confirmed by Western blot analysis of two APC substrates, the securin Pds1 and the cyclin B Clb2, which are normally degraded in anaphase and stabilised by the SAC. As expected, both proteins remained stable in wild-type cells treated with nocodazole, while they underwent proteolysis in mps1-1 and mps1-3 mutants (Fig 2B). Thus, the mps1-3 mutant is severely defective in SAC activation at high temperature.

The Mps1-3 mutant protein has enhanced kinase activity but has reduced levels at kinetochores

The catalytic activity of Mps1 is essential for both chromosome biorientation and SAC activation [START_REF] Jones | Chemical genetics reveals a role for Mps1 kinase in kinetochore attachment during mitosis[END_REF][START_REF] Maure | Mps1 kinase promotes sisterkinetochore bi-orientation by a tension-dependent mechanism[END_REF][START_REF] Storchová | Bub1, Sgo1, and Mps1 mediate a distinct pathway for chromosome biorientation in budding yeast[END_REF]15]. In order to understand if the defects of the mps1-3 mutant were due to impaired Mps1 kinase activity, we performed in vitro kinase assays using recombinant Mps1 and Mps1-3 proteins purified from bacteria. We measured the ability of these proteins to auto-phosphorylate and to phosphorylate the N-terminal part of Spc105 (aa 1-320) at different times of incubation in the presence of P 33 cATP at 34°C. Surprisingly, the Mps1-3 catalytic activity turned out to be more elevated than that of Mps1 at all time points (Fig 2C). This was confirmed by similar kinase assays in the presence of the exogenous substrate MBP and Mps1/ Mps1-3 proteins affinity-purified from yeast cells using galactoseinducible glutathione S-transferase (GST) fusion constructs [46] (Appendix Fig S2A). Contextually, Mps1-3 protein levels in yeast ◀ Figure 1. The mps1-3 mutant is defective in chromosome biorientation.

A

Serial dilutions of stationary phase cultures of the indicated strains were spotted on YEPD and incubated at the indicated temperatures. B Mps1 sequence alignment around Saccharomyces cerevisiae Ser635. Sc: Saccharomyces cerevisiae; Sp: Schizosaccharomyces pombe; Mm: Mus musculus: Hs: Homo sapiens; Dm: Drosophila melanogaster. C, D Wild-type, mps1-3 and mps1-1 mutant cells were synchronised in G1 with a-factor at permissive temperature (25°C) and then released at restrictive temperature (34°C, t = 0). Cells were collected at the indicated time points for FACS analysis of DNA contents (C) and for immunofluorescence using anti-tubulin antibodies in order to score metaphase and anaphase spindles (D). Budding and nuclear division were scored on the FACS samples. E

Frequencies of chromosome loss were quantified at permissive temperature (25°C). Since Mps1 levels at kinetochores increase when its kinase activity is inactivated [7,40,[48][49][50], it is conceivable that the elevated Mps1 kinase activity in mps1-3 cells accelerates Mps1 kinetochore turnover to a threshold that could be incompatible with SAC signalling and chromosome biorientation. To test this possibility, we tagged with three HA epitopes the MPS1 and mps1-3 genes at their genomic locus and checked the distribution of the corresponding proteins on chromosomes by ChIP-seq using cells pre-synchronised in G1 and released in nocodazole for 90 min at 34°C, time at which both MPS1 and mps1-3 cells were still in mitosis. Strikingly, while wild-type Mps1 clearly accumulated at the centromeric region of all 16 yeast chromosomes, Mps1-3 did not (Fig 2D and Appendix Fig S3), suggesting that its residence at kinetochores is severely impaired.

To definitely ascertain if reduced kinetochore levels are the main reason for the Mps1-3 loss of function, we artificially recruited to kinetochores GFP-tagged Mps1 and Mps1-3 in cells that co-expressed the kinetochore protein Mtw1 tagged with the high affinity GFPnanotrap (GFP-binding domain or GBD) [51]. This strategy led to constitutive association of Mps1 to the kinetochore in 91.3% of the cells and of Mps1-3 in 88.5% of the cells (n = 196 and n = 209, respectively). Cells also carried the deletion of MAD2 to obliterate SAC, which would be otherwise constitutively active following Mps1 tethering to kinetochores [49,52]. Notably, anchoring Mps1-3 to Mtw1 could partially restore proper chromosome segregation at 34°C, as shown by the bimodal distribution of DNA contents by FACS analysis (Fig 2E), suggesting that the chromosome biorientation defects of mps1-3 cells are largely accounted for by decreased Mps1 kinetochore levels.

A genetic screen for spontaneous suppressors of the mps1-3 mutation Since SAC is not essential in budding yeast [53,54], we reasoned that chromosome missegregation could be the main cause of the lethality of mps1-3 cells at high temperatures, which makes this mutant a unique genetic tool to study the role of Mps1 in chromosome biorientation. To gain insights into this process, we performed an unbiased genetic screen for spontaneous mutations suppressing the temperature sensitivity of mps1-3 cells at 34°C. Classical genetic analyses allowed us to establish if the suppressing mutations were recessive or dominant, distinguish the intragenic from the extragenic suppressors (i.e. suppressing mutation inside or outside the mps1-3 gene, respectively) and assess if the extragenic suppressors were allelic to one another. Through this screen, we could isolate 27 suppressors. Out of these, 17 turned out to be intragenic while 10 were extragenic suppressors. We focused on the extragenic suppressors, which could provide valuable insights into the molecular mechanism underlying the role of Mps1 in chromosome biorientation. Through genetic analyses and whole genome sequencing, we could establish that the suppressing mutations hit three different genes (Fig 3A andB): SPC105, encoding for the kinetochore protein Spc105/Knl1; GLC7, encoding for the catalytic subunit of the phosphatase PP1; and GRR1, encoding for an F-box protein of the E3 ubiquitin ligase complex SCF. Three GRR1 suppressors out of four carried also a mutation in RSC30, encoding for a subunit of the RSC chromatin-remodelling complex [START_REF] Huang | Tripin/hSgo2 recruits MCAK to the inner centromere to correct defective kinetochore attachments[END_REF]. The basis for the suppression by the GRR1 mutations, alone or in combination with the mutation in RSC30, was not immediately clear and will be investigated in the future.

Spc105 (Knl1 in metazoans) is a known target of Mps1 in the SAC [15]. Mps1-dependent phosphorylation of its MELT repeats is crucial for SAC signalling by recruiting Bub1 and Bub3 [15][16][START_REF] Shepperd | Phosphodependent recruitment of Bub1 and Bub3 to Spc7/KNL1 by Mph1 kinase maintains the spindle checkpoint[END_REF][START_REF] Yamagishi | MPS1/Mph1 phosphorylates the kinetochore protein KNL1/Spc7 to recruit SAC components[END_REF]. The PP1 Glc7 in turn dephosphorylates Spc105 MELT repeats by binding directly the consensus RV/IXF motif at the N-terminus of Spc105 (RVSF, aa [START_REF] García-Rodríguez | Aurora B-INCENP localization at centromeres/inner kinetochores is required for chromosome bi-orientation in budding yeast[END_REF][START_REF] Makrantoni | Efficient chromosome biorientation and the tension checkpoint in Saccharomyces cerevisiae both require Bir1[END_REF][START_REF] Fischböck-Halwachs | The COMA complex interacts with Cse4 and positions Sli15/Ipl1 at the budding yeast inner kinetochore[END_REF][START_REF] Kawashima | Phosphorylation of H2A by Bub1 prevents chromosomal instability through localizing shugoshin[END_REF] to release Bub1 and silence the checkpoint [15,[START_REF] Rosenberg | KNL1/Spc105 recruits PP1 to silence the spindle assembly checkpoint[END_REF]. Strikingly, all our suppressing mutations in SPC105 encompassed or were close to the PP1-binding motif, while both GLC7 suppressors carried a missense mutation of Phe256 (to Ile or Val), which resides in the hydrophobic groove involved in PP1 interaction with partners carrying the RV/IXF consensus motif [56] (Fig 3B). Collectively, these observations suggest that reduced PP1 interaction with Spc105 likely accounts for the rescue of the temperature sensitivity of mps1-3 cells. Interestingly, the spc105-V76G mutation was recessive and lethal in otherwise wild-type cells, consistent with the notion that mutations in the PP1-binding site of Spc105 are inviable due to constitutive SAC activation [START_REF] Rosenberg | KNL1/Spc105 recruits PP1 to silence the spindle assembly checkpoint[END_REF], while spc105-A79T and spc105-V82D were viable, suggesting that they might interfere with PP1 recruitment to kinetochores less severely than spc105-V76G. It is also worth noting that spc105-V76G suppressed more robustly than spc105-A79T and spc105-V82D the lethality of mps1-3 cells at high temperatures (Fig 3A).

◀ Figure 2. The mps1-3 mutant is defective in SAC signalling and does not localise Mps1 at kinetochores.

A Wild-type and mps1 mutant cells were synchronised in G1 with a-factor at 25°C and then released at 34°C in the presence of nocodazole (t = 0). Cells were collected at the indicated time points for FACS analysis of DNA contents. B Cells were treated as in (A), and after 2 h from the release, a-factor was re-added to prevent cells from undergoing a second cell cycle. Cell samples were collected at the indicated time points for Western blot analysis of the indicated proteins. Pgk1 was used as loading control. Cyc: cycling cells. C Top: in vitro kinase assays with recombinant Mps1 and Mps1-3 purified from Escherichia coli and incubated at 34°C for the indicated times in the presence of radioactive ATP and a recombinant Spc105 N-terminal fragment (aa 1-320) as substrate. Bottom left: kinase activity was quantified on the autoradiographs by ImageJ and normalised to the levels of the full-length protein on the Coomassie blue-stained gel (right). Bottom right: 1 lg of the recombinant Mps1 kinases used for kinase assays was loaded on SDS-PAGE and stained with Coomassie blue for normalisation of kinase assays. D Wild-type and mps1-3 cells were synchronised in G1 with a-factor at 25°C and then released at 34°C in the presence of nocodazole. Cells were collected after 90 min and fixed with formaldehyde for ChIP-seq analysis. ChIP sequence reads were normalised against sequence reads from corresponding input samples, and relative enrichment is plotted for chromosome III around the centromere (see the centromeric regions of all 16 yeast chromosomes in Appendix Consistent with the idea that lack of PP1 recruitment to Spc105 rescues the lethality of mps1-3 cells, the well-characterised RASA mutation in the PP1 binding motif of Spc105 [START_REF] Rosenberg | KNL1/Spc105 recruits PP1 to silence the spindle assembly checkpoint[END_REF] also suppressed the temperature sensitivity of mps1-3 cells (Fig 3C ; note that the spc105-RASA mutation is lethal unless SAC activity is abolished, e.g. by MAD2 deletion [START_REF] Rosenberg | KNL1/Spc105 recruits PP1 to silence the spindle assembly checkpoint[END_REF]). Additionally, fusing the PP1 catalytic subunit Glc7 to the N-terminus of the Spc105-RASA mutant protein [START_REF] Rosenberg | KNL1/Spc105 recruits PP1 to silence the spindle assembly checkpoint[END_REF] almost completely abolished its suppressing properties (Fig 3C), indicating that suppression stems only from loss of PP1 binding to Spc105.

The GLC7 mutations isolated in our screen were dominant and viable in otherwise wild-type cells. Since dominant mutations are often associated with gain of function, this observation was quite puzzling and prompted us to characterise PP1 activity in GLC7-24 cells. As a readout, we analysed the phosphorylation status of the well-characterised PP1 cytoplasmic substrate Snf1, which is hyperphosphorylated on T210 under low glucose conditions (1%) and gets dephosphorylated by Glc7 in high glucose (4%) [57]. No significant differences in the phosphorylation of Snf1 T210 were apparent in mps1-3 GLC7-24 and GLC7-24 cells relative to the wild type (Appendix Fig S4A), suggesting that the cytoplasmic activity of PP1 is not affected. To assess PP1 phosphatase activity at kinetochores in GLC7-24 cells, we monitored phosphorylation of Sli15, a subunit of the Aurora B complex that resides at kinetochores and on spindle microtubules and is involved in chromosome biorientation [45,58]. Sli15 is phosphorylated by Ipl1/Aurora B [59], whose activity is often reversed by PP1 [START_REF] Resnick | INCENP and Aurora B promote meiotic sister chromatid cohesion through localization of the Shugoshin MEI-S332 in Drosophila[END_REF]61]. Interestingly, Sli15 phosphorylation was more pronounced in cycling cells and slightly advanced during the cell cycle in the GLC7-24 mutant relative to the wild type (Appendix Fig S4B). Thus, Glc7 phosphatase activity is not globally elevated in GLC7-24 cells; rather, it might impair dephosphorylation of specific kinetochore substrates.

Suppressing mutations in SPC105 and GLC7 restore SAC activation and normal chromosome segregation in mps1-3 cells To investigate further the phenotypes of the suppressors, we analysed their ability to engage the SAC and undergo normal chromosome segregation. Pre-synchronised G1 cells were released at 34°C in the presence and absence of nocodazole and subjected to FACS analysis of DNA contents at different time points. Importantly, the spc105-18 and GLC7-24 mutations restored proficient SAC activation in mps1-3 cells (Fig 3D). In mps1-3 GLC7-24 cells, this was accompanied by Mad1 hyperphosphorylation, which requires Mps1 activity [62,63] and was abolished in mps1-3 cells (Fig 3E; note that we could not study Mad1 phosphorylation pattern in mps1-3 spc105-18 cells due to their synthetic lethality with HA-tagged Mad1). Since Mad1 is recruited to unattached kinetochores and is not part of soluble MCC [START_REF] Fraschini | Bub3 interaction with Mad2, Mad3 and Cdc20 is mediated by WD40 repeats and does not require intact kinetochores[END_REF][START_REF] Gillett | Spindle checkpoint proteins and chromosome-microtubule attachment in budding yeast[END_REF], these data further strengthen the idea that in GLC7-24 cells PP1 has impaired ability to dephosphorylate kinetochore substrates. Additionally, they raise the possibility that PP1 could dephosphorylate Mad1 directly.

Remarkably, the spc105-18 and GLC7-24 suppressors also rescued the chromosome segregation defects of mps1-3, as shown by the bimodal distribution of DNA contents after one complete cell cycle at 34°C (Fig 3F). Moreover, live cell imaging of mps1-3 spc105-18 and mps1-3 GLC7-24 cells with GFP-tagged CEN5 and expressing Tub1-mCherry showed nearly complete suppression of chromosome missegregation (Fig 3G and H). Importantly, the spc105-18 and GLC7-24 alleles did not restore normal levels of Mps1-3 at centromeres, as shown by ChIP-seq (Fig 3I and Appendix Fig S5). Furthermore, suppression of chromosome missegregation was not a consequence of restored SAC activity, which might impose sufficient time for error correction, because it remained unchanged upon MAD2 deletion (Fig 3H).

Thus, these data strongly argue that a common mechanism involving Mps1 and antagonised by PP1 simultaneously promotes error correction of faulty kinetochore attachments and SAC signalling.

The spc105-18 and GLC7-24 suppressors restore Spc105 phosphorylation in mps1-3 cells To further explore the possibility that Mps1 could promote SAC signalling and chromosome biorientation through the same molecular mechanism, we analysed the phosphorylation status of Spc105 in wild-type and mps1-3 cells at 34°C in presence or absence of nocodazole. As expected, Spc105 tagged with 3HA (Spc105-3HA) was promptly phosphorylated in wild-type cells treated with nocodazole, as indicated by its mobility shift in SDS-PAGE (times ◀ Figure 3. Characterisation of the extragenic suppressors of mps1-3 mutant cells.

A

Serial dilutions of stationary phase cultures of the indicated strains were spotted on YEPD and incubated at 25°C and 34°C.

B

List of extragenic suppressors found in the unbiased genetic screen. C Serial dilutions of stationary phase cultures of the indicated strains were spotted on YEPD and incubated at the indicated temperatures. D Cells with the indicated genotypes were synchronised in G1 with a-factor at 25°C and then released in fresh medium at 34°C in presence of nocodazole (t = 0). Cells were collected at the indicated time points for FACS analysis of DNA contents. E Cells were treated as in (D) and collected at the indicated time points for Western blot analysis of Mad1-3HA. Cyc: cycling cells. F Cells with the indicated genotypes were synchronised at 25°C and then released in fresh medium at 34°C (t = 0). Cells were collected at the indicated time points for FACS analysis of DNA contents. G, H Cells with the indicated genotypes carrying the TetO/TetR-GFP markers for CEN5 labelling and expressing mCherry-Tub1 were grown at 25°C and then shifted to 34°C for 1 h before filming. Cells were filmed at 34°C every A, B Wild-type and mps1-3 cells were synchronised in G1 with a-factor at 25°C and then released at 34°C in the presence (A) or absence (B) of nocodazole (t = 0). Cells were collected at the indicated time points for Western blot analysis of the indicated proteins. Equal amounts of protein extracts were loaded on two different gels, for Western blot of Spc105-3PK and Clb2/Pgk1, respectively. Clb2 was used as mitotic marker and Pgk1 as loading control. Cyc: cycling cells. C, D Cells with the indicated genotypes expressing Bub1-GFP and the kinetochore marker Mtw1-Tomato were grown in SD glu 2% and then shifted to 34°C for 1 h before filming; they were then filmed every 4 min by time-lapse fluorescence microscopy at 34°C. Note that BUB1-GFP is synthetic lethal with mps1-3. Thus, we used mps1-3 GALs-MPS1 BUB1-GFP cells that were grown in -His RG medium at 30°C; glucose was added to the culture for 30 min to shut off GALs-MPS1, followed by shifting cells to SD medium at 34°C for 1 h before imaging in the same medium. Bub1 recruitment to kinetochores is impaired in mps1-3 cells but is restored by the spc105-18 and GLC7-24 suppressing mutations

As mentioned above, Spc105/Knl1 phosphorylation primes Bub1 recruitment to kinetochores for SAC signalling [15][16][START_REF] Shepperd | Phosphodependent recruitment of Bub1 and Bub3 to Spc7/KNL1 by Mph1 kinase maintains the spindle checkpoint[END_REF][START_REF] Yamagishi | MPS1/Mph1 phosphorylates the kinetochore protein KNL1/Spc7 to recruit SAC components[END_REF]. Since Bub1 transiently associates with kinetochores also under unperturbed conditions [START_REF] Gillett | Spindle checkpoint proteins and chromosome-microtubule attachment in budding yeast[END_REF], we visualised by time-lapse video microscopy Bub1-GFP along with the kinetochore marker Mtw1-Tomato in wildtype and mps1-3 cells at 34°C. Under these conditions, Bub1 was transiently recruited to kinetochores in wild-type cells with a small and medium bud, i. In order to be fully functional for SAC activation, Bub1 also needs to be directly phosphorylated by Mps1, which in turn allows its binding to Mad1 [START_REF] Ji | A sequential multi-target Mps1 phosphorylation cascade promotes spindle checkpoint signaling[END_REF][START_REF] London | Mad1 kinetochore recruitment by Mps1mediated phosphorylation of Bub1 signals the spindle checkpoint[END_REF][START_REF] Hervas-Aguilar | Bub3-Bub1 binding to Spc7/KNL1 toggles the spindle checkpoint switch by licensing the interaction of Bub1 with Mad1-Mad2[END_REF]. Thus, we checked if Bub1 phosphorylation was also impaired in mps1-3 cells. Pre-synchronised G1 cells were released at 34°C in the presence or the absence of nocodazole. Although several forms of Bub1 with different electrophoretic mobility were present throughout the cell cycle, one particular isoform with slowest mobility appeared in the wild type at 20 0 after G1 release, i.e. when cells entered S phase, thus correlating with the timing of Bub1 kinetochore localisation (Fig 4E-H Thus, the mechanism underlying the suppression of mps1-3 mutant cells might be independent of the phosphorylation status of Bub1.

Since Bub1 has been involved in kinetochore biorientation in budding yeast [START_REF] Storchová | Bub1, Sgo1, and Mps1 mediate a distinct pathway for chromosome biorientation in budding yeast[END_REF]66], we conclude that lack of Bub1 recruitment to kinetochores is likely the main reason underlying the chromosome segregation defects of mps1-3 cells. Lowering PP1 levels at Spc105 in the suppressors favours phosphorylation of the MELT repeats of Spc105, thereby restoring Bub1 recruitment at kinetochores and chromosome biorientation.

Artificial targeting of Bub1 to Spc105 restores proper chromosome segregation and SAC response in mps1-3 cells If our above conclusions are correct, artificial tethering of Bub1 to kinetochores could rescue the chromosome segregation defects of mps1-3 cells at high temperatures. To test this hypothesis, we generated yeast strains expressing as sole source of Spc105 an Spc105 variant where we had inserted the GFP-binding domain upstream of the MELT repeats of Spc105 (Spc105-GBD). In the same strains, we co-expressed Bub1-GFP or Bub3-GFP (note that although Bub3 enhances Bub1 binding to Spc105 by directly reading its phosphorylated MELT repeats, it has no additional role in Bub1 activation [16,[START_REF] Breit | Role of intrinsic and extrinsic factors in the regulation of the mitotic checkpoint kinase Bub1[END_REF]). This strategy led to constitutive recruitment of Bub1 to kinetochores throughout the cell cycle (Fig 5A and Movie EV1), with no obvious impact on the proliferation rate of otherwise wildtype cells (Fig 5B ), indicating that prolonged Bub1 residence at kinetochores is insufficient to constitutively activate the SAC. Artificial recruitment of Bub1 to Spc105 only modestly, if at all, rescued the lethality of mps1-3 cells at high temperatures (Fig 5B). However, it did prolong the viability of mps1-3 cells after transient incubation at restrictive temperature (Fig 5C ), suggesting that proper chromosome segregation might be partially restored. In agreement with this hypothesis, FACS analysis of DNA contents in synchronised mps1-3 cells, either lacking or carrying the SPC105-GBD and BUB1-GFP or BUB3-GFP constructs, showed that the massive chromosome missegregation of mps1-3 cells at 34°C was partially (BUB3-GFP) or largely (BUB1-GFP) rescued, as indicated by the bimodal distribution of DNA contents (Appendix Fig S7A and Fig 5D). Interestingly, artificial anchoring of Bub1 to Spc105 also partially suppressed the SAC defects of mps1-3 cells (Appendix Fig S7B). Nevertheless, the rescue of chromosome missegregation was independent of restored SAC signalling, as it remained unaffected by MAD2 deletion (Fig 5C andD). Thus, the main reason underlying the chromosome biorientation, and possibly SAC defects, of mps1-3 cells is the lack of Bub1 at kinetochores.

The function of Mps1 and Bub1 in chromosome biorientation has been previously linked to pericentromeric localisation of Shugoshin, which in yeast requires Mps1 and Bub1 kinase activity [START_REF] Van Der Waal | Mps1 promotes rapid centromere accumulation of Aurora B[END_REF]66,[START_REF] Nerusheva | Tension-dependent removal of pericentromeric shugoshin is an indicator of sister chromosome biorientation[END_REF][START_REF] Peplowska | Sgo1 regulates both condensin and Ipl1/Aurora B to promote chromosome biorientation[END_REF]. Shugoshin senses the lack of kinetochore tension and engages the error correction machinery through kinetochore recruitment of the CPC complex (chromosome passenger complex) [START_REF] Nerusheva | Tension-dependent removal of pericentromeric shugoshin is an indicator of sister chromosome biorientation[END_REF][START_REF] Peplowska | Sgo1 regulates both condensin and Ipl1/Aurora B to promote chromosome biorientation[END_REF][70][START_REF] Kawashima | Shugoshin enables tension-generating attachment of kinetochores by loading Aurora to centromeres[END_REF][START_REF] Rivera | Xenopus Shugoshin 2 regulates the spindle assembly pathway mediated by the chromosomal passenger complex: XSgo2 regulates the spindle assembly pathway mediated by CPC[END_REF][START_REF] Vanoosthuyse | Shugoshin 2 regulates localization of the chromosomal passenger proteins in fission yeast mitosis[END_REF][START_REF] Verzijlbergen | Shugoshin biases chromosomes for biorientation through condensin recruitment to the pericentromere[END_REF]. Although the role of budding yeast Mps1 in chromosome biorientation seems independent of the CPC and Aurora B activity [START_REF] Maure | Mps1 kinase promotes sisterkinetochore bi-orientation by a tension-dependent mechanism[END_REF][START_REF] Storchová | Bub1, Sgo1, and Mps1 mediate a distinct pathway for chromosome biorientation in budding yeast[END_REF], we decided to investigate if the chromosome segregation defects of mps1-3 cells could be ascribed to poor Shugoshin activity at pericentromeres. Although co-localisation of Sgo1 (the only budding Shugoshin) with kinetochore clusters was partially compromised in mps1-3 cells at 34°C (Appendix Fig S8A), in agreement with previous observations [START_REF] Storchová | Bub1, Sgo1, and Mps1 mediate a distinct pathway for chromosome biorientation in budding yeast[END_REF] and with the loss of Bub1 at kinetochores, mps1-3 cells turned out to be synthetically lethal with SGO1 deletion even at 25°C (Appendix Fig S8B), strongly arguing against a linear pathway where Mps1 acts upstream of Sgo1.

The role of budding yeast Aurora B (Ipl1) in chromosome biorientation relies on its alternative targeting to centromeres or the inner kinetochore [START_REF] García-Rodríguez | Aurora B-INCENP localization at centromeres/inner kinetochores is required for chromosome bi-orientation in budding yeast[END_REF][START_REF] Makrantoni | Efficient chromosome biorientation and the tension checkpoint in Saccharomyces cerevisiae both require Bir1[END_REF][START_REF] Fischböck-Halwachs | The COMA complex interacts with Cse4 and positions Sli15/Ipl1 at the budding yeast inner kinetochore[END_REF]. While centromeric localisation depends on histone H2A phosphorylation by Bub1 and subsequent recruitment of Shugoshin and the Bir1 subunit of CPC (survivin in metazoans) [START_REF] Kawashima | Shugoshin enables tension-generating attachment of kinetochores by loading Aurora to centromeres[END_REF][START_REF] Fischböck-Halwachs | The COMA complex interacts with Cse4 and positions Sli15/Ipl1 at the budding yeast inner kinetochore[END_REF][START_REF] Kawashima | Phosphorylation of H2A by Bub1 prevents chromosomal instability through localizing shugoshin[END_REF], kinetochore localisation requires binding of Aurora B and the Sli15 subunit of CPC (INCENP in metazoans) to the COMA complex [START_REF] García-Rodríguez | Aurora B-INCENP localization at centromeres/inner kinetochores is required for chromosome bi-orientation in budding yeast[END_REF][START_REF] Fischböck-Halwachs | The COMA complex interacts with Cse4 and positions Sli15/Ipl1 at the budding yeast inner kinetochore[END_REF]. A truncated Sli15 lacking its N-terminal region (Sli15-DNterm) or alanine mutations in its CDK-dependent phosphorylation sites (Sli15-6A) might favour Ipl1 binding to the COMA complex and/or microtubules [START_REF] García-Rodríguez | Aurora B-INCENP localization at centromeres/inner kinetochores is required for chromosome bi-orientation in budding yeast[END_REF][START_REF] Fischböck-Halwachs | The COMA complex interacts with Cse4 and positions Sli15/Ipl1 at the budding yeast inner kinetochore[END_REF][START_REF] Pereira | Separase regulates INCENP-Aurora B anaphase spindle function through Cdc14[END_REF] and, concomitantly, rescue the lethality/growth defects of bir1D, bub1D and sgo1D cells [START_REF] Campbell | Tension sensing by Aurora B kinase is independent of survivin-based centromere localization[END_REF]. If the chromosome segregation defects of mps1-3 cells arose exclusively from poor activity of centromeric Sgo1/CPC, then they Finally, since Dam1 is a critical phosphorylation target of Ipl1 in the error correction process [START_REF] Kalantzaki | Kinetochore-microtubule error correction is driven by differentially regulated interaction modes[END_REF][START_REF] Cheeseman | Phospho-regulation of kinetochore-101[END_REF], we monitored its phosphorylation state in wild-type, mps1-3 and ipl1-321 cells during a synchronous release from G1 at 34°C. While the electrophoretic mobility of Dam1-3PK was dramatically altered by the ipl1-321 mutation, it was mainly unaffected in mps1-3 cells (Appendix Fig S8E ), consistent with the notion that phosphorylation of Dam1 by Mps1 is dispensable for chromosome biorientation [START_REF] Kalantzaki | Kinetochore-microtubule error correction is driven by differentially regulated interaction modes[END_REF]13] and suggesting that Ipl1 kinase activity is largely proficient in mps1-3 cells.

A C wild type mps1-3 SPC105-GBD mps1-3 SPC105-GBD SPC105-GBD BUB1-GFP mps1-3 SPC105-GBD BUB1-GFP SPC105-GBD BUB3-GFP mps1-3 SPC105-GBD BUB3-GFP 25°C 30°C 32°C 34°C
We therefore conclude that lack of Bub1 at kinetochores is mainly responsible for the chromosome biorientation flaws of mps1-3 mutant cells, at least partly independently of the role of Bub1 in Sgo1 kinetochore recruitment and CPC activation.

Discussion

The mps1-3 mutant as a novel genetic tool to study the role of Mps1 in chromosome segregation

The precise function of budding yeast Mps1 in chromosome biorientation has been elusive so far. We have shed light on this process through the characterisation of the conditional mps1-3 mutant that at 34°C is defective in this process but proficient at SPB duplication and bipolar spindle assembly. Our analysis of the segregation pattern of chromosome V in mps1-3 cells suggests that each pair of sister chromatids has equal chance to segregate in a bipolar or monopolar fashion. This random segregation applied to the 16 yeast chromosomes makes the likelihood for each single mps1-3 cell to segregate correctly all chromosomes extremely low (1.5 × 10 À5 ), which is in agreement with the massive chromosome missegregation we observe by FACS analysis. Being mps1-3 cells also SAC-deficient makes monopolar attachments undetected by the checkpoint, thus generating highly aneuploid cells.

We could establish through live cell imaging that mps1-3 cells are not defective in kinetochore-microtubule attachment, but rather in the error correction mechanism that converts monotelic or syntelic linkages into amphitelic attachments, in agreement with previous conclusions [START_REF] Maure | Mps1 kinase promotes sisterkinetochore bi-orientation by a tension-dependent mechanism[END_REF][START_REF] Storchová | Bub1, Sgo1, and Mps1 mediate a distinct pathway for chromosome biorientation in budding yeast[END_REF]. This function has been classically attributed to Aurora B and the CPC (reviewed in ref. [82]), but in more recent years Mps1 has also been implicated in this process. Mps1 was shown to impact on Aurora B activity through phosphorylation of the CPC subunit borealin [START_REF] Jelluma | Mps1 phosphorylates borealin to control Aurora B activity and chromosome alignment[END_REF], as well as to act downstream of Aurora B [7], suggesting a complex interplay between the two kinases. In contrast, in budding yeast Mps1 and Aurora B (Ipl1) were proposed to operate independently [START_REF] Maure | Mps1 kinase promotes sisterkinetochore bi-orientation by a tension-dependent mechanism[END_REF][START_REF] Storchová | Bub1, Sgo1, and Mps1 mediate a distinct pathway for chromosome biorientation in budding yeast[END_REF].

While ipl1 mutants missegregate chromosomes preferentially towards the old SPB, presumably because the new SPB must undergo a maturation time before becoming fully able to nucleate microtubules [45], we find different results in mps1-3 cells depending on the experimental conditions. In particular, during the first cell cycle at restrictive temperature mps1-3 cells missegregate chromosomes preferentially towards the bud (i.e. towards the old SPB) similar to ipl1 mutant cells. In contrast, during the following cell cycles this bias seems to be progressively lost. These results can be rationalised by surmising that Mps1 and Ipl1 are both involved in error correction of kinetochore attachments, but Mps1 could be additionally required for SPB maturation. In other words, when Mps1 activity is disabled, young SPBs fail to age properly and SPB asymmetry is obliterated, thus explaining the loss of missegregation bias after the second cell cycle at restrictive temperature. It is worth noting that apparently contradictory data exist in the literature as to whether in mps1 mutants chromosomes missegregate preferentially towards the bud [START_REF] Jones | Chemical genetics reveals a role for Mps1 kinase in kinetochore attachment during mitosis[END_REF][START_REF] Maure | Mps1 kinase promotes sisterkinetochore bi-orientation by a tension-dependent mechanism[END_REF]83]. The above hypothesis offers a plausible explanation to reconcile these data.

Although the mps1-3 Ser635Phe mutation lies in the kinase domain, the in vitro kinase activity of the corresponding protein is surprisingly enhanced, rather than impaired, at high temperature. The crystal structure of the Mps1 kinase domain has been solved [84] and reveals that Ser635 is semi-buried, thus precluding any straightforward prediction of the possible outcome of its mutation on Mps1 folding or activity. Importantly, the Mps1-3 protein does not accumulate at kinetochores, suggesting that lack of phosphorylation of specific kinetochore substrates underlies the chromosome biorientation and SAC defects of mps1-3 cells. For what concerns SAC signalling, although MPS1 overexpression is able to hyperactivate SAC in the absence of functional kinetochores [85], our data imply that endogenous levels of Mps1 must accumulate at kinetochores for a productive SAC response, in agreement with previous reports [9,[86][87][88]. Mps1 kinetochore recruitment depends on its N-terminus that interacts with the kinetochore protein Ndc80/ Hec1 [86,87]. Additionally, Mps1 kinase activity promotes its own turnover at kinetochores [7,40,49,50]. It is therefore tempting to speculate that the enhanced kinase activity of Mps1-3 might accelerate its own turnover and account for its low levels at kinetochores. The characterisation of the intragenic suppressors identified in our genetic screen might help shedding light on this hypothesis.

Mps1 promotes SAC signalling and chromosome biorientation through a common molecular mechanism

Our unbiased screen for extragenic suppressors of mps1-3 has been particularly revealing and points at the antagonism between Mps1 and PP1 at kinetochores as critical for the correction of faulty kinetochore-microtubule attachments and correct chromosome segregation. Our data strongly argue that the same mechanism implicated in SAC signalling, i.e. Spc105 phosphorylation and subsequent Bub1 recruitment, is a crucial function of Mps1 also in chromosome biorientation and independently of SAC activation. Several lines of evidence are consistent with this conclusion. First, the spc105 and GLC7 suppressors that we have identified can re-establish both proper chromosome segregation and SAC signalling in mps1-3 cells without bringing back Mps1 to kinetochores. Second, Spc105 phosphorylation and Bub1 kinetochore localisation, which are severely impaired in mps1-3 mutant cells, are restored in the suppressors. Third, constitutive binding of Bub1 to Spc105 is sufficient to rescue considerably chromosome missegregation and partly SAC signalling in mps1-3 cells at restrictive temperature. The incomplete rescue of SAC activation is not surprising, given that Mps1 plays additional roles in the SAC, such as promoting Bub1 and Mad1 phosphorylation [START_REF] Ji | A sequential multi-target Mps1 phosphorylation cascade promotes spindle checkpoint signaling[END_REF][START_REF] London | Mad1 kinetochore recruitment by Mps1mediated phosphorylation of Bub1 signals the spindle checkpoint[END_REF]. Along this line, it is worth noting that the spc105-18 and GLC7-24 suppressors, which restore a normal SAC response in mps1-3 cells, also restore wild-type Mad1 (but not Bub1) hyperphosphorylation upon nocodazole treatment.

The idea that a common molecular mechanism based on Knl1/ Spc105 phosphorylation would underlie the role of Mps1 in both SAC and chromosome biorientation was previously proposed in fission yeast. Indeed, phospho-mimicking mutations in the MELT repeats of Spc7 (the fission yeast homologue of Knl1/Spc105) were shown to partially suppress the chromosome biorientation defects of mps1D cells [START_REF] Yamagishi | MPS1/Mph1 phosphorylates the kinetochore protein KNL1/Spc7 to recruit SAC components[END_REF]. However, in fission yeast Mps1 is not essential for viability, and mps1D cells experience only modest chromosome missegregation [START_REF] Yamagishi | MPS1/Mph1 phosphorylates the kinetochore protein KNL1/Spc7 to recruit SAC components[END_REF]. Thus, our data gathered in an organism where Mps1 is critical for chromosome biorientation and with the aid of an unbiased genetic screen largely extend and strengthen this idea.

Despite our data strongly argue that Spc105 is a critical Mps1 substrate for the error correction pathway, we cannot rule out that other Mps1 substrates contribute to this process. Further experiments will help refining the precise mechanism underlying the control of chromosome segregation by Mps1.

The N-terminal region of Knl1 and Bub1 was previously shown to stimulate Aurora B kinase activity at kinetochores and increase the turnover of erroneous kinetochore-microtubule attachments [START_REF] Kemmler | Mimicking Ndc80 phosphorylation triggers spindle assembly checkpoint signalling[END_REF][90][91]. Furthermore, Bub1 has been involved in chromosome biorientation through phosphorylation of histone H2A and Sgo1 accumulation at pericentromeres [66,[START_REF] Kawashima | Phosphorylation of H2A by Bub1 prevents chromosomal instability through localizing shugoshin[END_REF]92,[START_REF] Vanneste | The role of Hklp2 in the stabilization and maintenance of spindle bipolarity[END_REF]. Sgo1, in turn, enhances CPC activity in the error correction mechanism [START_REF] Kawashima | Shugoshin enables tension-generating attachment of kinetochores by loading Aurora to centromeres[END_REF][START_REF] Verzijlbergen | Shugoshin biases chromosomes for biorientation through condensin recruitment to the pericentromere[END_REF]94]. Consistent with the idea that Mps1 is required for Bub1 recruitment to kinetochores, Sgo1 co-localisation with kinetochores is compromised in mps1 mutants (ref. [START_REF] Storchová | Bub1, Sgo1, and Mps1 mediate a distinct pathway for chromosome biorientation in budding yeast[END_REF] and our data). Our data suggest, however, that impaired Sgo1 kinetochore localisation does not fully account for the chromosome biorientation defects of mps1-3 cells. Indeed, Sgo1 centromeric recruitment is weakened, but not abolished, in mps1-3 cells and the mps1-3 mutation and SGO1 deletion are synthetically lethal. Furthermore, mutations in the CPC centromere-targeting subunit Sli15 that rescue the sickness of sgo1D and bub1D cells [START_REF] Campbell | Tension sensing by Aurora B kinase is independent of survivin-based centromere localization[END_REF] have no ameliorating effects on the lethality and chromosome segregation defects of mps1-3 cells at high temperature. Finally, phosphorylation of the Aurora B kinetochore target Dam1 is unaffected in mps1-3 cells, suggesting that the error correction defects of this mutant are not caused by reduced Aurora B kinase activity. Thus, although Mps1 could promote chromosome biorientation partly through the Bub1-Sgo1-CPC axis, it must have at least one additional function in this process. Consistent with this hypothesis, Bub1 and Sgo1 are not essential proteins, unlike Mps1, and their deletion leads to milder chromosome segregation defects than mps1-3 cells at restrictive temperature. One intriguing possibility that is supported by our genetic results is that Mps1 could directly harness PP1 activity at kinetochores. In this scenario, Mps1 would master two opposing activities, acting indirectly on the CPC and perhaps more directly on PP1, thereby allowing the highest accuracy of bipolar attachments. This hypothesis would also explain why the spc105 and GLC7 suppressors isolated in our screen rescue the lethality and chromosome segregation defects of mps1-3 cells considerably better than artificial recruitment of Bub1 to Spc105. Whether and how Mps1 controls PP1 activity at kinetochores will be an important subject for future investigations.

In sum, our data provide experimental support to the hypothesis that a single sensory device may be used for both error correction of faulty kinetochore-microtubule attachments and SAC signalling [START_REF] Kemmler | Mimicking Ndc80 phosphorylation triggers spindle assembly checkpoint signalling[END_REF]95]. Given the evolutionary conservation of the players involved, it is very likely that Mps1 promotes SAC activation and chromosome biorientation through a common mechanism also in other organisms.

Materials and Methods

Yeast strains, plasmids and growth conditions

All yeast strains (Appendix Table S1) are congenic to or at least four times backcrossed to W303 (ade2-1, trp1-1, leu2-3,112, his3-11 and 15 ura3).

One-step tagging techniques were used to generate 3HA-, 3PK-, GFP-and mCherry-tagged proteins at the C-terminus. The CEN5:: tetO2X224 (12.6 kB) tetR-GFP strain was a generous gift from T. Tanaka [43]; BUB1-GFP from P. de Wulf; MTW1-Tomato from A. Marston [START_REF] Nerusheva | Tension-dependent removal of pericentromeric shugoshin is an indicator of sister chromosome biorientation[END_REF]; GFP-TUB1 from A. Straight [START_REF] Gama | Molecular mechanism of dynein recruitment to kinetochores by the Rod-Zw10-Zwilch complex and Spindly[END_REF]; mCherry-TUB1 from A. Amon [97]; PDS1-myc18 from K. Nasmyth [START_REF] Yamagishi | MPS1/Mph1 phosphorylates the kinetochore protein KNL1/Spc7 to recruit SAC components[END_REF]; spc105-RASA and GLC7-spc105-RASA mutants from H. Funabiki and F. Cross [START_REF] Rosenberg | KNL1/Spc105 recruits PP1 to silence the spindle assembly checkpoint[END_REF]; SGO1-GFP and CEN5::tetO2X112 (1.4 kB) tetR-GFP from E. Schwob; and ura3::URA3::tetOX224 (35 kB) tetR-GFP from K. Nasmyth. The sli15-DNterm (sli15D2-228) and sli15-6A plasmids (pCC253 and pCC297) were a gift of C. Campbell [START_REF] Campbell | Tension sensing by Aurora B kinase is independent of survivin-based centromere localization[END_REF] and were integrated at the SLI15 locus by cutting with HindII and SalI, respectively.

The mps1-3 mutant was obtained by hydroxylamine-induced mutagenesis of an MPS1-bearing plasmid and selection of temperature-sensitive mutants through plasmid shuffling [99]. The mps1-3 allele (entire coding sequence) together with 480 bp of 5 0 UTR and 900 bp of 3 0 UTR was cloned as an EcoRI/SalI fragment in the integrative plasmid pFL34 to generate pSP1041 and integrated at the MPS1 locus by the Pop-in/Pop-out method [100].

The SPC105-GBD-bearing plasmid (pSP1530) was generated by cloning in KpnI/HindIII of Yiplac204 a KpnI/PfoI fragment containing SPC105 promoter and part of the coding region from plasmid SB1884 [15] together with a PfoI/HindIII SPC105 synthetic gene (aa 61-386 of Spc105) carrying the GBD domain after aa 130 (pSP1516). SPC105-GBD was integrated at the SPC105 locus by digesting pSP1530 with BglII. This generates a partial duplication of SPC105, with full-length SPC105-GBD gene followed by a truncated spc105 allele (aa 1-386).

The GALs-MPS1 plasmid (pSP149) was constructed by amplifying a DNA fragment comprising 30 bp of MPS1 5 0 UTR, its CDS and 105 bp of 3 0 UTR by PCR from W303 genomic DNA with primers SP127 and SP129, followed by its cloning into EcoRI/XhoI of a pRS416 harbouring the attenuated GALs promoter [101]. Growth of wild-type cells carrying the GALs-MPS1 construct is unaffected (data not shown), in contrast to high-level overexpression of MPS1 that causes a SAC-dependent metaphase arrest [62].

The GAL1-GST-MPS1 plasmid (pSP228) was constructed by amplifying MPS1 by PCR with primers SP238 and SP239 and cloning it into pEG(KT) [102] after cutting SmaI/HindIII. The GAL1-GST-mps1-3 plasmid (pSP1202) was generated by replacing a BspEI/XbaI fragment of pSP228 internal to MPS1 coding region by the same fragment containing the mps1-3 mutation from pSP1041.

The plasmids for expression of His-tagged Mps1 and Mps1-3 in bacteria (pSP1575, pSP1576) were generated by amplifying the coding sequence of MPS1/mps1-3 by PCR with primers MP1124-MP1125, followed by cloning into EcoRI/HindIII of pPROEX-HTa.

The plasmid for expression of His-tagged Spc105 N-terminus (aa 1-320) in Escherichia coli (pSP1571) was generated by PCR-amplifying part of the coding sequence of SPC105 with primers MP1126-1127, followed by cloning into BamHI/PstI of pPROEX-HTa.

Yeast cultures were grown at 25-30°C, in either SD medium (6.7 g/l yeast nitrogen base without amino acids) supplemented with the appropriate nutrients or YEP (1% yeast extract, 2% bactopeptone, 50 mg/l adenine) medium. Raffinose was supplemented to 2% (SD-raffinose or YEPR), glucose to 2% (SD-glucose or YEPD), and galactose to 1% (SD-raffinose/galactose or YEPRG). Cells were synchronised in G1 by alpha factor (4 lg/ml) in YEP medium containing the appropriate sugar at 25°C. G1 arrest was monitored under a transmitted light microscope, and cells were released in fresh medium (typically after 120-135 min of alpha factor treatment) at 34°C, unless otherwise specified, after being collected by centrifugation at 2,000 g followed by one wash with YEP containing the appropriate sugar. Nocodazole was used at 15 lg/ml. To detect spindle formation and elongation, alpha-tubulin immunostaining was performed on formaldehyde-fixed cells using the YOL34 monoclonal antibody (1:100; MCA78S AbDSerotec, Raleigh, NC), followed by indirect immunofluorescence using CY2-conjugated anti-rat antibody (1:100;31645 Pierce Chemical Co.).

For time-lapse video microscopy, cells were mounted on 1% agarose pads in SD medium on Fluorodishes (World Precision Instruments) and filmed at controlled temperature with a 100× 1.45 NA oil immersion objective mounted on a Spinning disk CSU-X1 Andor Nikon Eclipse Ti microscope coupled to an iXon Ultra camera controlled by the Andor iQ3 software. Z-stacks of 10 planes were acquired every 2-4 min with a step size of 0. Budding index was scored under a transmitted light microscope on cells fixed with 70% ethanol and treated for FACS analysis. Percentage of nuclear division was determined on the same samples after DNA staining with propidium iodide under a fluorescence microscope using a CY3 filter.

FACS analysis of DNA contents

For DNA quantification by fluorescence-activated cell sorting (FACS), 1-2 × 10 7 cells were collected, spun at 10,000 g and fixed with 1 ml of 70% ethanol for at least 30 min at RT. After one wash with 50 mM Tris-Cl pH 7.5, cells were resuspended in 0.5 ml of the same buffer containing 0.05 ml of a preboiled 10 mg/ml RNAse solution and incubated overnight at 37°C. The next day cells were spun at 10,000 g and resuspended in 0.5 ml of 5 mg/ml pepsin freshly diluted in 55 mM HCl. After 30-min incubation at 37°C, cells were washed with FACS buffer (200 mM Tris-Cl pH 7.5, 200 mM NaCl, 78 mM MgCl 2 ) and resuspended in the same buffer containing 50 lg/ml propidium iodide. After a short sonication, samples were diluted (1:20-1:10) in 1 ml of 50 mM Tris-Cl pH 7.5 and analysed with a FACSCalibur device (BD Biosciences). 10,000 events were scored for each sample and plotted after gating out the debris.

Protein extracts, Western blotting and kinase assays

For TCA protein extracts, 10-15 ml of cell culture in logarithmic phase (OD 600 = 0.5-1) were collected by centrifugation at 2,000 g, washed with 1 ml of 20% TCA and resuspended in 100 ll of 20% TCA before breakage of cells with glass beads (diameter 0.5-0.75 mm) on a Vibrax VXR (IKA). After addition of 400 ll of 5% TCA, lysates were centrifuged for 10 min at 845 g. Protein precipitates were resuspended in 100 ll of 3× SDS sample buffer (240 mM Tris-Cl pH 6.8, 6% SDS, 30% glycerol, 2.28 M b-mercaptoethanol, 0.06% bromophenol blue), denatured at 99°C for 3 min and loaded on SDS-PAGE after elimination of cellular debris by centrifugation (5 min at 20,000 g).

Proteins were wet-transferred on Protran membranes (Schleicher and Schuell) overnight at 0.2 A and probed with monoclonal anti-HA 12CA5 (1:5,000); anti-PK (alias anti-V5; MCA1360 AbDSerotec, 1:3,000); polyclonal anti-Cdc5 (sc-6733 Santa Cruz, 1:3,000); anti-Clb2 (a generous gift from W. Zachariae, 1:2,000); anti-Pgk1 (Invitrogen Molecular Probes, 1:40,000); monoclonal anti-myc 9E10 (1:5,000); anti-phospho-AMPK (Thr172) (Cell Signalling, 1:1,000); and anti-GST (1:2,000, 27-4577-01 GE Healthcare). Antibodies were diluted in 5% low-fat milk (Regilait) dissolved in TBST. Secondary antibodies were purchased from GE Healthcare, and proteins were detected by a home-made enhanced chemiluminescence system.

For kinase assays with recombinant Mps1 proteins, Mps1/Mps1-3 and Spc105 have been purified as follows: BL21 DE3 cells transformed with pSP1571, pSP1575 or pSP1576 were grown in LB containing 50 lg/ml of ampicillin, 34 lg/ml of chloramphenicol and 1% glucose and induced with 0.2 mM IPTG at 16°C overnight. Cells were lysed in 50 mM Tris-Cl pH 8, 300 mM NaCl, 0.1% Tween 20 containing 1 mg/ml of lysozyme and a cocktail of protease inhibitors (Complete EDTA-free Roche). Cells were then sonicated 3× with 1 0 30″ cycles of 8″ pulses/8″ ice and extracts cleared at 30,000 g for 30 0 at 4°C. Lysates were incubated 2 h with 1 ml of Ni-NTA agarose beads (Qiagen), pre-washed three times with 50 mM Tris-Cl pH 8, 300 mM NaCl, 0.1% Tween 20 and 10 mM imidazole at 4°C. After incubation with the extracts, the slurry was washed three times with 50 mM Tris-Cl pH 8, 300 mM NaCl, 0.1% Tween 20 and 20 mM imidazole and loaded on Polyprep columns (Bio-Rad). Fractions of 0.5 ml were eluted with 50 mM Tris-Cl pH 8, 300 mM NaCl and 250 mM imidazole and quantified by Nanodrop. The most concentrated fractions were washed and concentrated through the use of Amicon Ultra filter units (10 kDa cut-off for Spc105 and 50 kDa cut-off for Mps1) to finally have them in 50 mM Tris-Cl pH 8, 50 mM NaCl. Glycerol was added to the final concentration of 20%. Protein concentration was quantified by loading them on SDS-PAGE along with a BSA standard curve followed by staining with Coomassie blue. Kinase assays were performed at 34°C for different times in 25 ll of a reaction mix containing 50 mM Tris-Cl pH 7.5, 10 mM MgCl 2 , 0.5 mM DTT, 10 lM ATP, 1 ll ATP-c P33 3,000 Ci/mmol and 80 lg/ml MBP. 75 ng of Spc105 N-term fragment and 1 lg Mps1/Mps1-3 kinases were used for each assay. Reactions were stopped using 7 ll of 3× SDS sample buffer (described in TCA extracts). Samples were then boiled 3 min at 95°C and loaded on SDS-PAGE. The gel was then dried and exposed on autoradiography films.

Native yeast protein extracts for kinase assays were performed by collecting cells in logarithmic phase (OD 600 = 1) grown in -Ura RG at 30°C, centrifuging at 2,000 g and washing with 1 ml of cold 10 mM Tris-Cl pH 7.5. Cells were then broken in 50 mM Tris-Cl pH 7.5, 50 mM NaCl, 0.2% Triton X-100 containing a cocktail of protease inhibitors (Complete EDTA-free Roche) and phosphatase inhibitors (PhosSTOP Roche). Lysates were cleared at 20,000 g for 10 min at 4°C. The equivalent of 6 OD 600 /assay was incubated on a nutator for 2 h at 4°C with 50 ll of Glutathione Sepharose 4 Fast Flow (GE Healthcare) beads pre-washed with the above buffer. After incubation with the extracts, beads were spun down at 4°C at 845 g, washed twice with the same buffer without protease and phosphatase inhibitors, twice with the same buffer containing 0.1% of Triton X-100, twice with the same buffer without Triton X-100 and once with 50 mM Tris-Cl pH 7.5, 10 mM MgCl 2 and 0.5 mM DTT. Kinase assays were performed as above using 2 lg of MBP as exogenous substrate.

Chromosome loss assay

This assay is based on the loss of heterozygosity of the MAT locus, carried by chromosome III [42]. Briefly, wild-type diploid cells are heterozygous MATa/MATalpha and cannot mate unless they lose one copy of the MAT locus through mutations, chromosome rearrangements or chromosome loss. Cells were grown overnight at 25°C, and their concentration was measured at the time of inoculation and the day after to calculate the number of cell divisions. 2 × 10 7 diploid Met + cells were incubated 4 h at RT with an excess (2 × 10 8 ) of MATalpha met4 tester cells in 400 ll of YEPD. 10 ll of the suspension was then diluted to plate 500 cells on ÀMet plates (where only the Met + diploids can form colonies) to calculate the percentage of viable (i.e. colony-forming); the remaining cells were plated on minimal SD plates (MIN), where only triploids, generated from the mating of diploids with the haploid tester cells, can grow. Plates have been incubated at 25°C. Frequency of chromosome loss has been calculated as follows: {[(no. of colonies on MIN/2 × 10 7 )/ fraction of viable cells]/no. of cellular divisions}. We note that with this assay we likely underestimate the real frequencies of chromosome loss because our measurements do not take into account the frequency of mating for each strain, which probably is much lower than 100%.

Suppressor screen

2.6 × 10 7 mps1-3 cells were plated on YEPD and incubated at 34°C for 3-5 days until colonies' appearance. The isolated spontaneous suppressors were then characterised by classical genetic tests to distinguish intragenic from extragenic mutations, recessive from dominant mutations, and to group the suppressors in complementation/allelic groups. Genomic DNA was extracted from one representative suppressor for each complementation/allelic group and sent for whole genome sequencing to identify the mutated genes. The identity of the suppressing mutations was confirmed by classical genetics. Targeted gene re-sequencing approaches were used to identify the suppressing mutations in the other extragenic suppressors.

Viability test

Cells were grown at 25°C to 2 × 10 6 cells/ml and then serially diluted to plate 200 cells/plate in duplicate (wild-type, mps1-3 SPC105-GBD BUB1-GFP and mps1-3 SPC105-GBD BUB3-GFP) or 200, 500 and 1,000 cells/plate in duplicate for mps1-3 cells (t = 0). Cells were then shifted to 34°C, withdrawn every hour, serially diluted to plate 200 cells/plate in duplicate (wild-type, mps1-3 SPC105-GBD BUB1-GFP and mps1-3 SPC105-GBD BUB3-GFP) or 200, 500 and 1,000 cells/plate in duplicate for mps1-3 cells. Plates were incubated at 25°C until colony appearance. The number of colonies at each time point (average values of the duplicates) was divided by the number of colonies at t = 0 to obtain percentages of viability.

ChIP-seq analysis 100 ml of cells (concentration 10 7 cells/ml) was fixed in 1% formaldehyde for 30 0 at RT with shaking and then transferred ON at 4°C. Cells were then spun down 3 min at 2,000 g at 4°C and washed three times with TBS. Cells were resuspended in 0.4 ml of 50 mM Hepes-KOH pH 7.5, 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% Na-deoxycholate containing a cocktail of protease inhibitors (Complete EDTA-free Roche) and 1 mM PMSF, followed by cell breakage through a Multi-beads shaker (Yasui-Kikai) at 0°C. Lysates were eluted in 15 ml Falcon tubes (centrifuged twice at 3,000 rpm for 1 min at 4°C), transferred in 1.5 Eppendorf tube, centrifuged at 2,500 g 1 min at 4°C and sonicated through a Sonifier Branson 2508 (5-7 cycles of 15 s, tune 1.5 with centrifugation of 5,000 rpm for 1 min between each cycle). Sonicated lysates were then centrifuged for 5 min at 21,000 g at 4°C. Supernatants were recovered. 10 ll of lysates was withdrawn at this stage (=Inputs). For immunoprecipitations (IPs), 60 ll of Protein G-dynabeads pre-washed twice with PBS containing 5 mg/ml BSA and pre-adsorbed with 3 lg Babco 16B12 anti-HA antibody was added to each lysate and incubated overnight at 4°C on a nutator. IPs were then washed twice with icecold 50 mM Hepes-KOH pH 7.5, 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, and 0.1% Na-deoxycholate; twice with ice-cold 50 mM Hepes-KOH pH 7.5, 500 mM NaCl, 1 mM EDTA, 1% Triton X-100, and 0.1% Na-deoxycholate; twice with ice-cold 10 mM Tris-Cl pH 8, 250 mM LiCl, 0.5% NP-40, 0.5% Na-deoxycholate and 1 mM EDTA; and once with ice-cold TE (50 mM Tris-Cl pH 8, 1 mM EDTA pH 8). IPs were eluted at 65°C for 15 min with 50 ll of 50 mM Tris-Cl pH 8, 10 mM EDTA, 1% SDS, resuspended in 50 ll of TE/1% SDS and incubated overnight at 65°C to reverse crosslinking. IPs were then incubated at 37°C for 1 h with 100 ll of TE containing 10 lg of RNAseA and 2 h at 37°C with 2 ll of Protei-naseK 50 mg/ml. DNA was then purified with the Qiagen PCR purification kit. Libraries for Illumina sequencing have been then prepared following the NEBNext Ultra II DNA Library Prep Kit for Illumina. Sequencing results have been analysed using the DROMPA software [103].

CEN1 CEN2 CEN2 CEN4 CEN4 CEN5 CEN5 CEN6 CEN6 CEN7 CEN7 CEN8 CEN8 CEN9 CEN9 CEN10 CEN10 CEN11 CEN11 CEN12 CEN12 CEN13 CEN13 CEN14 CEN14 CEN15 CEN15 CEN16 CEN16
wild type mps1-3 wild type mps1-3 wild type mps1-3 The SCF Grr1 complex could be a new player in the regulation of chromosome segregation at kinetochores

Through the genetic screen for spontaneous suppressors of the temperature-sensitivity of the mps1-3 mutant I found four mutations in the GRR1 gene (Fig. 29 and Table 2), encoding for an F-box protein of the SCF (SKP1-cullin-F-Box protein) complex [START_REF] Kishi | Grr1 functions in the ubiquitin pathway in Saccharomyces cerevisiae through association with Skp1[END_REF]F. N. Li and Johnston, 1997).

Three of them, suppressor 17, 19 and 26, also carry a mutation in RSC30, encoding for a subunit of the RSC chromatin-remodeling complex [START_REF] Angus-Hill | A Rsc3/Rsc30 Zinc Cluster Dimer Reveals Novel Roles for the Chromatin Remodeler RSC in Gene Expression and Cell Cycle Control[END_REF][START_REF] Cairns | RSC, an Essential, Abundant Chromatin-Remodeling Complex[END_REF]Fig. 29). All four suppressors were able to form visible colonies at restrictive temperatures of (30°C-34°C), albeit to different degrees (Fig. 29). Indeed, suppressors 19 and 20 could partially rescue the temperaturesensitivity up to 34°C, whereas suppressor 17 only suppressed up to 32°C. Moreover, suppressor 26

showed a poor suppression already at 32°C. Through further genetic analyses (see Materials and Methods), I could attribute the suppression to the grr1 mutations, while the rsc30 mutation could simply increase the fitness of grr1 mutant cells. Since these grr1 mutations were all recessive (and as such probably linked to loss of function), we assumed that decreased activity of the SCF Grr1 complex underlies the suppression. SCF is a multi-subunit RING finger ubiquitin ligase complex composed of four proteins: Skp1, Cul1, Hrt1 and an F-box protein that confer substrate specificity. At the center of the complex there is the RING finger domain-containing the protein Hrt1. Cul1 interacts with Skp1 to recruit one F-box protein that directly interacts with the substrate (reviewed in [START_REF] Hegde | 4.26 -Proteolysis and Synaptic Plasticity[END_REF].

In budding yeast there are seventeen different F-box proteins that in principle can form a corresponding number of SCF complexes [START_REF] Kipreos | The F-box protein family[END_REF]. The SCF complex is implicated in the ubiquitylation and subsequent degradation of many substrates that participate in a variety of cellular functions, including regulation of CDK activity, activation of transcription, signal transduction, kinetochore assembly and DNA replication (reviewed in [START_REF] Deshaies | SCF and Cullin/RING H2-Based Ubiquitin Ligases[END_REF]. When discovered in yeast, grr1 mutants were showing phenotypes related to deregulated glucose metabolism [START_REF] Bailey | Isolation and characterization of a pleiotropic glucose repression resistant mutant of Saccharomyces cerevisiae[END_REF][START_REF] Flick | GRR1 of Saccharomyces cerevisiae is required for glucose repression and encodes a protein with leucine-rich repeats[END_REF]. Notably, Grr1 targets have been identified among regulators of glycolysis [START_REF] Benanti | A proteomic screen reveals SCF Grr1 targets that regulate the glycolytic-gluconeogenic switch[END_REF] and regulators of hexose transporters [START_REF] Flick | Grr1-dependent Inactivation of Mth1 Mediates Glucose-induced Dissociation of Rgt1 from HXT Gene Promoters[END_REF][START_REF] Ozcan | Three different regulatory mechanisms enable yeast hexose transporter (HXT) genes to be induced by different levels of glucose[END_REF][START_REF] Vallier | Altered regulatory responses to glucose are associated with a glucose transport defect in grr1 mutants of Saccharomyces cerevisiae[END_REF]. Additional targets are amino acids permeases [START_REF] Bernard | Ubiquitin and the SCFGrr1 ubiquitin ligase complex are involved in the signalling pathway activated by external amino acids in Saccharomyces cerevisiae[END_REF]. Furthermore, Grr1 also regulates degradation of the G1 cyclins Cln1 and Cln2 [START_REF] Skowyra | Reconstitution of G1 cyclin ubiquitination with complexes containing SCFGrr1 and Rbx1[END_REF][START_REF] Willems | Cdc53 targets phosphorylated G1 cyclins for degradation by the ubiquitin proteolytic pathway[END_REF], whose accumulation causes a typical elongated morphology of cells carrying grr1 mutations [START_REF] Flick | GRR1 of Saccharomyces cerevisiae is required for glucose repression and encodes a protein with leucine-rich repeats[END_REF].

The meaning of the grr1 mutations in the suppression of the temperature-sensitivity of mps1-3 cells is not immediately clear, since to date there are no published data linking the SCF Grr1 complex to kinetochore-related processes. However, the SCF subunit Skp1 physically associates with the protein Sgt1, which is important for the assembly of the yeast kinetochore complex CBF3 through activation of its subunit Ctf13 [START_REF] Kitagawa | SGT1 Encodes an Essential Component of the Yeast Kinetochore Assembly Pathway and a Novel Subunit of the SCF Ubiquitin Ligase Complex[END_REF]. Interestingly, Bub1 was found to interact with Skp1 and this is required for the mitotic delay induced by kinetochore tension defects, but not for the arrests induced by spindle depolymerization, kinetochore assembly defects or Mps1 overexpression [START_REF] Kitagawa | Requirement of Skp1-Bub1 Interaction for Kinetochore-Mediated Activation of the Spindle Checkpoint[END_REF]. Grr1 itself has never been found at kinetochores but there is circumstantial evidence of a possible interaction with Mps1, since phosphorylated Grr1 peptides have been identified in Mps1 purifications from yeast cells in a high throughput study of yeast kinases and phosphatases interactions based on mass spectrometry [START_REF] Breitkreutz | A Global Protein Kinase and Phosphatase Interaction Network in Yeast[END_REF]. This suggests that Grr1 might be a phosphorylation target of Mps1.

Table 2 : Mutations in grr1 and rsc30 found in the suppressor screen.

I then investigated if the grr1 suppressors were able to activate the SAC and to correctly segregate chromosomes. For this purpose, I synchronized cells in G1, released them at restrictive temperature (34°C) in presence and absence of nocodazole and analyzed DNA contents by FACS. In the presence of nocodazole, the suppressor mps1-3 grr1-17 mostly arrested in mitosis with a ~2C DNA content, unlike mps1-3 cells, although a moderate ability to escape the cell cycle arrest was apparent compared to wild type cells (Fig. 30A). In the absence of nocodazole, the same suppressor showed an approximately bimodal distribution of DNA contents, similar to wild type cells and unlike mps1-3 cells, indicating that proper chromosome segregation is quite efficiently restored (Fig. 30B).

Figure 30 : grr1 mutations restore SAC signaling and proper chromosome segregation.

Wild-type, mps1-3 and mps1-grr1-17 mutant cells were synchronised in G1 with a-factor at permissive temperature (25°C) and then released at restrictive temperature (34°C, t = 0) in presence (A) or absence (B) of nocodazole. Cells were collected at the indicated time points for FACS analysis of DNA contents.

As mentioned above, all grr1 suppressing mutations were recessive, suggesting that they might be loss of function. To check if they destabilize Grr1 protein, I tagged Grr1 at the C-terminus with three tags PK (Grr1-3PK) and checked its protein levels in wild type cells, as well as in the mps1-3 grr1-17 and mps1-3 grr1-20 suppressors during a synchronous cell cycle at 34°C in the presence of nocodazole. The results showed that in both suppressors Grr1 was expressed at reduced levels compared to wild type cells, suggesting that it is less stable (Fig. 31). Therefore, the grr1 mutations might destabilize Grr1, thereby impacting the binding of specific target proteins. Cells with the indicated genotypes were synchronized in G1 with α-factor at 25°C and then released in fresh medium at 34°C (t = 0). Cells were collected at the indicated time points for western blot analysis of Grr1-3PK and Pgk1. Pgk1 has been used as loading control. Cyc: cycling cells.

Grr1 has been found in the cytoplasm, in the nucleus and at the bud neck (Blondel et al., 2000). However, whether it localizes at kinetochores is not known. During my one-month stay in the laboratory of Prof. K. Shirahige at the University of Tokyo, I investigated by ChIP-seq the localization of Grr1-3PK along the chromosomes of wild type and mps1-3 cells. Interestingly, my preliminary data showed that Grr1 accumulates at centromeres in wild type cells, either asynchronously growing at 25°C or synchronised in G1 and released at 34°C for 90 minutes, to enrich for mitotic cells (Fig. 32). Moreover, in the latter conditions Grr1 does not accumulate at centromeric regions in mps1-3 cells (Fig. 32), suggesting that SCF Grr1 recruitment to centromeres or kinetochores might require Mps1. Unfortunately, during my stay in Japan I did not have the time to repeat these data, which therefore remain preliminary. In the future, they could also be confirmed by studying the co-localization of the Grr1-3PK with a kinetochore protein in chromosome spreads. Wild type and mps1-3 cells were synchronized in G1 with α-factor at 25°C and then released at 34°C for 90 minutes. Cells were collected before the α-factor arrest (25°C) and after 90 minutes from the release and processed for ChIP-seq analysis. ChIP sequence reads were normalized against sequence reads from corresponding input samples, and relative enrichment is plotted for chromosome VIII around the centromere (CEN8). Y-axis shows enrichment values (linear scale, range is 0-25). Values below 1.5 are shown in grey, and values above 1.5 (i.e. sequences enriched in ChIP samples) are red-coloured. Similar data were obtained for the other 15 yeast chromosomes (data not shown).

These data suggest that SCF Grr1 could play a role at centromeres or kinetochores. To explain how reduced activity of SCF Grr1 could suppress the SAC and chromosome segregation defects of mps1-3 cells, we hypothesized that the SCF Grr1 complex could directly regulate Mps1 stability. During a normal cell cycle progression, Mps1 is degraded in anaphase by the APC Cdc20 and APC Cdh1 ubiquitin ligase complexes, and this is also an important feedback regulatory mechanism to silence the SAC [START_REF] Cui | Degradation of the Human Mitotic Checkpoint Kinase Mps1 Is Cell Cycle-regulated by APC-cCdc20 and APC-cCdh1 Ubiquitin Ligases[END_REF][START_REF] Palframan | Anaphase Inactivation of the Spindle Checkpoint[END_REF]. However, no data so far link Mps1 degradation to SCF complexes. To assess this possibility, I checked the levels of Mps1 throughout the cell cycle in wild type and grr1-20 single mutant cells (i.e. without the mps1-3 mutation in order to better see a possible

We then hypothesized that a protein other than Mps1 could be targeted by SCF Grr1 and accumulate in the grr1 suppressors. Since my published data indicate that PP1 antagonizes Mps1 in both SAC signaling and regulation of chromosome biorientation (Benzi et al., 2020), we considered the possibility that in the grr1 suppressors the PP1 phosphatase activity might be lowered, thus reestablishing a correct balance between kinase and phosphatase activity at the kinetochore. We therefore hypothesized that the grr1 suppressors could fail to degrade a putative PP1 inhibitor.

Ypi1 is a regulatory subunit of PP1 that is most similar to I3, a protein of the Inhibitor 3 family in human cells. Ypi1 was found to act in vivo both as a positive and a negative regulator of the phosphatase [START_REF] Bharucha | Ypi1, a Positive Regulator of Nuclear Protein Phosphatase Type 1 Activity in Saccharomyces cerevisiae[END_REF][START_REF] García-Gimeno | Molecular Characterization of Ypi1, a Novel Saccharomyces cerevisiae Type 1 Protein Phosphatase Inhibitor[END_REF]. Sds22 is another regulatory subunit of Glc7/PP1 that targets the phosphatase to substrates involved in the cell cycle control [START_REF] Mackelvie | The Saccharomyces cerevisiae gene SDS22 encodes a potential regulator of the mitotic function of yeast type 1 protein phosphatase[END_REF][START_REF] Peggie | Essential functions of Sds22p in chromosome stability and nuclear localization of PP1[END_REF]. Interestingly, both Sds22 and Ypi1 together interact with Glc7 in yeast cells and form an inhibitory complex for the phosphatase [START_REF] Pedelini | YPI1 and SDS22 Proteins Regulate the Nuclear Localization and Function of Yeast Type 1 Phosphatase Glc7[END_REF]. Consistently, in human cells Sds22 forms a heterotrimeric complex with PP1 and I3 that inhibits PP1 phosphatase activity [START_REF] Lesage | A complex of catalytically inactive protein phosphatase-1 sandwiched between Sds22 and inhibitor-3[END_REF]. I have therefore checked the stability of Ypi1 and Sds22 in synchronized wild type, mps1-3 and mps1-3 grr1-17 cells released at 34°C in presence of nocodazole. Ypi1 and Sds22 protein levels did not change significantly throughout the cell cycle and did not accumulate in the mps1-3 grr1-17 suppressor (Fig. 35A-B), suggesting that these proteins are unlikely ubiquitylation targets of the SCF Grr1 complex. Of course, we cannot exclude that only a small pool of these proteins, residing perhaps at kinetochores, could be controlled by SCF Grr1 . However, testing this hypothesis would be technically challenging. A-B: Cells with the indicated genotypes were synchronized in G1 with α-factor at 25°C and then released in fresh medium at 34°C in presence of nocodazole (t = 0). Cells were collected at the indicated time points for western blot analysis of Ypi1-3PK or Sds22-3PK, Clb2 and Pgk1. Clb2 has been used as mitotic marker and Pgk1 has been used as loading control. Cyc: cycling cells.

Other PP1 regulatory subunits could be tested in the future as putative SCF Grr1 targets. Among them, Gac1 is a PP1-interacting protein whose overexpression causes glucose derepression, similar to mutants affecting the PP1 catalytic subunit (Wu et al., 2001). Thus, Gac1 could act as a negative regulator of PP1. Glc8 is another regulatory subunit of PP1, homolog of mammalian Inhibitor-2, which functions both as a positive and a negative regulator of the phosphatase PP1 and was found to regulate chromosome segregation [START_REF] Tung | Regulation of chromosome segregation by Glc8p, a structural homolog of mammalian inhibitor 2 that functions as both an activator and an inhibitor of yeast protein phosphatase 1[END_REF]Böhm and Buchberger, 2013). Therefore, it could be another good candidate to be tested in the grr1 mutants.

Obviously, these are only some hypothesis on the possible role of Grr1 at kinetochores and that could explain the possible mechanism through which these mutants suppress the mps1-3 mutant phenotypes. Further work will be required to define the relationship between SCF Grr1 and Mps1 activity at kinetochores.

Characterization of internal suppressors to decipher the phenotypic defects of mps1-3 cells

The genetic screen for spontaneous suppressors of the temperature-sensitivity of the mps1-3 mutation allowed me also to find intragenic suppressors, which carried a second, missense mutation in the kinase domain of the MPS1 gene (Fig. 36 A-B). A notable exception was the mps1-sup22 suppressor, where the serine originally mutated to phenylalanine in the mps1-3 mutant was converted to tyrosine (Fig. 36 A-B). The suppressors could divide and form visible colonies at restrictive temperature (30°C-34°C), albeit to different extents. In particular, suppressors carrying any of the following mutations (Pro603Thr, Gly449Ser, Phe483Leu, Leu548Met and Ser635Tyr) could grow at 34°C, although less efficiently than wild type cells. In contrast, the suppressor carrying the mutation Ser451Ala could divide only up to 32°C, whereas at 34°C it was temperature-sensitive (Fig. 36B). I first verified if the suppression of the temperature-sensitivity correlated with the re-establishment of SAC signalling and proper chromosome segregation. For this purpose, I synchronized cells in G1, released them at restrictive temperature (34°C) in presence and absence of nocodazole and analysed DNA contents by FACS. In the presence of nocodazole, all mutants but one (mps1-sup27) mostly arrested in mitosis with ~2C DNA contents, although a moderate ability to escape the cell cycle arrest was apparent in mps1-sup5 cells compared to wild type cells. Under these conditions, no suppression could be observed for mps1-sup27 cells (Fig. 37A). This was not surprising, given that mps1-sup27 cells were temperature-sensitive at 34°C (Fig. 35B). Thus, we concluded that the suppressors mostly restored proficient SAC signalling. In the absence of nocodazole, these mutants showed a bimodal distribution of DNA contents, similar to wild type cells and unlike mps1-3 cells, indicating that chromosome bi-orientation was mostly restored (Fig. 37B). However, mps1-sup2 showed a partial suppression, while mps1-sup27 cells, showed no suppression of the mps1-3 mutant phenotype like observed for SAC activation, suggesting that these cells should be tested at lower temperatures (e.g.

A B

32°C).

Figure 37 : A second mutation in MPS1 leads to suppression of the chromosome segregation and SAC defects of the mps1-3 mutant. Wild-type, mps1-3 and mps1-sup mutant cells were synchronised in G1 with a-factor at permissive temperature (25°C) and then released at restrictive temperature (34°C, t = 0) in the presence (A) or absence (B) of nocodazole. Cells were collected at the indicated time points for FACS analysis of DNA contents. Note that for technical reasons mps1-sup7 could not be tested.

The phenotypes of the mps1-3 mutant stem from the reduced levels of the Mps1-3 protein at kinetochores, which in turn impact on the phosphorylation of specific substrates (Benzi et al., 2020).

It was therefore important to establish if the intragenic suppressors were able to restore kinetochore localization of the Mps1-3 protein. To address this question, centromeric enrichment of Mps1 was analysed in the suppressors through ChIP-seq by our collaborators (Y. Atsunori and K. Shirahige, University of Tokyo). The experiment was conducted on cells synchronized in G1 and released in the presence of nocodazole for 90 minutes, at either 34°C or 32°C depending on the suppressor. Indeed, the insertion of the 3HA tagging cassette before the stop codon of the mps1-sup genes decreased the 123 maximal permissive temperature for one suppressor (mps1-sup22) and completely abolished suppression at any temperature for two of them (mps1-sup2 and mps1-sup27) (Fig. 38), which therefore were not analysed. In addition, due to technical problems, mps1-sup7 remains to be tested.

The ChIP-seq analysis showed that in the suppressors analysed so far Mps1 centromeric localization was re-established in all the 16 yeast chromosomes (Fig. 39 with chromosome II as an example and data not shown), although not to wild type levels. These preliminary experiments therefore suggest that the intragenic suppressors might restore at least partial Mps1 kinetochore localization, thus explaining the re-gain of chromosome bi-orientation and SAC signalling. These experiments will be repeated shortly at 32°C for all the strains, since at this temperature all the 3HA-tagged suppressor proteins suppress efficiently the growth defects of mps1-3 cells. Wild type, mps1-3 and mps1-3-sup cells were synchronized in G1 with α-factor at 25°C and then released at 34°C (wild type, mps1-3, mps1-sup1 and mps1-sup5) or at 32°C (mps1-sup22) in presence of nocodazole. Cells were collected after 90 minutes and processed for ChIP-seq analysis. ChIP sequence reads were normalized against sequence reads from corresponding input samples, and relative enrichment is plotted for chromosome II around the centromere. Y-axis shows enrichment values (linear scale, range is 0-8). Values below 1.5 are shown in grey, and values above 1.5 (i.e. sequences enriched in ChIP samples) are red coloured.

Since all the suppressing mutations hit the MPS1 kinase domain, we wished to identify their position in the 3D structure of human Mps1 kinase domain (sequence ID: 3HMN_A). This analysis has been carried out by Andrey Kajava, a computational scientist at CRBM, who found that the mps1-sup2 and mps1-sup27 mutations (Gly449Ser and Ser451Ala) hit the ATP-binding domain of the kinase, whereas the mutation of mps1-sup5 (Phe483Leu) fell in the substrate-binding domain (data not shown). The other mutations were either in regions of the protein for which there was no structural information or did not give any obvious clue about their possible impact on Mps1 kinase activity.

Nevertheless, the available information allowed us to formulate an interesting hypothesis. Since the Mps1-3 mutant protein bears increased kinase activity compared to its wild type counterpart (Benzi et al., 2020), we hypothesised that the intragenic suppressors could decrease Mps1 kinase activity to levels that could be more similar to those of the wild type protein. In turn, since Mps1 regulates its own kinetochore turnover through autophosphorylation (Dou et al., 2015;Hayward et al., 2019b;Hewitt et al., 2010;Jelluma et al., 2010;Koch et al., 2019;Santaguida et al., 2010;Sliedrecht et al., 2010;X. Wang et al., 2014), we speculated that the intragenic suppressors could re-establish Mps1 kinetochore localization by lowering the kinase activity of the Mps1-3 protein. To address this possibility, I performed in vitro kinase assays on wild type, Mps1-3, and Mps1-sup proteins. For these analysis I used different conditions. I first used affinity-purified proteins from yeast cells using galactose-inducible glutathione S-transferase (GST) fusion constructs [START_REF] Lauzé | Yeast spindle pole body duplication gene MPS1 encodes an essential dual specificity protein kinase[END_REF] and verified the incorporation of P 33 γATP at 34°C. Through this approach I could observe that most suppressing mutations, and particularly Mps1-supp2 and Mps1-supp27, were able to lower the kinase activity of the Mps1-3 protein (Fig. 40) for what concerns the phosphorylation of the exogenous substrate MBP. Mps1 autophosphorylation followed a similar pattern in most cases, with the notable exception of Mps1-sup5 (Fig. 40). However, in all cases except Mps1-sup2 and Mps1-27, auto-and MBP phosphorylation remained higher than Mps1-3 levels. GST-tagged Mps1, Mps1-3, Mps1-1 and Mps1-sup proteins expressed in yeast cells from the GAL1 promoter [START_REF] Lauzé | Yeast spindle pole body duplication gene MPS1 encodes an essential dual specificity protein kinase[END_REF] were affinity-purified and incubated in vitro with radioactive ATP and myelin basic protein (MBP) for 4 minutes.

Since Mps1 kinase activity influences the stability of the Mps1 protein in yeast (Dou et al., 2015;Hayward et al., 2019b;Hewitt et al., 2010;Jelluma et al., 2010;Koch et al., 2019;Santaguida et al., 2010;Sliedrecht et al., 2010;X. Wang et al., 2014), it was difficult to reliably normalise the results above with the amount of Mps1 used in the kinase assays. Therefore, I also performed the in vitro kinase assays using recombinant wild type, Mps1-3 and Mps1-sup proteins purified from bacterial cells. For these assays I used as substrate the recombinant N-terminal part of Spc105 (aa1-320, Benzi et al., 2020) instead of the non-physiological substrate MBP. Based on these preliminary data, all suppressors seemed to reduce phosphorylation of Spc105 relative to the Mps1-3 but not to the wild type protein (Fig. 41). In contrast, autophosphorylation did not seem significantly affected by most suppressing mutations except for Mps1-sup2, which displayed very poor kinase activity (Fig. 41).

Thus, these data suggest that the internal suppressors might impact the kinase activity of the Mps1-3 protein on exogenous substrates but not their autophosphorylation. In vitro kinase assays with recombinant Mps1, Mps1-3 and Mps1-sup proteins purified from E. coli and incubated at 34°C for 1 or 5 minutes in the presence of radioactive gP 33 ATP and recombinant Spc105 Nterminal fragment (aa 1-320) as substrate.

To further extend these data, I also performed in vitro kinase assays in the same conditions using recombinant wild type, Mps1-3 and proteins carrying the single suppressing mutations without the mps1-3 mutation. Interestingly, all mutations reduced Mps1 kinase activity on Spc105 relative to Mps1-3, most often to lower levels than wild type Mps1 (except for Mps1-S635Y). Additionally, three mutant proteins also reduced their autophosphorylation to levels comparable to or lower than wild type Mps1 (i.e. Mps1-P603T, Mps1-G449S and Mps1-L548M), while the remaining mutant proteins behaved similar to Mps1-3 (Fig. 42).

Altogether, these data suggest that the suppressing mutations impact Mps1 kinase activity and could therefore reduce the ability of the Mps1-3 to phosphorylate at least exogenous substrates. In vitro kinase assays with recombinant Mps1, Mps1-3 and various Mps1 mutant proteins incubated at 34°C for 1 or 5 minutes in the presence of γP 33 ATP and a recombinant Spc105 N-terminal fragment (aa 1-320) as substrate.

Since Mps1 is an essential gene and kinase-dead mutants are lethal [START_REF] Lauzé | Yeast spindle pole body duplication gene MPS1 encodes an essential dual specificity protein kinase[END_REF], I asked if yeast strains carrying the suppressing mutations alone (i.e. without the original mps1-3 mutation) were viable. I therefore constructed yeast plasmids expressing the mutant alleles above from the MPS1 promoter and asked if they were able to rescue the lethality of mps1Δ cells. All of them, except for mps1-G449S that was semi-lethal, were able to complement MPS1 deletion. Cell proliferation assays at different temperature (25°C, 30°C, 32°C, 34°C and 37°C) or in presence of benomyl showed that most mutants were able to divide at all the temperatures, except for mps1-S635T, which turned out to be temperature-sensitive above 34°C and benomyl-sensitive, although to a lesser extent than mps1-3 (S635P) cells (Fig. 43). Furthermore, mps1-G449S cells were partially inviable already at 25°C and completely inviable at higher temperatures or in presence of benomyl (Fig. 43). These results support the importance of G449 in the ATP-binding domain for Mps1 kinase activity. For the other mutants, obviously the decreased levels of kinase activity that we observe in vitro are compatible with normal proliferation and viability in the presence of low doses of benomyl. In the future it will be interesting to better characterise the phenotypes of these single mutants. We conclude that the intragenic suppressors re-establish the kinetochore localization of the Mps1-3 protein, perhaps by lowering its kinase activity towards exogenous substrates. Since the suppressor proteins carrying the mps1-3 mutation mostly display elevated autophosphorylation levels, similar to Mps1-3, it is difficult to reconcile these data with the notion that Mps1 autophosphorylation controls its own residence time at kinetochores (Dou et al., 2015;Hayward et al., 2019b;Hewitt et al., 2010;Jelluma et al., 2010;Koch et al., 2019;Santaguida et al., 2010;Sliedrecht et al., 2010;X. Wang et al., 2014). Rather, phosphorylation of an unknown kinetochore substrate could influence Mps1 recruitment to Ndc80 or other proteins at kinetochores. Further work will be required to explore this exciting possibility.

Introduction

Chromosome segregation is a vulnerable, error-prone process that must be tightly regulated in time and space. Several requirements must be fulfilled for daughter cells to get an equal chromosome complement. First, chromosomes must be faithfully duplicated into identical sister chromatids through DNA replication. Second, sister chromatids must be glued together by sister chromatid cohesion, which allows cells to distinguish genetically identical from distinct chromosomes. Third, sister chromatids need to attach via their kinetochores to microtubules emanating from opposite spindle poles, a process referred to as chromosome biorientation (reviewed in Bloom and Yeh 2010). If this fails, a surveillance mechanism called spindle assembly checkpoint (SAC) temporarily halts cell cycle progression in metaphase to allow error correction. Conversely, when all chromosomes are bipolarly attached, SAC is satisfied and anaphase can shortly ensue.

SAC detects the lack of attachment between spindle microtubules and kinetochores (reviewed in Maresca and Salmon 2010), which are large protein assemblies residing at the centromere of each chromosome (reviewed in Musacchio and Desai 2017). Unattached kinetochores generate an alert signal that ultimately leads to the formation of a mitotic checkpoint complex (MCC) made of Bub3, Mad2, BubR1 (Mad3 in yeast) and Cdc20, which binds to and inhibits the E3 ubiquitin ligase Anaphase Promoting Complex bound to its activator Cdc20 (APC Cdc20 ). In turn, stabilization of APC Cdc20 targets, such as securin and cyclin B, prevents sister chromatid separation and mitotic exit, thereby imposing a reversible metaphase arrest that provides the time for correction of improper kinetochore-microtubule connections (reviewed in Musacchio 2015;[START_REF] Sacristan | Joined at the hip: kinetochores, microtubules, and spindle assembly checkpoint signaling[END_REF].

The dual-specificity mitotic kinase Mps1 (Monopolar spindle 1) is a key player in SAC signaling. Mps1 was first identified in budding yeast (Winey et al. 1991) due to its involvement in the duplication of spindle pole bodies (SPBs), the functional equivalents of centrosomes in animal cells.

While the involvement of Mps1 in centrosome duplication in eukaryotic cells other than yeast is controversial (Stucke et al. 2002;[START_REF] Fisk | Human Mps1 protein kinase is required for centrosome duplication and normal mitotic progression[END_REF]Kwiatkowski et al. 2010), Mps1 is almost universally implicated in SAC activation (reviewed in [START_REF] Pachis | Leader of the SAC: molecular mechanisms of Mps1/TTK regulation in mitosis[END_REF]. Mps1 works at the apex of SAC signaling through phosphorylation of the kinetochore protein Knl1/Spc105 on its N terminal MELT repeats, which in turn is essential for the kinetochore recruitment of the Bub1-Bub3 complex (London et al. 2012;Shepperd et al. 2012;Yamagishi et al. 2012;Primorac et al. 2013). Mps1 also phosphorylates Bub1 and Mad1, allowing their interaction and kinetochore recruitment of Mad2 that acts as a catalyzer for MCC assembly [START_REF] Faesen | Basis of catalytic assembly of the mitotic checkpoint complex[END_REF]Ji et al. 2018;London and Biggins, 2014;Mora-Santos et al. 2016;Moyle et al. 2014).

Coupling chromosome biorientation with the spindle assembly checkpoint

During the SAC-induced metaphase arrest, cells rectify the tensionless kinetochore-microtubule attachments with the aim of establishing chromosome biorientation. An error correction mechanism involves the continuous detachment of improper attachments to provide kinetochores with further opportunities to capture microtubules (reviewed in Lampson and Grishchuk 2017). When tension across kinetochores is finally established, bipolar attachments are stabilized and chromosomes congress to the cell's equator, due to the equilibrium between pulling and cohesive forces (by microtubules and sister chromatid cohesion, respectively).

A key player in the error correction pathway is the chromosomal passenger complex (CPC). The CPC is composed of the Aurora B kinase, the inner centromere protein INCENP, Survivin and Borealin (Ipl1, Sli15, Bir1 and Nbl1, respectively, in budding yeast) (reviewed in Carmena et al. 2012). Aurora B detaches improper kinetochore-microtubule connections by progressively phosphorylating Ndc80/Hec1, the subunit of the NDC80 complex that directly interacts with microtubules (Cheeseman et al. 2006;DeLuca et al. 2006;Ciferri et al. 2008;Alushin et al. 2010;[START_REF] Tooley | The Ndc80 complex uses a tripartite attachment point to couple microtubule depolymerization to chromosome movement[END_REF]Zaytsev et al. 2014Zaytsev et al. , 2015)). Moreover, Aurora B/Ipl1 phosphorylates the DAM1 complex in budding yeast and its functional homolog SKA complex in mammalian cells (Chan et al. 2012;Cheeseman et al. 2002;Kalantzaki et al. 2015;Lampert et al. 2010;Tien et al. 2010), which couples chromosome mouvement to microtubule depolymerization (Abad et al. 2014;Asbury et al. 2006;Grishchuk et al. 2008;Lampert et al. 2010;Schmidt et al. 2012;Tien et al. 2010;Welburn et al. 2009;Westermann et al. 2006).

Besides correcting improper kinetochore-microtubule connections, Aurora B is also part of SAC signaling (reviewed in Krenn and Musacchio 2015). On one side, it generates unattached kinetochores that are sensed by SAC [START_REF] Pinsky | The Ipl1-Aurora protein kinase activates the spindle checkpoint by creating unattached kinetochores[END_REF]). On the other, it facilitates the rapid recruitment of Mps1 to kinetochores (Santaguida et al. 2011;Saurin et al. 2011) and phosphorylates the RVSF motif in KNL1 (Liu et al. 2010). The latter hampers the binding of the PP1 phosphatase, which is required for SAC silencing (Liu et al. 2010;[START_REF] Pinsky | The Ipl1-Aurora protein kinase activates the spindle checkpoint by creating unattached kinetochores[END_REF]Rosenberg et al. 2011;Vanoosthuyse and Hardwick 2009). Thus, Aurora B contributes to chromosome biorientation by both promoting error correction and sustaining SAC signaling.

Similarly, The Mps1 kinase has been involved in the correction of faulty kinetochore-microtubule attachments, in addition to triggering SAC activity (Jones et al. 2005;Maure et al. 2007;Santaguida et al. 2010;Hewitt et al. 2010;Maciejowski et al. 2010). However, its role in the error correction pathway is far from being understood. While in mammalian cells Mps1 seems to work in concert with Aurora B (Vigneron et al. 2004;Jelluma et al. 2008;Saurin et al. 2011;Santaguida et al. 2011), in yeast the two kinases appear to play independent roles (Maure et al. 2007;Storchová et al. 2011). In addition, while in mammalian cells the SKA complex has been proposed to be a relevant Mps1 substrate in the error correction pathway (Maciejowski et al. 2017), in the budding yeast S. cerevisiae the critical targets of Mps1 in this process remain to be identified. Indeed, although S.c.Mps1 phosphorylates Dam1 and Ndc80 on several residues, none of these phosphorylations is required for chromosome biorientation (Shimogawa et al. 2006;Kemmler et al. 2009;Kalantzaki et al. 2015). The identification of the critical targets of S.c.Mps1 in chromosome biorientation is not an easy task, and is complicated by the essential function of S.c.Mps1 in SPB duplication and spindle assembly (Weiss and Winey 1996). Indeed, the initial discovery that Mps1 is involved in the error correction pathway was based on a complex experimental setup (Maure et al. 2007).

A novel genetic tool to dissect the function of Mps1 in chromosome biorientation

We have recently characterized a novel temperature-sensitive mutant, named mps1-3, which bears a single substitution of serine 635 to phenylalanine. The mps1-3 mutant is lethal at temperatures above 32 °C, and at temperatures ranging from 32 to 34 °C is severely defective in chromosome biorientation but proficient in SPB duplication and spindle elongation (Benzi et al. 2020), unlike the majority of mps1 mutants (Weiss and Winey 1996;Schutz and Winey 1998;Castillo et al. 2002;Araki et al. 2010). Contextually, mps1-3 cells are also SAC deficient, which allows them to progress through the cell cycle in the face of chromosome misalignment, thus accumulating massive aneuploidy. We could attribute lack of chromosome biorientation in mps1-3 cells to faulty correction of improper attachments, rather than to a failure to establish kinetochore-microtubule connections, in agreement with earlier conclusions on Mps1 inhibition through an analog-sensitive mutant (Jones et al. 2005;Maure et al. 2007). Consistent with defects in the error correction pathway, in mps1-3 cells sister chromatids co-segregate preferentially toward the bud (Benzi et al. 2020), where the old SPB usually migrates during anaphase (Pereira et al. 2001). This phenotype had been previously described for ipl1 (Aurora B) mutants and is likely due to a delay in the gain of microtubule-nucleating activity of the new versus the old SPB, thereby biasing kinetochore attachment to the old, mature SPB (Tanaka et al. 2002).

To our surprise, the mps1-3 mutation, which hits the kinase domain of the protein (aa 440-720), increases the in vitro kinase activity of Mps1 relative to the wild-type protein. However, the Mps1-3 does not localize at kinetochores at restrictive temperatures. Although Mps1 can sustain SAC signaling away from kinetochores under non-physiological conditions (Maciejowski et al. 2017;Fraschini et al. 2001;[START_REF] Yuan | Generation of a spindle checkpoint arrest from synthetic signaling assemblies[END_REF], its kinetochore recruitment clearly contributes to proficient chromosome biorientation and SAC signaling in normal cells (Jelluma et al. 2010;Saurin et al. 2011;[START_REF] Heinrich | Mph1 kinetochore localization is crucial and upstream in the hierarchy of spindle assembly checkpoint protein recruitment to kinetochores[END_REF]Nijenhuis et al. 2013;Zhu et al. 2013;[START_REF] Aravamudhan | Dual mechanisms regulate the recruitment of spindle assembly checkpoint proteins to the budding yeast kinetochore[END_REF]. Thus, the SAC and chromosome segregation defects of mps1-3 mutant cells likely stem from the lack of phosphorylation of critical Mps1 substrates at kinetochores. Consistently, phosphorylation of known Mps1 substrates at kinetochores, such as Spc105/Knl1, Bub1, and Mad1 (Shepperd et al. 2012;Yamagishi et al. 2012;Primorac et al. 2013;London and Biggins 2014;Moyle et al. 2014;Mora-Santos et al. 2016;[START_REF] Faesen | Basis of catalytic assembly of the mitotic checkpoint complex[END_REF][START_REF] Qian | An Attachment-independent biochemical timer of the spindle assembly checkpoint[END_REF]Ji et al. 2018) is markedly compromised in mps1-3 mutant cells. Additionally, artificial anchoring of Mps1 to the kinetochore protein Mtw1 partially restores proper chromosome segregation in mps1-3 cells (Benzi et al. 2020).

The domain responsible for Mps1 kinetochore localization lies at the N-terminus of the protein, far from the catalytic domain [START_REF] Ji | Kinetochore attachment sensed by competitive Mps1 and microtubule binding to Ndc80[END_REF]Liu et al. 2003;Stucke et al. 2004;Kemmler et al. 2009;Araki et al. 2010;Hached et al. 2011;Nijenhuis et al. 2013;Hiruma et al. 2015;Maciejowski et al. 2017). Thus, it is unlikely that the mps1-3 mutation interferes directly with Mps1 binding to its kinetochore receptor(s). Nonetheless, Mps1 has been proposed to accelerate its own kinetochore turnover through autophosphorylation (Sliedrecht et al. 2010;Kwiatkowski et al. 2010;Hewitt et al. 2010;Santaguida et al. 2010;Jelluma et al. 2010;Wang et al. 2014;Dou et al. 2015;Koch et al. 2019;Hayward et al. 2019). Furthermore, activation of the Mps1 kinase leads to extensive conformational changes in the protein (Combes et al. 2018), which in turn could mask its kinetochore-binding regions. Thus, the increased kinase activity of Mps1-3 could reduce its retention time at kinetochores, thereby impairing SAC signaling and chromosome biorientation. We attempted to test directly this hypothesis by introducing into the mps1-3 allele the analog-sensitive mps1-as1 mutation, which inhibits the kinase in the presence of the ATP analog inhibitor 1-NM-PP1 (Jones et al. 2005). However, the double mutant turned out to be lethal even in the absence of the analog (our unpublished data), making this approach inconclusive. The characterization of the internal suppressors that we identified (see below) might provide us with alternative tools to test the contribution of the hyperactivity of the Mps1-3 kinase to its own kinetochore turnover.

The Spc105/Knl1 kinetochore sca old as a critical target of Mps1 in chromosome biorientation beyond SAC signaling

Out of the two processes that are affected by the mps1-3 mutation, i.e. SAC signaling and chromosome biorientation, only the latter is essential for cell viability and accounts for the lethality of mps1-3 cells at high temperatures. This makes the mps1-3 mutant a valuable genetic tool to underpin the molecular bases underlying the involvement of Mps1 in the error correction pathway. We therefore exploited the potential of budding yeast as a powerful genetic tool to identify spontaneous suppressors that enable mps1-3 cells to divide at 34 °C. This unbiased genetic screen was instrumental to our current model envisioning a common Mps1 substrate (Spc105/Knl1, i.e. the stone) in the control of both chromosome biorientation and SAC (i.e. the two birds).

Through our genetic screen, we isolated several intragenic suppressors that carried second site mutations within the mps1-3 allele. These were all missense mutations in the catalytic kinase domain. Their future characterization might reveal important insights into the control of Mps1 activity and kinetochore recruitment. We also isolated extragenic suppressors that bore, instead, a suppressing mutation in genes other than MPS1. Strikingly, all of them rescued both chromosome segregation and SAC defects of mps1-3 cells, suggesting that a common mechanism underlies the role of Mps1 in these processes. Among these suppressors, we found mutations in SPC105, which codes for the kinetochore protein Spc105/Knl1, and GLC7, which codes for the catalytic subunit of the protein phosphatase PP1. Importantly, the spc105 suppressing mutations hit the motif (RVSF) for Spc105 binding to PP1, while the GLC7 suppressors carried a missense mutation of Phe256, which resides in the hydrophobic groove involved in PP1 interaction with partners carrying the RV/IXF consensus motif [START_REF] Egloff | Structural basis for the recognition of regulatory subunits by the catalytic subunit of protein phosphatase 1[END_REF]Wu and Tatchell 2001). As mentioned above, Mps1-dependent phosphorylation of Spc105/Knl1 at its MELT repeats recruits the Bub3-Bub1 complex and is critical for SAC signaling (London et al. 2012;Shepperd et al. 2012;Yamagishi et al. 2012;Primorac et al. 2013). Upon chromosome biorientation, Spc105/Knl1 phosphorylation is reversed by the phosphatase PP1, which binds the RVSF motif and silences the SAC (Pinsky et al. 2009;Vanoosthuyse and Hardwick 2009;Liu et al. 2010;Rosenberg et al. 2011;Meadows et al. 2011;London et al. 2012;Moura et al. 2017). Importantly, the spc105 and GLC7 suppressors that we isolated did not restore Mps1 kinetochore localization, strongly suggesting that PP1 opposes Mps1 activity at kinetochores for both proper chromosome biorientation and SAC signaling. The antagonism between Mps1 and PP1 has been well characterized for SAC, while its involvement in the error correction pathway had not been established. Altogether, our data have two important implications: first, the same Mps1-dependent sensory device may be used for both correction of improper kinetochore-microtubule attachments and SAC signaling, in line with previous proposals [START_REF] Musacchio | Spindle assembly checkpoint: the third decade[END_REF][START_REF] Caldas | KNL1 facilitates phosphorylation of outer kinetochore proteins by promoting Aurora B kinase activity[END_REF]; second, PP1 is expected to stabilize kinetochore-microtubule attachments in yeast, as it does in human cells (Liu et al. 2010).

On the basis of our results, we wondered if Spc105 phosphorylation and subsequent Bub1 recruitment could be the main function of Mps1 in the error correction pathway, as it is in SAC activation. Consistent with this idea, Bub1 is no longer recruited to kinetochores in mps1-3 mutant cells, while it regains kinetochore binding in the suppressors. Furthermore, artificial tethering of Bub1 to Spc105 is sufficient to restore balanced chromosome segregation and partial SAC signaling in mps1-3 cells (Benzi et al. 2020). Thus, our data strongly imply that Mps1 promotes the error correction pathway and SAC activity via a common mechanism that relies on Spc105/Knl1 phosphorylation (Fig. 1). Along this line, phosphorylation of other Mps1 kinetochore targets, such as Dam1 and Ndc80, turned out to be dispensable for equal chromosome segregation (Shimogawa et al. 2006;Kemmler et al. 2009;Kalantzaki et al. 2015).

The main conclusion of our paper is consistent with several published observations: an spc105-6A yeast mutant with unphosphorylatable MELT repeats is defective in Bub1 kinetochore recruitment and displays chromosome (London et al. 2012); phospho-mimicking mutations in the MELT repeats of Spc7 (the fission yeast homolog of Spc105/Knl1) partially suppress the chromosome biorientation defects of fission yeast mps1Δ cells, though in fission yeast Mps1 only modestly contributes to chromosome biorientation (Yamagishi et al. 2012). Finally, Knl1 depletion in human cells impairs Aurora B activity and chromosome alignment on the metaphase plate [START_REF] Caldas | KNL1 facilitates phosphorylation of outer kinetochore proteins by promoting Aurora B kinase activity[END_REF].

Is Bub1 kinetochore recruitment the only function of Mps1 in chromosome biorientation?

Bub1 has been extensively involved in chromosome biorientation through phosphorylation of T120 of histone H2A (S121 in yeast), which in turn recruits shugoshin proteins (Sgo1 in budding yeast) to pericentromeres (Fernius and Hardwick 2005;Kawashima et al. 2010;Yamagishi et al. 2010;[START_REF] Liu | Mitotic transcription installs Sgo1 at centromeres to coordinate chromosome segregation[END_REF]. Shugoshins, in turn, promote recruitment of the CPC and biorientation of sister chromatids (Indjeian et al. 2005;Kawashima et al. 2007;[START_REF] Huang | Tripin/hSgo2 recruits MCAK to the inner centromere to correct defective kinetochore attachments[END_REF][START_REF] Kiburz | Shugoshin promotes sister kinetochore biorientation in Saccharomyces cerevisiae[END_REF]Tsukahara et al. 2010;Yamagishi et al. 2010;Verzijlbergen et al. 2014;[START_REF] Liu | Mitotic transcription installs Sgo1 at centromeres to coordinate chromosome segregation[END_REF]. Consistent with Bub1 being required for Sgo1 accumulation at pericentromeres, localization of Sgo1 at kinetochores is compromised in mps1-3 mutant cells. However, the synthetic lethality between the mps1-3 mutation and SGO1 deletion suggests that Mps1 could have at least one additional function in chromosome biorientation, besides recruiting Bub1 to kinetochores. Indeed, Bub1 and Sgo1 are not essential proteins, in contrast to Mps1. Furthermore, artificial tethering of Bub1 to Spc105 cannot fully rescue the chromosome biorientation defects and temperature sensitivity of mps1-3 cells (Benzi et al. 2020), suggesting that other Mps1 substrates and/or other factors binding to Spc105 might intervene. One intriguing hypothesis, which would be supported by our genetic data, is that Mps1 could keep PP1 inhibited at kinetochores. PP1, in turn, stabilizes bipolar attachments (Liu et al. 2010).

Another interesting candidate that might play a function complementary to Bub1 in chromosome biorientation, downstream of Mps1, is the microtubule rescue factor Stu1, which is a member of the CLASP family (Al-Bassam and Chang 2011). At kinetochores, Stu1 stabilizes microtubules, and, consistently, gets sequestered at unattached kinetochores to facilitate their own capturing [START_REF] Funk | A TOGL domain specifically targets yeast CLASP to kinetochores to stabilize kinetochore microtubules[END_REF]). Stu1 sequestration at unattached kinetochores requires Mps1 and Spc105 phosphorylation of its MELT repeats, but not Bub1 [START_REF] Kolenda | Unattached kinetochores drive their own capturing by sequestering a CLASP[END_REF], suggesting that Stu1 and Bub1 might cooperate at Spc105 to achieve successful bipolar attachment. Stu1 recruits Stu2, a microtubule-associated protein of the XMAP215 family, to polymerize kinetochore-microtubules [START_REF] Vasileva | Molecular mechanisms facilitating the initial kinetochore encounter with spindle microtubules[END_REF]). Remarkably, Stu2 has been recently proposed to mediate an error correction pathway independent of Aurora B/Ipl1 in budding yeast [START_REF] Miller | A TOG protein confers tension sensitivity to kinetochore-microtubule attachments[END_REF][START_REF] Miller | Kinetochore-associated Stu2 promotes chromosome biorientation in vivo[END_REF]. Whether and how Mps1 modulates Stu1 and/or Stu2 function at kinetochores to achieve chromosome biorientation is an important question that deserves future investigations.

Another important question that remains open is whether an interplay between S.c.Mps1 and the CPC in the error correction pathway can be completely ruled out. As already mentioned, in budding yeast, Mps1 and Ipl1 were proposed to operate independently of one another (Maure et al. 2007;Storchová et al. 2011). Our data are consistent with this view. First, phosphorylation of the known kinetochore substrate of Ipl1/ Aurora B Dam1 is not affected in mps1-3 cells (Benzi et al. 2020). Similarly, Mps1 inhibition does not perturb phosphorylation of Aurora B substrates in human cells (Hewitt et al. 2010;Maciejowski et al. 2010;Santaguida et al. 2010). Second, the temperature sensitivity of mps1-3 cells cannot be suppressed by either the NDC80-13D phospho-mimicking mutant allele where all Mps1-dependent phosphorylation sites of Ndc80 have been replaced by aspartate (Kemmler et al. 2009), or the DAM1-S221F allele (also known as DAM1-765, (Shimogawa et al. 2006), which was previously found to suppress the temperature sensitivity of ipl1-321 mutant cells [START_REF] Shimogawa | Laterally attached kinetochores recruit the checkpoint protein Bub1, but satisfy the spindle checkpoint[END_REF]our unpublished data). Third, mutations in the CPC subunit Sli15 that activate Ipl1/Aurora B and rescue the sickness of bir1Δ, bub1Δ and sgo1Δ cells (Campbell and Desai 2013) do not suppress the lethality and chromosome segregation defects of mps1-3 cells at high temperature (Benzi et al. 2020). Thus, we favor the hypothesis that Mps1 sets in motion an error correction mechanism that does not involve Aurora B/Ipl1. However, it remains possible that, while differing in their respective upstream targets, the Mps1-and Ipl1-dependent error correction pathways ultimately converge on shared downstream effectors to destabilize flawed attachments. Future work will certainly shed light on this exciting possibility.

The SCF Grr1 complex: a novel player into the complex regulation of chromosome segregation at kinetochores?

The unbiased genetic screen identified suppressing mutations in the F-box protein Grr1 that is part of the SCF ubiquitin ligase complex [START_REF] Kishi | Grr1 functions in the ubiquitin pathway in Saccharomyces cerevisiae through association with Skp1[END_REF]F N Li and Johnston, 1997). Although some of them carried a second mutation in the RSC30 gene, encoding for a subunit of the RSC chromatinremodeling complex [START_REF] Angus-Hill | A Rsc3/Rsc30 Zinc Cluster Dimer Reveals Novel Roles for the Chromatin Remodeler RSC in Gene Expression and Cell Cycle Control[END_REF][START_REF] Cairns | RSC, an Essential, Abundant Chromatin-Remodeling Complex[END_REF], I could establish that the suppression was due to the grr1 mutation. The grr1 suppressors, like those hitting SPC105 and GLC7, restored proper chromosome segregation and SAC signaling in the mps1-3 mutant. However, the Mps1-3 protein was not restored at kinetochores in these suppressors, suggesting that suppression relies on a different mechanism.

An obvious impact of the two grr1 suppressing mutations that I studied in further detail (grr1-17 and grr1-20) was an apparent destabilization of the Grr1 protein, which led to decreased Grr1 levels. Grr1 protein levels do not seem to change during the cell cycle in wild type cells, suggesting that, unlike other F-box proteins, Grr1 is unlikely a target of SCF. Therefore, by lowering Grr1 protein levels in the grr1 suppressing mutants, the SCF Grr1 could be less active and fail to efficiently target its substrates to degradation.

The genetic interactions linking Grr1 to Mps1 are at the moment very hard to interpret, since not many published data link the two proteins, except for the finding that phosphorylated Grr1 peptides were identified in Mps1 immunoprecipitates through a mass spectrometry screen [START_REF] Breitkreutz | A Global Protein Kinase and Phosphatase Interaction Network in Yeast[END_REF].

Importantly, my preliminary ChIP-seq data show a centromeric enrichment of Grr1, which suggests that Grr1 could play a role at centromeres/kinetochores. These data still needs further confirmation also by fluorescence imaging of GFP-tagged Grr1. We first hypothesized that a possible kinetochore function for Grr1 could be directly related to Mps1 activity, but we found that in the grr1 suppressors neither the protein levels nor the kinetochore localization of Mps1 is altered. Therefore, other target(s) could be regulated by Grr1 and their lack of degradation in the suppressors could rescue the defects of mps1-3 cells.

Since PP1 counteracts Mps1 in both SAC signaling and chromosome bi-orientation (Benzi et al., 2020), we hypothesized that the grr1 suppressors might lower PP1 activity at kinetochores through accumulation of a PP1 inhibitor. I therefore checked the levels of different proteins that were shown to act as negative regulators of PP1, such as Yip1 and Sds22 [START_REF] Bharucha | Ypi1, a Positive Regulator of Nuclear Protein Phosphatase Type 1 Activity in Saccharomyces cerevisiae[END_REF][START_REF] García-Gimeno | Molecular Characterization of Ypi1, a Novel Saccharomyces cerevisiae Type 1 Protein Phosphatase Inhibitor[END_REF][START_REF] Lesage | A complex of catalytically inactive protein phosphatase-1 sandwiched between Sds22 and inhibitor-3[END_REF][START_REF] Mackelvie | The Saccharomyces cerevisiae gene SDS22 encodes a potential regulator of the mitotic function of yeast type 1 protein phosphatase[END_REF][START_REF] Pedelini | YPI1 and SDS22 Proteins Regulate the Nuclear Localization and Function of Yeast Type 1 Phosphatase Glc7[END_REF][START_REF] Peggie | Essential functions of Sds22p in chromosome stability and nuclear localization of PP1[END_REF].

However, the levels of these two proteins did not increase in grr1 mutants, indicating that they are unlikely targets of the SCF Grr1 complex. Of course, we cannot rule out the possibility that only a small pool of these proteins, perhaps residing at kinetochores, is controlled by SCF Grr1 , a hypothesis that would be quite challenging to test. Although these results discouraged us from pursuing this model, there could be other PP1 regulators to test as putative substrates of SCF Grr1 . In particular, we think that the PP1-interactor Gac1 is a promising candidate. Although Gac1 has been proposed to be a positive regulator of PP1 [START_REF] François | GAC1 may encode a regulatory subunit for protein phosphatase type 1 in Saccharomyces cerevisiae[END_REF]Wu et al., 2001), its overexpression causes glucose derepression, similar to mutants affecting the PP1 catalytic subunit (Wu et al., 2001). Thus, Gac1, like many phosphatase regulators, could act as both positive and negative regulator of PP1.

Interestingly, Gac1 has been found in a proteomic screen for proteins increasing in levels in grr1Δ mutant cells [START_REF] Mark | Ubiquitin Ligase Trapping Identifies an SCFSaf1 Pathway Targeting Unprocessed Vacuolar/Lysosomal Proteins[END_REF], suggesting that Gac1 could be target of the SCF Grr1 complex. Thus, in the future it will be interesting to investigate if increased levels of Gac1 in the grr1 mutants are responsible for the suppression.

Bub1 interacts with Skp1, another component of the SCF complex, and this interaction is important for centromeric localization of Bub1 [START_REF] Kitagawa | Requirement of Skp1-Bub1 Interaction for Kinetochore-Mediated Activation of the Spindle Checkpoint[END_REF]. We have shown that Mps1 regulates both SAC activation and the error correction process through kinetochore recruitment of Bub1 (Benzi et al., 2020). We could therefore speculate that the SCF Grr1 complex, by binding to Bub1, could lead to its ubiquitylation and subsequent degradation. In this view, in the grr1 suppressing mutants, where the ubiquitylation complex is likely less active, Bub1 would be less degraded and this could balance its strongly reduced kinetochore localization in the mps1-3 mutant. However, this idea would be inconsistent with published data proposing that the Skp1-Bub1 interaction is important to activate SAC in response to lack of tension. Moreover, Bub1 is known to be degraded in an APC-dependent manner [START_REF] Qi | KEN-Box-dependent Degradation of the Bub1 Spindle Checkpoint Kinase by the Anaphase-promoting Complex/Cyclosome[END_REF]. Therefore, this hypothesis remains quite unlikely.

Another interesting aspect comes from the preliminary evidence that Grr1 seems to localize at centromeric regions in wild type but not mps1-3 cells. Although these data need to be confirmed, together with the observation that phosphorylated Grr1 peptides were found associated to Mps1 in a proteomic study [START_REF] Breitkreutz | A Global Protein Kinase and Phosphatase Interaction Network in Yeast[END_REF] they suggest that Mps1 could control directly or indirectly the centromeric/kinetochore localization of Grr1. However, the meaning of this regulation, if any, and its relationship with Mps1 is at the moment hard to interpret. Indeed, the suppression of the

Relationship between Mps1 kinase activity and kinetochore localization

The Mps1-3 protein exhibits lack of kinetochore localization and increased catalytic activity (Benzi et al., 2020). The domain responsible for Mps1 binding to kinetochores resides in its N-terminal 300 aminoacids (Araki et al., 2010;Hached et al., 2011;Liu et al., 2003;Maciejowski et al., 2010;Stucke et al., 2004) and therefore how a mutation in the kinase domain could affect Mps1 kinetochore recruitment is not immediately clear. However, since Mps1 accelerates its own kinetochore turnover through autophosphorylation (Dou et al., 2015;Hayward et al., 2019;Hewitt et al., 2010;Jelluma et al., 2010;Koch et al., 2019;Kwiatkowski et al., 2010;Santaguida et al., 2010;Sliedrecht et al., 2010;Wang et al., 2014), an intriguing hypothesis is that the higher kinase activity of Mps1-3 accelerates its own turnover and causes its displacement from kinetochores. To test directly this possibility, I tried to introduce an analogue-sensitive mutation (M516G, also referred to as mps1-as1; (Jones et al., 2005)) in the mps1-3 allele. Indeed, this strategy renders the kinase exquisitely sensitive to bulky ATP analogs, which therefore inhibit the kinase in a dose-dependent manner [START_REF] Bishop | A chemical switch for inhibitor-sensitive alleles of any protein kinase[END_REF].

Unfortunately, this approach was inconclusive, as the M516G mutation was lethal in combination with the S635P substitution of the mps1-3 mutant allele (data not shown). I therefore turned my attention to the intragenic suppressors that I isolated in the genetic screen, as they could help deciphering the links between Mps1 kinase activity and kinetochore localization. Intragenic suppressors bear a second mutation in the kinase domain of the mps1-3 allele and the ones analyzed so far seem to partially restore Mps1 kinetochore localization. Interestingly, some of them carry a mutation in the ATP-binding or substrate-binding domain, strongly suggesting that they could suppress the defects of the mps1-3 mutant by decreasing auto and/or substrate phosphorylation. Surprisingly, autophosphorylation of the Mps1 proteins carrying the suppressing mutation was not consistently lowered, while phosphorylation of exogenous substrates (Spc105 in particular) seemed almost invariably affected. Although these data are still preliminary, they are at odds with the idea that a reduction in the levels of autophosphorylation could restore Mps1 residence at kinetochores and suppress the mitotic defects of mps1-3 cells. It is still possible, however, that suppression stems from impaired phosphorylation of an exogenous substrate, e.g. a regulator of the Mps1 kinetochore localization. In humans, localization of Mps1 at kinetochores relies on binding to the NDC80 complex through two interaction interfaces (Hiruma et al., 2015;[START_REF] Ji | Kinetochore attachment sensed by competitive Mps1 and microtubule binding to Ndc80[END_REF][START_REF] Martin-Lluesma | Role of Hec1 in spindle checkpoint signaling and kinetochore recruitment of Mad1/Mad2[END_REF]Santaguida et al., 2011;Saurin et al., 2011;Zhu et al., 2013) However, the existence of additional

Conclusions

Altogether, the data presented in this thesis have shed light on the mechanism through which Mps1 could regulate chromosome segregation in budding yeast. In particular, I have characterized a novel mps1 mutant exhibiting increased kinase activity that is specifically defective in chromosome biorientation and, as such, represents a valuable tool to dissect the role of Mps1 in this process. Through an unbiased genetic screen, we could demonstrate that a common molecular mechanism based on Spc105 phosphorylation and subsequent Bub1 kinetochore recruitment is underlying the function of Mps1 in both SAC and error correction process (Benzi et al., 2020;Benzi and Piatti, 2020). Therefore, these two processes are likely under the control of a single sensory device involving Mps1 and, presumably, Aurora B. Our genetic data also strongly suggest that Aurora B and Mps1 would act independently from one another, although we cannot exclude that the two pathways converge on the same downstream targets (Benzi et al., 2020;Benzi and Piatti, 2020). Finally, the finding of four independent mutants in grr1 suppressing the mitotic phenotypes of the mps1-3 mutant, suggests a new role for the SCF Grr1 ubiquitin ligase complex as a possible partner of Mps1 in the regulation of chromosome segregation, although these conjectures needs further investigation.

Many of the mechanisms here described are conserved in the eukaryotic kingdom. Although in mammalian cells Mps1 seems to have more defined targets in the regulation of chromosome biorientation and its relationship with Aurora B seems to be more direct than in yeast, our findings are likely applicable to other systems.

Mps1 is overexpressed in several cancers and, given its implication in chromosome segregation and SAC signaling, is an attractive therapeutic target. Consistently, several Mps1 inhibitors have been developed for cancer treatment, although a more detailed elucidation of the relationship between the mitotic functions of Mps1 roles and tumorigenesis is needed for the development of more potent and selective inhibitors (reviewed in [START_REF] Wang | Molecular design and anticancer activities of small-molecule monopolar spindle 1 inhibitors: A Medicinal chemistry perspective[END_REF]. Hence, a better understanding of the control of chromosome bi-orientation by Mps1 could help designing novel strategies to fight cancer progression. MATa cells were grown in YEP containing the appropriate sugar, diluted to 5 x 10 6 cells/mL and incubated at 25°C for 2 hours and 15 minutes with 4 µg/mL α factor. G1-arrest was monitored using a transmitted light microscope. Cells were washed with fresh medium to remove the pheromone and resuspended in fresh medium to release them in the appropriate conditions.

Media for E.coli

Nocodazole arrest

Cells were released from G1 arrest in YEP pre-heated at a specific temperature (32°C, 25°C, 34°C or 37°C) and containing the appropriate sugar and 15 µg/mL Nocodazole (Sigma) freshly dissolved in DMSO. The final concentration of DMSO in the cell cultures was 1%.

E.coli transformation

50 µL of DH5α or BL21 Rosetta competent cells, stocked at -80°C, were unfrozen in ice. After incubation with 1-10 ng of DNA for 30 min in ice, cells were heat-shocked at 42°C for 1 minute and put back in ice. 1 mL of LD medium was added to each reaction tube and cells were incubated 90 minutes at 37°C before plating on selective medium and incubation at 37°C for 15-24 hours.

Yeast transformation

Cells in stationary phase grown at 25°C-30°C in YEP containing the appropriate sugar or selective medium were centrifuged and washed with 1 M CH3COOLi and resuspended in 1 M CH3COOLi to obtain a 1:1 suspension. 1-2 µg of DNA were added to the suspension together with 30 µg of salmon sperm DNA (carrier) and 45 µL of 50% PEG 4000. Cells were incubated 30-60 minutes (30 minutes for diploid cells, 60 minutes for haploid cells) at RT. Subsequently, 6 µL of 60% glycerol was added to the cell suspension and cells were again incubated for the same incubation time. Cells were then heat shocked 2-10 minutes (2 minutes for temperature-sensitive and diploid strains and 10 minutes for other strains) at 42°C and then plated on selective medium (for DNA cassettes containing resistance to drugs or plasmids transformations) or grown in YEPD for 6 hours before plating on selective medium (for DNA cassettes complementing auxotrophies).

Proliferation assays on plates (Drop tests)

Cell cultures were grown overnight to stationary phase and then serial dilutions of the cultures were made to 1.25 x 10 6 , 1.25 x 10 5 , 1.25 x 10 4 , 1.25 x 10 3 cells/mL. For each dilution 5 µL of suspension was spotted on plate and then incubated at the indicated temperatures.

Chromosome loss assay

This assay is based on the loss of heterozygosity of the MAT locus, carried by chromosome III [START_REF] Andersen | A Genetic Screen for Increased Loss of Heterozygosity in Saccharomyces cerevisiae[END_REF]. Briefly, wild type diploid cells are heterozygous MATa/MATalpha and cannot mate unless they lose one copy of the MAT locus through mutations, chromosome re-arrangements or chromosome loss. Cells were grown overnight at 25°C and their concentration was measured at the time of inoculation and the day after to calculate the number of cell divisions. 2x10 7 diploid Met + cells were incubated 4 hours at RT with an excess (2x10 8 ) MATalpha met4 tester cells in 400 µL of YEPD. 10 µL of the suspension was then diluted to plate 500 cells on -Met plates (where only the Met + diploids can form colonies) to calculate the percentage of viable (i.e. colony-forming); the remaining cells were plated on minimal SD plates (MIN), where only triploids, generated from the mating of diploids with the haploid tester cells, can grow. Plates have been incubated at 25°C. Frequency of chromosome loss has been calculated as follows: {[(n° of colonies on MIN / 2x10 7 ) / fraction of viable cells] / n° of cellular divisions}. We note that with this assay we likely underestimate the real frequencies of chromosome loss because our measurements do not take into account the frequency of mating for each strain, which probably is much lower than 100%.

Suppressor screen

2.6x10 7 mps1-3 cells were plated on YEPD and incubated at 34°C for 3-5 days until colonies appearance. The isolated spontaneous suppressors were then characterised by classical genetic tests to distinguish intragenic from extragenic mutations, recessive from dominant mutations, and to group the suppressors in complementation/allelic groups. Genomic DNA was extracted from one representative suppressor for each complementation/allelic group and sent for whole genome sequencing to identify the mutated genes. The identity of the suppressing mutations was confirmed by classical genetics. Targeted gene re-sequencing approaches were used to identify the supressing mutations in the other extragenic suppressors.

Images of Sgo1-GFP (Fig. S8A) have been deconvolved using the software Huygens using the following settings: background=200; ratio signal/noise=4; iterations=5.

Budding index was scored under a transmitted light microscope on cells fixed with 70% ethanol and treated for FACS analysis. Percentage of nuclear division was determined on the same samples after DNA staining with propidium iodide under a fluorescence microscope using a CY3 filter.

FACS analysis of DNA contents

For DNA quantification by fluorescence-activated cell sorting (FACS), 1-2 x 10 7 cells were collected, spun at 10000g and fixed with 1 mL of 70% ethanol for at least 30 min at RT. After one wash with 50 mM Tris-Cl pH 7.5, cells were resuspended in 0.5 mL of the same buffer containing 0.05 mL of a preboiled 10 mg/mL RNAse solution and incubated overnight at 37 °C. The next day cells were spun at 10,000g and resuspended in 0.5 mL of 5 mg/mL pepsin freshly diluted in in 55 mM HCl. After 30 min incubation at 37 °C cells were washed with FACS buffer (200 mM Tris-Cl pH 7.5, 200 mM NaCl, 78 mM MgCl2) and resuspended in the same buffer containing 50 μg/mL propidium iodide.

After a short sonication samples were diluted (1:20-1:10) in 1 mL of 50 mM Tris-Cl pH 7.5 and analysed with a FACSCalibur device (BD Biosciences). 10000 events were scored for each sample and plotted after gating out the debris.

Protein extracts, western blotting and kinase assays

For TCA protein extracts, 10-15 mL of cell culture in logarithmic phase (OD600 = 0.5-1) were collected by centrifugation at 2000g, washed with 1 mL of 20% TCA and resuspended in 100 µL of 20% TCA before breakage of cells with glass beads (diameter 0.5-0.75 mm) on a Vibrax VXR (IKA).

After addition of 400 µL of 5% TCA, lysates were centrifuged for 10 min at 845g. Protein precipitates were resuspended in 100 µL of 3× SDS sample buffer (240 mM Tris-Cl pH6.8, 6% SDS, 30% glycerol, 2.28 M β-mercaptoethanol, 0.06% bromophenol blue), denatured at 99°C for 3 min and loaded on SDS-PAGE after elimination of cellular debris by centrifugation (5 min at 20000g).

Proteins were wet-transferred on Protran membranes (Schleicher and Schuell) overnight at 0.2 A and probed with: monoclonal anti-HA 12CA5 (1:5000); anti-PK (alias anti-V5; MCA1360 AbD Serotec, 1:3000); polyclonal anti-Cdc5 (sc-6733 Santa Cruz, 1:3000); anti-Clb2 (a generous gift from W.Zachariae, 1:2000); anti-Pgk1 (Invitrogen Molecular Probes, 1:40000); monoclonal antimyc 9E10 (1:5000); anti-phospho-AMPK (Thr172) (Cell Signalling, 1:1000); anti-GST (1:2000, 27-4577-01 GE Healthcare). Antibodies were diluted in 5% low-fat milk (Regilait) dissolved in TBST.

Secondary antibodies were purchased from GE Healthcare and proteins were detected by a homemade enhanced chemiluminescence system.

For kinase assays with recombinant Mps1 proteins, Mps1/Mps1-3/Mps1-suppressors and Spc105 have been purified as follows: BL21 DE3 cells transformed with pSP1571, pSP1575 or pSP1576 were grown in LB containing 50µg/mL of ampicillin, 34µg/mL of cloramphenicol and 1% glucose and induced with 0.2 mM IPTG at 16°C overnight. Cells were lysed in 50mM Tris-Cl pH 8, 300mM NaCl, 0.1% Tween20 containing 1mg/mL of lysozyme and a cocktail of protease inhibitors (Complete EDTA-free Roche). Cells were then sonicated 3x with 1'30'' cycles of 8'' pulses/8'' ice and extracts cleared at 30000g for 30' at 4°C. Lysates were incubated 2h with 1mL of Ni-NTA agarose beads (Qiagen), pre-washed 3 times with 50mM Tris-Cl pH 8, 300mM NaCl, 0.1% Tween20, 10mM imidazole at 4°C. After incubation with the extracts, the slurry was washed 3 times with 50mM

Tris-Cl pH 8, 300mM NaCl, 0.1% Tween20, 20mM imidazole and loaded on Polyprep columns (BioRad). Fractions of 0.5mL were eluted with 50mM Tris-Cl pH 8, 300mM NaCl 250mM imidazole and quantified by Nanodrop. The most concentrated fractions were washed and concentrated through the use of Amicon Ultra filter units (10 KDa cut-off for Spc105 and 50 KDa cut-off for Mps1) to finally have them in 50mM Tris-Cl pH 8, 50mM NaCl. Glycerol was added to the final concentration of 20%. Protein concentration was quantified by loading them on SDS-page along with a BSA standard curve followed by staining with Coomassie blue. Kinase assays were performed at 34°C for different times in 20 µL of a reaction mix containing 50mM Tris-Cl pH7.5, 10mM MgCl2, 0.5mM DTT, 10µM ATP, 1 µL ATP-γ P33 3000 Ci/mmol, 80µg/mL MBP. 75 ng of Spc105 N-term fragment and 1µg Mps1/Mps1-3/Mps1-suppressor kinases were used for each assay. Reactions were stopped using 7 µL of 3x SDS sample buffer (described in TCA extracts). Samples were then boiled 3 minutes at 95°C and loaded on SDS-PAGE. The gel was then dried and exposed on autoradiography films.

Native yeast protein extracts for kinase assays were performed by collecting cells in logarithmic phase (OD600 = 1) grown in -Ura RG at 30°C, centrifuging at 2000g and washing with 1 mL of cold 10 mM Tris-Cl pH 7.5. Cells were then broken in 50mM Tris-Cl pH 7.5, 50mM NaCl, 0.2% TritonX100 containing a cocktail of protease inhibitors (Complete EDTA-free Roche) and phosphatase inhibitors (PhosSTOP Roche). Lysates were cleared at 20000g for 10 min at 4 °C. The equivalent of 6 OD600/assay was incubated on a nutator for 2h at 4 °C with 50 µL of Glutathione Sepharose 4 Fast Flow (GE Healthcare) beads pre-washed with the above buffer. After incubation with the extracts, beads were spun down at 4°C at 845g, washed twice with the same buffer without protease and phosphatase inhibitors, twice with the same buffer containing 0.1% of Triton X100, twice with the DNA was prepared by resuspending cells in 200mM CH3COOLi/1% SDS and lysis by heating 15minutes at 70°C. After addition of 300 µL of 100% EtOH, DNA was collected by centrifugation and resuspended in 100 µL of TE.

For genome sequencing DNA from 30mL of stationary phase cells were prepared using the Quiagen Genomic-tip 500/G kit. Southern blot 5mL (YEPD) or 10 mL (selective medium) of stationary phase cells were washed with water and then resuspended in 200 µL 1M sorbitol/0.1M HOC(COONa)(CH2COONa)2/0.06M EDTA containing 0.2-0.4 mg of zymolase and 1.6 µL of β-mercaptoethanol to digest the cell wall and get spheroplasts.

Once sheroplasting was complete, 2% SDS/0.1M Tris-HCl pH 9.0/0.05M EDTA was added and cells were heated 5 minutes at 65°C. 0.2 mL of 5M CH3COOK was added and cells were cool down in ice for 20 minutes. 0.2 mL of 5M CH3CO2NH4 and 1 mL of isopropanol were added to the supernatant, samples were centrifuged and supernatant completely removed. Pellets were dissolved in 90 µL of TE.10 µL of 5M CH3CO2NH4 and 200 µL of isopropanol were added. Samples were centrifuged and the supernatant was eliminated, pellets were rinsed with 80% of EtOH, the supernatant removed and pellets dissolved in 50 µL of TE.

Southern Blot analysis

Yeast DNA was prepared as described above and digested with the appropriate restriction enzyme (HindIII for integration at the URA3 locus). The resulting DNA fragments were separated by gel electrophoresis in a 0.6% agarose gel and transferred to Amersham HybondTM-N+ nylon membrane (GE healthcare), followed by crosslinking of DNA to the membrane using an UV crosslinker. Then, the membrane was hybridized ON at 50°C with the appropriate DIG-labelled probe (URA3) in 50% formamide, 5x SSC, 0.1% N-Lauroylsarcosine, 0.02% SDS 2% Blocking reagent 0.1M Maleic acid 0.15M NaCl 0.3% tween. After two washes with 5x SSC, one with 1x SSC 0.1% SDS, one with 0.1x SSC 0.1% SDS, one with 0.1M Maleic acid 0.15M NaCl 0.3% tween, one with Blocking Reagent 3.3.1.1 3,7 ]decan}-4-yl)phenyl phosphate), allowing the detection by a chemiluminescence reaction, which was then revealed by exposing the blot to X-ray film.

PCR (Polymerase Chain Reaction)

PCR was performed on plasmid DNA or on genomic yeast DNA. A DNA fragment amplification requires two oligonucleotides flanking the region of amplification, one forward and one reverse, working as primers for the DNA polymerase. Different DNA polymerases were used: Vent 1) denaturation 3min at 95°C

2) denaturation 30sec at 95°C

3) annealing 30sec at variable temperature (50-55°C) depending on primers' Tm 4) extension time and temperature depending on the DNA polymerase 5) steps 2-4 repeated 30 cycles

6) final extension 10min

Primers' melting temperature (Tm) was calculated using NEB calculator on the NEB website.

Lots of people have been fundamental during these last years.

First of all, nothing of what I am now, as a scientist, could exist without my supervisor, Simonetta. I should write a thesis for thanking you for everything but… thank you for the way you have taught me, for the way you allowed me to always discuss with you as a mature scientist, for transferring me your passion for science, for pushing me to do always my best, for believing in my future and my possibilities even when I wasn't, for fighting for my paper, for being a friend when I needed, for being patient when I was exaggerating, stressing, panicking, for supporting me in all my choices. I will incredibly miss you and is going to be hard to find a chief like you.

I want to thank my family, and in particular my mum, who always have supported me, believed in me, and helped me when I have needed, and my brother who now has become the first person I call for any need, doubt, problem, crisis. I feel that your support and guidance are now essential for me to grow.

Friendship has been fundamental during these past years and I won't be the same without my best friends balloons Anna, Serena and Otta who are always there, no matter any distance. And I am sure it will always be like that. Broti, part of your wisdom is always with me! Montpellier allowed me to find a family, that became more and more small during the past years, but that includes many people that crossed my road and helped me to become who I am. First of all, Ale&Anto, I am so lucky to have found you and I will incredibly miss you. You were always there during these past years and you became essential to me. All the other people who left or not: Carole, Anna Laura, Luca, Antonella, Davide, Matilde, Lara, it has been a pleasure to share this trip with you.

I want to thank all the past and present members of the Piatti's lab, for all the time we spent together, for being there for any help. Sandy, Maritzaida, Alain, Ingrid, Magda, Ilaria, Leon, Selene, Davide, Antonella, Stefano, everybody has been incredibly important somehow for me. A particular thank to Sandy, who helped me a lot with French summaries of the thesis.

I also want to thank all the people of the CRBM, in particular the members of the Helmlinger lab, Peggy, who always has a good advice; Celine, Pascal, for the lunches we spent together. Many other people of the institute, particularly Thierry, Virginie, Tony, who have been particularly kind to me during these years, but all the people of the institute contributed to my personal growth.

I would like to thank my mentor, Barbara, who was able to understand me and pushing me to give me another chance with academia as soon as we met. You have literally changed my life!

Figure 1 :

 1 Figure 1 : Budding yeast mitosis. .................................................................................................................Figure 2 : A diagram of the main mitotic events in mammalian cells. .....................................................Figure 3 : The γ-tubulin small complex (γ-TuSC). .....................................................................................Figure 4 : The spindle pole body in budding yeast. ....................................................................................Figure 5 : SPB duplication. ...........................................................................................................................Figure 6 : The centrosome. ...........................................................................................................................Figure 7 : The centrosome cycle. ..................................................................................................................Figure 8 : The γ-tubulin template model. ....................................................................................................Figure 9 : Condensin architecture. ..............................................................................................................Figure 10 : Loop extrusion induced by a single condensin complex. ........................................................Figure 11 : Effects of condensin I and II on mitotic chromosome condensation. ....................................Figure 12 : Structure of the cohesin ring. ....................................................................................................Figure 13 : Models proposed for chromatin entrapment by cohesin. .......................................................Figure 14 : The cohesin cycle in human cells. .............................................................................................Figure 15 : Schematic kinetochore structure. .............................................................................................Figure 16 : The end-on conversion process. ................................................................................................Figure 17 : kinetochore orientation in mitosis and meiosis I. ....................................................................Figure 18 : Types of kinetochore-microtubule attachments. .....................................................................Figure 19 : SAC principles. ...........................................................................................................................Figure 20 : Domain organisation of human MPS1. ....................................................................................Figure 21 : Regulation of Mps1 during mitosis in humans. .......................................................................Figure 22 : Spindle Assembly Checkpoint activation. ................................................................................Figure 23 : The Mad2 template model. ........................................................................................................Figure 24 : The Chromosomal Passenger Complex and its recruitment to centromeres. ......................Figure 25 : The tension-related model. ........................................................................................................Figure 26 : The "dog leash" model for CPC function. ...............................................................................Figure 27 : Regulation of PP1 kinetochore recruitment. ...........................................................................Figure 28 : Mechanisms of Spindle Assembly Checkpoint silencing. .......................................................Figure 29 : grr1 mutations suppress the temperature-sensitivity of mps1-3 cells. .................................Figure 30 : grr1 mutations restore SAC signaling and proper chromosome segregation. ....................Figure 31 : Grr1 is degraded in the grr1 suppressors. .............................................................................Figure 32 : Grr1 localizes at kinetochores of wild type but not of mps1-3 cells. ....................................Figure 33 : Mps1 is present at similar levels in wild type and grr1-20 mutant cells. .............................Figure 34 : The Mps1-3 protein does not localize at kinetochores in the grr1-20 suppressor. .............Figure 35 : Ypi1 and Sds22 protein levels are not controlled by the SCF Grr1 complex. ........................Figure 36 : A second mutation in the mps1-3 allele suppresses the temperature-sensitivity of mps1-3 cells. ...............................................................................................................................................................Figure 37 : A second mutation in MPS1 leads to suppression of the chromosome segregation and SAC defects of the mps1-3 mutant. .....................................................................................................................Figure 38 : A 3HA tag at the C-terminus of Mps1 decreases suppressive effects of the intragenic suppressors. ..................................................................................................................................................Figure 39 : The intragenic suppressors restore Mps1 kinetochore localization. ...................................Figure 40 : The Mps1-sup proteins bear somewhat decreased kinase activity. .....................................Figure 41 : Recombinant Mps1-sup kinases might bear reduced kinase activity on Spc105 compared to Mps1-3. .........................................................................................................................................................Figure 42 : The intragenic suppressing mutations lower Mps1 kinase activity on Spc105. .................Figure 43 : Cells containing suppressing mutations proliferate normally, except for mps1-G449S. ...

Figure 2 :

 2 Figure 1 : Budding yeast mitosis. .................................................................................................................Figure 2 : A diagram of the main mitotic events in mammalian cells. .....................................................Figure 3 : The γ-tubulin small complex (γ-TuSC). .....................................................................................Figure 4 : The spindle pole body in budding yeast. ....................................................................................Figure 5 : SPB duplication. ...........................................................................................................................Figure 6 : The centrosome. ...........................................................................................................................Figure 7 : The centrosome cycle. ..................................................................................................................Figure 8 : The γ-tubulin template model. ....................................................................................................Figure 9 : Condensin architecture. ..............................................................................................................Figure 10 : Loop extrusion induced by a single condensin complex. ........................................................Figure 11 : Effects of condensin I and II on mitotic chromosome condensation. ....................................Figure 12 : Structure of the cohesin ring. ....................................................................................................Figure 13 : Models proposed for chromatin entrapment by cohesin. .......................................................Figure 14 : The cohesin cycle in human cells. .............................................................................................Figure 15 : Schematic kinetochore structure. .............................................................................................Figure 16 : The end-on conversion process. ................................................................................................Figure 17 : kinetochore orientation in mitosis and meiosis I. ....................................................................Figure 18 : Types of kinetochore-microtubule attachments. .....................................................................Figure 19 : SAC principles. ...........................................................................................................................Figure 20 : Domain organisation of human MPS1. ....................................................................................Figure 21 : Regulation of Mps1 during mitosis in humans. .......................................................................Figure 22 : Spindle Assembly Checkpoint activation. ................................................................................Figure 23 : The Mad2 template model. ........................................................................................................Figure 24 : The Chromosomal Passenger Complex and its recruitment to centromeres. ......................Figure 25 : The tension-related model. ........................................................................................................Figure 26 : The "dog leash" model for CPC function. ...............................................................................Figure 27 : Regulation of PP1 kinetochore recruitment. ...........................................................................Figure 28 : Mechanisms of Spindle Assembly Checkpoint silencing. .......................................................Figure 29 : grr1 mutations suppress the temperature-sensitivity of mps1-3 cells. .................................Figure 30 : grr1 mutations restore SAC signaling and proper chromosome segregation. ....................Figure 31 : Grr1 is degraded in the grr1 suppressors. .............................................................................Figure 32 : Grr1 localizes at kinetochores of wild type but not of mps1-3 cells. ....................................Figure 33 : Mps1 is present at similar levels in wild type and grr1-20 mutant cells. .............................Figure 34 : The Mps1-3 protein does not localize at kinetochores in the grr1-20 suppressor. .............Figure 35 : Ypi1 and Sds22 protein levels are not controlled by the SCF Grr1 complex. ........................Figure 36 : A second mutation in the mps1-3 allele suppresses the temperature-sensitivity of mps1-3 cells. ...............................................................................................................................................................Figure 37 : A second mutation in MPS1 leads to suppression of the chromosome segregation and SAC defects of the mps1-3 mutant. .....................................................................................................................Figure 38 : A 3HA tag at the C-terminus of Mps1 decreases suppressive effects of the intragenic suppressors. ..................................................................................................................................................Figure 39 : The intragenic suppressors restore Mps1 kinetochore localization. ...................................Figure 40 : The Mps1-sup proteins bear somewhat decreased kinase activity. .....................................Figure 41 : Recombinant Mps1-sup kinases might bear reduced kinase activity on Spc105 compared to Mps1-3. .........................................................................................................................................................Figure 42 : The intragenic suppressing mutations lower Mps1 kinase activity on Spc105. .................Figure 43 : Cells containing suppressing mutations proliferate normally, except for mps1-G449S. ...

Figure 3 :

 3 Figure 1 : Budding yeast mitosis. .................................................................................................................Figure 2 : A diagram of the main mitotic events in mammalian cells. .....................................................Figure 3 : The γ-tubulin small complex (γ-TuSC). .....................................................................................Figure 4 : The spindle pole body in budding yeast. ....................................................................................Figure 5 : SPB duplication. ...........................................................................................................................Figure 6 : The centrosome. ...........................................................................................................................Figure 7 : The centrosome cycle. ..................................................................................................................Figure 8 : The γ-tubulin template model. ....................................................................................................Figure 9 : Condensin architecture. ..............................................................................................................Figure 10 : Loop extrusion induced by a single condensin complex. ........................................................Figure 11 : Effects of condensin I and II on mitotic chromosome condensation. ....................................Figure 12 : Structure of the cohesin ring. ....................................................................................................Figure 13 : Models proposed for chromatin entrapment by cohesin. .......................................................Figure 14 : The cohesin cycle in human cells. .............................................................................................Figure 15 : Schematic kinetochore structure. .............................................................................................Figure 16 : The end-on conversion process. ................................................................................................Figure 17 : kinetochore orientation in mitosis and meiosis I. ....................................................................Figure 18 : Types of kinetochore-microtubule attachments. .....................................................................Figure 19 : SAC principles. ...........................................................................................................................Figure 20 : Domain organisation of human MPS1. ....................................................................................Figure 21 : Regulation of Mps1 during mitosis in humans. .......................................................................Figure 22 : Spindle Assembly Checkpoint activation. ................................................................................Figure 23 : The Mad2 template model. ........................................................................................................Figure 24 : The Chromosomal Passenger Complex and its recruitment to centromeres. ......................Figure 25 : The tension-related model. ........................................................................................................Figure 26 : The "dog leash" model for CPC function. ...............................................................................Figure 27 : Regulation of PP1 kinetochore recruitment. ...........................................................................Figure 28 : Mechanisms of Spindle Assembly Checkpoint silencing. .......................................................Figure 29 : grr1 mutations suppress the temperature-sensitivity of mps1-3 cells. .................................Figure 30 : grr1 mutations restore SAC signaling and proper chromosome segregation. ....................Figure 31 : Grr1 is degraded in the grr1 suppressors. .............................................................................Figure 32 : Grr1 localizes at kinetochores of wild type but not of mps1-3 cells. ....................................Figure 33 : Mps1 is present at similar levels in wild type and grr1-20 mutant cells. .............................Figure 34 : The Mps1-3 protein does not localize at kinetochores in the grr1-20 suppressor. .............Figure 35 : Ypi1 and Sds22 protein levels are not controlled by the SCF Grr1 complex. ........................Figure 36 : A second mutation in the mps1-3 allele suppresses the temperature-sensitivity of mps1-3 cells. ...............................................................................................................................................................Figure 37 : A second mutation in MPS1 leads to suppression of the chromosome segregation and SAC defects of the mps1-3 mutant. .....................................................................................................................Figure 38 : A 3HA tag at the C-terminus of Mps1 decreases suppressive effects of the intragenic suppressors. ..................................................................................................................................................Figure 39 : The intragenic suppressors restore Mps1 kinetochore localization. ...................................Figure 40 : The Mps1-sup proteins bear somewhat decreased kinase activity. .....................................Figure 41 : Recombinant Mps1-sup kinases might bear reduced kinase activity on Spc105 compared to Mps1-3. .........................................................................................................................................................Figure 42 : The intragenic suppressing mutations lower Mps1 kinase activity on Spc105. .................Figure 43 : Cells containing suppressing mutations proliferate normally, except for mps1-G449S. ...

Figure 4 :

 4 Figure 1 : Budding yeast mitosis. .................................................................................................................Figure 2 : A diagram of the main mitotic events in mammalian cells. .....................................................Figure 3 : The γ-tubulin small complex (γ-TuSC). .....................................................................................Figure 4 : The spindle pole body in budding yeast. ....................................................................................Figure 5 : SPB duplication. ...........................................................................................................................Figure 6 : The centrosome. ...........................................................................................................................Figure 7 : The centrosome cycle. ..................................................................................................................Figure 8 : The γ-tubulin template model. ....................................................................................................Figure 9 : Condensin architecture. ..............................................................................................................Figure 10 : Loop extrusion induced by a single condensin complex. ........................................................Figure 11 : Effects of condensin I and II on mitotic chromosome condensation. ....................................Figure 12 : Structure of the cohesin ring. ....................................................................................................Figure 13 : Models proposed for chromatin entrapment by cohesin. .......................................................Figure 14 : The cohesin cycle in human cells. .............................................................................................Figure 15 : Schematic kinetochore structure. .............................................................................................Figure 16 : The end-on conversion process. ................................................................................................Figure 17 : kinetochore orientation in mitosis and meiosis I. ....................................................................Figure 18 : Types of kinetochore-microtubule attachments. .....................................................................Figure 19 : SAC principles. ...........................................................................................................................Figure 20 : Domain organisation of human MPS1. ....................................................................................Figure 21 : Regulation of Mps1 during mitosis in humans. .......................................................................Figure 22 : Spindle Assembly Checkpoint activation. ................................................................................Figure 23 : The Mad2 template model. ........................................................................................................Figure 24 : The Chromosomal Passenger Complex and its recruitment to centromeres. ......................Figure 25 : The tension-related model. ........................................................................................................Figure 26 : The "dog leash" model for CPC function. ...............................................................................Figure 27 : Regulation of PP1 kinetochore recruitment. ...........................................................................Figure 28 : Mechanisms of Spindle Assembly Checkpoint silencing. .......................................................Figure 29 : grr1 mutations suppress the temperature-sensitivity of mps1-3 cells. .................................Figure 30 : grr1 mutations restore SAC signaling and proper chromosome segregation. ....................Figure 31 : Grr1 is degraded in the grr1 suppressors. .............................................................................Figure 32 : Grr1 localizes at kinetochores of wild type but not of mps1-3 cells. ....................................Figure 33 : Mps1 is present at similar levels in wild type and grr1-20 mutant cells. .............................Figure 34 : The Mps1-3 protein does not localize at kinetochores in the grr1-20 suppressor. .............Figure 35 : Ypi1 and Sds22 protein levels are not controlled by the SCF Grr1 complex. ........................Figure 36 : A second mutation in the mps1-3 allele suppresses the temperature-sensitivity of mps1-3 cells. ...............................................................................................................................................................Figure 37 : A second mutation in MPS1 leads to suppression of the chromosome segregation and SAC defects of the mps1-3 mutant. .....................................................................................................................Figure 38 : A 3HA tag at the C-terminus of Mps1 decreases suppressive effects of the intragenic suppressors. ..................................................................................................................................................Figure 39 : The intragenic suppressors restore Mps1 kinetochore localization. ...................................Figure 40 : The Mps1-sup proteins bear somewhat decreased kinase activity. .....................................Figure 41 : Recombinant Mps1-sup kinases might bear reduced kinase activity on Spc105 compared to Mps1-3. .........................................................................................................................................................Figure 42 : The intragenic suppressing mutations lower Mps1 kinase activity on Spc105. .................Figure 43 : Cells containing suppressing mutations proliferate normally, except for mps1-G449S. ...

Figure 5 :

 5 Figure 1 : Budding yeast mitosis. .................................................................................................................Figure 2 : A diagram of the main mitotic events in mammalian cells. .....................................................Figure 3 : The γ-tubulin small complex (γ-TuSC). .....................................................................................Figure 4 : The spindle pole body in budding yeast. ....................................................................................Figure 5 : SPB duplication. ...........................................................................................................................Figure 6 : The centrosome. ...........................................................................................................................Figure 7 : The centrosome cycle. ..................................................................................................................Figure 8 : The γ-tubulin template model. ....................................................................................................Figure 9 : Condensin architecture. ..............................................................................................................Figure 10 : Loop extrusion induced by a single condensin complex. ........................................................Figure 11 : Effects of condensin I and II on mitotic chromosome condensation. ....................................Figure 12 : Structure of the cohesin ring. ....................................................................................................Figure 13 : Models proposed for chromatin entrapment by cohesin. .......................................................Figure 14 : The cohesin cycle in human cells. .............................................................................................Figure 15 : Schematic kinetochore structure. .............................................................................................Figure 16 : The end-on conversion process. ................................................................................................Figure 17 : kinetochore orientation in mitosis and meiosis I. ....................................................................Figure 18 : Types of kinetochore-microtubule attachments. .....................................................................Figure 19 : SAC principles. ...........................................................................................................................Figure 20 : Domain organisation of human MPS1. ....................................................................................Figure 21 : Regulation of Mps1 during mitosis in humans. .......................................................................Figure 22 : Spindle Assembly Checkpoint activation. ................................................................................Figure 23 : The Mad2 template model. ........................................................................................................Figure 24 : The Chromosomal Passenger Complex and its recruitment to centromeres. ......................Figure 25 : The tension-related model. ........................................................................................................Figure 26 : The "dog leash" model for CPC function. ...............................................................................Figure 27 : Regulation of PP1 kinetochore recruitment. ...........................................................................Figure 28 : Mechanisms of Spindle Assembly Checkpoint silencing. .......................................................Figure 29 : grr1 mutations suppress the temperature-sensitivity of mps1-3 cells. .................................Figure 30 : grr1 mutations restore SAC signaling and proper chromosome segregation. ....................Figure 31 : Grr1 is degraded in the grr1 suppressors. .............................................................................Figure 32 : Grr1 localizes at kinetochores of wild type but not of mps1-3 cells. ....................................Figure 33 : Mps1 is present at similar levels in wild type and grr1-20 mutant cells. .............................Figure 34 : The Mps1-3 protein does not localize at kinetochores in the grr1-20 suppressor. .............Figure 35 : Ypi1 and Sds22 protein levels are not controlled by the SCF Grr1 complex. ........................Figure 36 : A second mutation in the mps1-3 allele suppresses the temperature-sensitivity of mps1-3 cells. ...............................................................................................................................................................Figure 37 : A second mutation in MPS1 leads to suppression of the chromosome segregation and SAC defects of the mps1-3 mutant. .....................................................................................................................Figure 38 : A 3HA tag at the C-terminus of Mps1 decreases suppressive effects of the intragenic suppressors. ..................................................................................................................................................Figure 39 : The intragenic suppressors restore Mps1 kinetochore localization. ...................................Figure 40 : The Mps1-sup proteins bear somewhat decreased kinase activity. .....................................Figure 41 : Recombinant Mps1-sup kinases might bear reduced kinase activity on Spc105 compared to Mps1-3. .........................................................................................................................................................Figure 42 : The intragenic suppressing mutations lower Mps1 kinase activity on Spc105. .................Figure 43 : Cells containing suppressing mutations proliferate normally, except for mps1-G449S. ...

Figure 6 :

 6 Figure 1 : Budding yeast mitosis. .................................................................................................................Figure 2 : A diagram of the main mitotic events in mammalian cells. .....................................................Figure 3 : The γ-tubulin small complex (γ-TuSC). .....................................................................................Figure 4 : The spindle pole body in budding yeast. ....................................................................................Figure 5 : SPB duplication. ...........................................................................................................................Figure 6 : The centrosome. ...........................................................................................................................Figure 7 : The centrosome cycle. ..................................................................................................................Figure 8 : The γ-tubulin template model. ....................................................................................................Figure 9 : Condensin architecture. ..............................................................................................................Figure 10 : Loop extrusion induced by a single condensin complex. ........................................................Figure 11 : Effects of condensin I and II on mitotic chromosome condensation. ....................................Figure 12 : Structure of the cohesin ring. ....................................................................................................Figure 13 : Models proposed for chromatin entrapment by cohesin. .......................................................Figure 14 : The cohesin cycle in human cells. .............................................................................................Figure 15 : Schematic kinetochore structure. .............................................................................................Figure 16 : The end-on conversion process. ................................................................................................Figure 17 : kinetochore orientation in mitosis and meiosis I. ....................................................................Figure 18 : Types of kinetochore-microtubule attachments. .....................................................................Figure 19 : SAC principles. ...........................................................................................................................Figure 20 : Domain organisation of human MPS1. ....................................................................................Figure 21 : Regulation of Mps1 during mitosis in humans. .......................................................................Figure 22 : Spindle Assembly Checkpoint activation. ................................................................................Figure 23 : The Mad2 template model. ........................................................................................................Figure 24 : The Chromosomal Passenger Complex and its recruitment to centromeres. ......................Figure 25 : The tension-related model. ........................................................................................................Figure 26 : The "dog leash" model for CPC function. ...............................................................................Figure 27 : Regulation of PP1 kinetochore recruitment. ...........................................................................Figure 28 : Mechanisms of Spindle Assembly Checkpoint silencing. .......................................................Figure 29 : grr1 mutations suppress the temperature-sensitivity of mps1-3 cells. .................................Figure 30 : grr1 mutations restore SAC signaling and proper chromosome segregation. ....................Figure 31 : Grr1 is degraded in the grr1 suppressors. .............................................................................Figure 32 : Grr1 localizes at kinetochores of wild type but not of mps1-3 cells. ....................................Figure 33 : Mps1 is present at similar levels in wild type and grr1-20 mutant cells. .............................Figure 34 : The Mps1-3 protein does not localize at kinetochores in the grr1-20 suppressor. .............Figure 35 : Ypi1 and Sds22 protein levels are not controlled by the SCF Grr1 complex. ........................Figure 36 : A second mutation in the mps1-3 allele suppresses the temperature-sensitivity of mps1-3 cells. ...............................................................................................................................................................Figure 37 : A second mutation in MPS1 leads to suppression of the chromosome segregation and SAC defects of the mps1-3 mutant. .....................................................................................................................Figure 38 : A 3HA tag at the C-terminus of Mps1 decreases suppressive effects of the intragenic suppressors. ..................................................................................................................................................Figure 39 : The intragenic suppressors restore Mps1 kinetochore localization. ...................................Figure 40 : The Mps1-sup proteins bear somewhat decreased kinase activity. .....................................Figure 41 : Recombinant Mps1-sup kinases might bear reduced kinase activity on Spc105 compared to Mps1-3. .........................................................................................................................................................Figure 42 : The intragenic suppressing mutations lower Mps1 kinase activity on Spc105. .................Figure 43 : Cells containing suppressing mutations proliferate normally, except for mps1-G449S. ...

Figure 7 :

 7 Figure 1 : Budding yeast mitosis. .................................................................................................................Figure 2 : A diagram of the main mitotic events in mammalian cells. .....................................................Figure 3 : The γ-tubulin small complex (γ-TuSC). .....................................................................................Figure 4 : The spindle pole body in budding yeast. ....................................................................................Figure 5 : SPB duplication. ...........................................................................................................................Figure 6 : The centrosome. ...........................................................................................................................Figure 7 : The centrosome cycle. ..................................................................................................................Figure 8 : The γ-tubulin template model. ....................................................................................................Figure 9 : Condensin architecture. ..............................................................................................................Figure 10 : Loop extrusion induced by a single condensin complex. ........................................................Figure 11 : Effects of condensin I and II on mitotic chromosome condensation. ....................................Figure 12 : Structure of the cohesin ring. ....................................................................................................Figure 13 : Models proposed for chromatin entrapment by cohesin. .......................................................Figure 14 : The cohesin cycle in human cells. .............................................................................................Figure 15 : Schematic kinetochore structure. .............................................................................................Figure 16 : The end-on conversion process. ................................................................................................Figure 17 : kinetochore orientation in mitosis and meiosis I. ....................................................................Figure 18 : Types of kinetochore-microtubule attachments. .....................................................................Figure 19 : SAC principles. ...........................................................................................................................Figure 20 : Domain organisation of human MPS1. ....................................................................................Figure 21 : Regulation of Mps1 during mitosis in humans. .......................................................................Figure 22 : Spindle Assembly Checkpoint activation. ................................................................................Figure 23 : The Mad2 template model. ........................................................................................................Figure 24 : The Chromosomal Passenger Complex and its recruitment to centromeres. ......................Figure 25 : The tension-related model. ........................................................................................................Figure 26 : The "dog leash" model for CPC function. ...............................................................................Figure 27 : Regulation of PP1 kinetochore recruitment. ...........................................................................Figure 28 : Mechanisms of Spindle Assembly Checkpoint silencing. .......................................................Figure 29 : grr1 mutations suppress the temperature-sensitivity of mps1-3 cells. .................................Figure 30 : grr1 mutations restore SAC signaling and proper chromosome segregation. ....................Figure 31 : Grr1 is degraded in the grr1 suppressors. .............................................................................Figure 32 : Grr1 localizes at kinetochores of wild type but not of mps1-3 cells. ....................................Figure 33 : Mps1 is present at similar levels in wild type and grr1-20 mutant cells. .............................Figure 34 : The Mps1-3 protein does not localize at kinetochores in the grr1-20 suppressor. .............Figure 35 : Ypi1 and Sds22 protein levels are not controlled by the SCF Grr1 complex. ........................Figure 36 : A second mutation in the mps1-3 allele suppresses the temperature-sensitivity of mps1-3 cells. ...............................................................................................................................................................Figure 37 : A second mutation in MPS1 leads to suppression of the chromosome segregation and SAC defects of the mps1-3 mutant. .....................................................................................................................Figure 38 : A 3HA tag at the C-terminus of Mps1 decreases suppressive effects of the intragenic suppressors. ..................................................................................................................................................Figure 39 : The intragenic suppressors restore Mps1 kinetochore localization. ...................................Figure 40 : The Mps1-sup proteins bear somewhat decreased kinase activity. .....................................Figure 41 : Recombinant Mps1-sup kinases might bear reduced kinase activity on Spc105 compared to Mps1-3. .........................................................................................................................................................Figure 42 : The intragenic suppressing mutations lower Mps1 kinase activity on Spc105. .................Figure 43 : Cells containing suppressing mutations proliferate normally, except for mps1-G449S. ...

Figure 8 :

 8 Figure 1 : Budding yeast mitosis. .................................................................................................................Figure 2 : A diagram of the main mitotic events in mammalian cells. .....................................................Figure 3 : The γ-tubulin small complex (γ-TuSC). .....................................................................................Figure 4 : The spindle pole body in budding yeast. ....................................................................................Figure 5 : SPB duplication. ...........................................................................................................................Figure 6 : The centrosome. ...........................................................................................................................Figure 7 : The centrosome cycle. ..................................................................................................................Figure 8 : The γ-tubulin template model. ....................................................................................................Figure 9 : Condensin architecture. ..............................................................................................................Figure 10 : Loop extrusion induced by a single condensin complex. ........................................................Figure 11 : Effects of condensin I and II on mitotic chromosome condensation. ....................................Figure 12 : Structure of the cohesin ring. ....................................................................................................Figure 13 : Models proposed for chromatin entrapment by cohesin. .......................................................Figure 14 : The cohesin cycle in human cells. .............................................................................................Figure 15 : Schematic kinetochore structure. .............................................................................................Figure 16 : The end-on conversion process. ................................................................................................Figure 17 : kinetochore orientation in mitosis and meiosis I. ....................................................................Figure 18 : Types of kinetochore-microtubule attachments. .....................................................................Figure 19 : SAC principles. ...........................................................................................................................Figure 20 : Domain organisation of human MPS1. ....................................................................................Figure 21 : Regulation of Mps1 during mitosis in humans. .......................................................................Figure 22 : Spindle Assembly Checkpoint activation. ................................................................................Figure 23 : The Mad2 template model. ........................................................................................................Figure 24 : The Chromosomal Passenger Complex and its recruitment to centromeres. ......................Figure 25 : The tension-related model. ........................................................................................................Figure 26 : The "dog leash" model for CPC function. ...............................................................................Figure 27 : Regulation of PP1 kinetochore recruitment. ...........................................................................Figure 28 : Mechanisms of Spindle Assembly Checkpoint silencing. .......................................................Figure 29 : grr1 mutations suppress the temperature-sensitivity of mps1-3 cells. .................................Figure 30 : grr1 mutations restore SAC signaling and proper chromosome segregation. ....................Figure 31 : Grr1 is degraded in the grr1 suppressors. .............................................................................Figure 32 : Grr1 localizes at kinetochores of wild type but not of mps1-3 cells. ....................................Figure 33 : Mps1 is present at similar levels in wild type and grr1-20 mutant cells. .............................Figure 34 : The Mps1-3 protein does not localize at kinetochores in the grr1-20 suppressor. .............Figure 35 : Ypi1 and Sds22 protein levels are not controlled by the SCF Grr1 complex. ........................Figure 36 : A second mutation in the mps1-3 allele suppresses the temperature-sensitivity of mps1-3 cells. ...............................................................................................................................................................Figure 37 : A second mutation in MPS1 leads to suppression of the chromosome segregation and SAC defects of the mps1-3 mutant. .....................................................................................................................Figure 38 : A 3HA tag at the C-terminus of Mps1 decreases suppressive effects of the intragenic suppressors. ..................................................................................................................................................Figure 39 : The intragenic suppressors restore Mps1 kinetochore localization. ...................................Figure 40 : The Mps1-sup proteins bear somewhat decreased kinase activity. .....................................Figure 41 : Recombinant Mps1-sup kinases might bear reduced kinase activity on Spc105 compared to Mps1-3. .........................................................................................................................................................Figure 42 : The intragenic suppressing mutations lower Mps1 kinase activity on Spc105. .................Figure 43 : Cells containing suppressing mutations proliferate normally, except for mps1-G449S. ...

Figure 9 :

 9 Figure 1 : Budding yeast mitosis. .................................................................................................................Figure 2 : A diagram of the main mitotic events in mammalian cells. .....................................................Figure 3 : The γ-tubulin small complex (γ-TuSC). .....................................................................................Figure 4 : The spindle pole body in budding yeast. ....................................................................................Figure 5 : SPB duplication. ...........................................................................................................................Figure 6 : The centrosome. ...........................................................................................................................Figure 7 : The centrosome cycle. ..................................................................................................................Figure 8 : The γ-tubulin template model. ....................................................................................................Figure 9 : Condensin architecture. ..............................................................................................................Figure 10 : Loop extrusion induced by a single condensin complex. ........................................................Figure 11 : Effects of condensin I and II on mitotic chromosome condensation. ....................................Figure 12 : Structure of the cohesin ring. ....................................................................................................Figure 13 : Models proposed for chromatin entrapment by cohesin. .......................................................Figure 14 : The cohesin cycle in human cells. .............................................................................................Figure 15 : Schematic kinetochore structure. .............................................................................................Figure 16 : The end-on conversion process. ................................................................................................Figure 17 : kinetochore orientation in mitosis and meiosis I. ....................................................................Figure 18 : Types of kinetochore-microtubule attachments. .....................................................................Figure 19 : SAC principles. ...........................................................................................................................Figure 20 : Domain organisation of human MPS1. ....................................................................................Figure 21 : Regulation of Mps1 during mitosis in humans. .......................................................................Figure 22 : Spindle Assembly Checkpoint activation. ................................................................................Figure 23 : The Mad2 template model. ........................................................................................................Figure 24 : The Chromosomal Passenger Complex and its recruitment to centromeres. ......................Figure 25 : The tension-related model. ........................................................................................................Figure 26 : The "dog leash" model for CPC function. ...............................................................................Figure 27 : Regulation of PP1 kinetochore recruitment. ...........................................................................Figure 28 : Mechanisms of Spindle Assembly Checkpoint silencing. .......................................................Figure 29 : grr1 mutations suppress the temperature-sensitivity of mps1-3 cells. .................................Figure 30 : grr1 mutations restore SAC signaling and proper chromosome segregation. ....................Figure 31 : Grr1 is degraded in the grr1 suppressors. .............................................................................Figure 32 : Grr1 localizes at kinetochores of wild type but not of mps1-3 cells. ....................................Figure 33 : Mps1 is present at similar levels in wild type and grr1-20 mutant cells. .............................Figure 34 : The Mps1-3 protein does not localize at kinetochores in the grr1-20 suppressor. .............Figure 35 : Ypi1 and Sds22 protein levels are not controlled by the SCF Grr1 complex. ........................Figure 36 : A second mutation in the mps1-3 allele suppresses the temperature-sensitivity of mps1-3 cells. ...............................................................................................................................................................Figure 37 : A second mutation in MPS1 leads to suppression of the chromosome segregation and SAC defects of the mps1-3 mutant. .....................................................................................................................Figure 38 : A 3HA tag at the C-terminus of Mps1 decreases suppressive effects of the intragenic suppressors. ..................................................................................................................................................Figure 39 : The intragenic suppressors restore Mps1 kinetochore localization. ...................................Figure 40 : The Mps1-sup proteins bear somewhat decreased kinase activity. .....................................Figure 41 : Recombinant Mps1-sup kinases might bear reduced kinase activity on Spc105 compared to Mps1-3. .........................................................................................................................................................Figure 42 : The intragenic suppressing mutations lower Mps1 kinase activity on Spc105. .................Figure 43 : Cells containing suppressing mutations proliferate normally, except for mps1-G449S. ...

Figure 10 :

 10 Figure 1 : Budding yeast mitosis. .................................................................................................................Figure 2 : A diagram of the main mitotic events in mammalian cells. .....................................................Figure 3 : The γ-tubulin small complex (γ-TuSC). .....................................................................................Figure 4 : The spindle pole body in budding yeast. ....................................................................................Figure 5 : SPB duplication. ...........................................................................................................................Figure 6 : The centrosome. ...........................................................................................................................Figure 7 : The centrosome cycle. ..................................................................................................................Figure 8 : The γ-tubulin template model. ....................................................................................................Figure 9 : Condensin architecture. ..............................................................................................................Figure 10 : Loop extrusion induced by a single condensin complex. ........................................................Figure 11 : Effects of condensin I and II on mitotic chromosome condensation. ....................................Figure 12 : Structure of the cohesin ring. ....................................................................................................Figure 13 : Models proposed for chromatin entrapment by cohesin. .......................................................Figure 14 : The cohesin cycle in human cells. .............................................................................................Figure 15 : Schematic kinetochore structure. .............................................................................................Figure 16 : The end-on conversion process. ................................................................................................Figure 17 : kinetochore orientation in mitosis and meiosis I. ....................................................................Figure 18 : Types of kinetochore-microtubule attachments. .....................................................................Figure 19 : SAC principles. ...........................................................................................................................Figure 20 : Domain organisation of human MPS1. ....................................................................................Figure 21 : Regulation of Mps1 during mitosis in humans. .......................................................................Figure 22 : Spindle Assembly Checkpoint activation. ................................................................................Figure 23 : The Mad2 template model. ........................................................................................................Figure 24 : The Chromosomal Passenger Complex and its recruitment to centromeres. ......................Figure 25 : The tension-related model. ........................................................................................................Figure 26 : The "dog leash" model for CPC function. ...............................................................................Figure 27 : Regulation of PP1 kinetochore recruitment. ...........................................................................Figure 28 : Mechanisms of Spindle Assembly Checkpoint silencing. .......................................................Figure 29 : grr1 mutations suppress the temperature-sensitivity of mps1-3 cells. .................................Figure 30 : grr1 mutations restore SAC signaling and proper chromosome segregation. ....................Figure 31 : Grr1 is degraded in the grr1 suppressors. .............................................................................Figure 32 : Grr1 localizes at kinetochores of wild type but not of mps1-3 cells. ....................................Figure 33 : Mps1 is present at similar levels in wild type and grr1-20 mutant cells. .............................Figure 34 : The Mps1-3 protein does not localize at kinetochores in the grr1-20 suppressor. .............Figure 35 : Ypi1 and Sds22 protein levels are not controlled by the SCF Grr1 complex. ........................Figure 36 : A second mutation in the mps1-3 allele suppresses the temperature-sensitivity of mps1-3 cells. ...............................................................................................................................................................Figure 37 : A second mutation in MPS1 leads to suppression of the chromosome segregation and SAC defects of the mps1-3 mutant. .....................................................................................................................Figure 38 : A 3HA tag at the C-terminus of Mps1 decreases suppressive effects of the intragenic suppressors. ..................................................................................................................................................Figure 39 : The intragenic suppressors restore Mps1 kinetochore localization. ...................................Figure 40 : The Mps1-sup proteins bear somewhat decreased kinase activity. .....................................Figure 41 : Recombinant Mps1-sup kinases might bear reduced kinase activity on Spc105 compared to Mps1-3. .........................................................................................................................................................Figure 42 : The intragenic suppressing mutations lower Mps1 kinase activity on Spc105. .................Figure 43 : Cells containing suppressing mutations proliferate normally, except for mps1-G449S. ...

Figure 11 :

 11 Figure 1 : Budding yeast mitosis. .................................................................................................................Figure 2 : A diagram of the main mitotic events in mammalian cells. .....................................................Figure 3 : The γ-tubulin small complex (γ-TuSC). .....................................................................................Figure 4 : The spindle pole body in budding yeast. ....................................................................................Figure 5 : SPB duplication. ...........................................................................................................................Figure 6 : The centrosome. ...........................................................................................................................Figure 7 : The centrosome cycle. ..................................................................................................................Figure 8 : The γ-tubulin template model. ....................................................................................................Figure 9 : Condensin architecture. ..............................................................................................................Figure 10 : Loop extrusion induced by a single condensin complex. ........................................................Figure 11 : Effects of condensin I and II on mitotic chromosome condensation. ....................................Figure 12 : Structure of the cohesin ring. ....................................................................................................Figure 13 : Models proposed for chromatin entrapment by cohesin. .......................................................Figure 14 : The cohesin cycle in human cells. .............................................................................................Figure 15 : Schematic kinetochore structure. .............................................................................................Figure 16 : The end-on conversion process. ................................................................................................Figure 17 : kinetochore orientation in mitosis and meiosis I. ....................................................................Figure 18 : Types of kinetochore-microtubule attachments. .....................................................................Figure 19 : SAC principles. ...........................................................................................................................Figure 20 : Domain organisation of human MPS1. ....................................................................................Figure 21 : Regulation of Mps1 during mitosis in humans. .......................................................................Figure 22 : Spindle Assembly Checkpoint activation. ................................................................................Figure 23 : The Mad2 template model. ........................................................................................................Figure 24 : The Chromosomal Passenger Complex and its recruitment to centromeres. ......................Figure 25 : The tension-related model. ........................................................................................................Figure 26 : The "dog leash" model for CPC function. ...............................................................................Figure 27 : Regulation of PP1 kinetochore recruitment. ...........................................................................Figure 28 : Mechanisms of Spindle Assembly Checkpoint silencing. .......................................................Figure 29 : grr1 mutations suppress the temperature-sensitivity of mps1-3 cells. .................................Figure 30 : grr1 mutations restore SAC signaling and proper chromosome segregation. ....................Figure 31 : Grr1 is degraded in the grr1 suppressors. .............................................................................Figure 32 : Grr1 localizes at kinetochores of wild type but not of mps1-3 cells. ....................................Figure 33 : Mps1 is present at similar levels in wild type and grr1-20 mutant cells. .............................Figure 34 : The Mps1-3 protein does not localize at kinetochores in the grr1-20 suppressor. .............Figure 35 : Ypi1 and Sds22 protein levels are not controlled by the SCF Grr1 complex. ........................Figure 36 : A second mutation in the mps1-3 allele suppresses the temperature-sensitivity of mps1-3 cells. ...............................................................................................................................................................Figure 37 : A second mutation in MPS1 leads to suppression of the chromosome segregation and SAC defects of the mps1-3 mutant. .....................................................................................................................Figure 38 : A 3HA tag at the C-terminus of Mps1 decreases suppressive effects of the intragenic suppressors. ..................................................................................................................................................Figure 39 : The intragenic suppressors restore Mps1 kinetochore localization. ...................................Figure 40 : The Mps1-sup proteins bear somewhat decreased kinase activity. .....................................Figure 41 : Recombinant Mps1-sup kinases might bear reduced kinase activity on Spc105 compared to Mps1-3. .........................................................................................................................................................Figure 42 : The intragenic suppressing mutations lower Mps1 kinase activity on Spc105. .................Figure 43 : Cells containing suppressing mutations proliferate normally, except for mps1-G449S. ...

Figure 12 :

 12 Figure 1 : Budding yeast mitosis. .................................................................................................................Figure 2 : A diagram of the main mitotic events in mammalian cells. .....................................................Figure 3 : The γ-tubulin small complex (γ-TuSC). .....................................................................................Figure 4 : The spindle pole body in budding yeast. ....................................................................................Figure 5 : SPB duplication. ...........................................................................................................................Figure 6 : The centrosome. ...........................................................................................................................Figure 7 : The centrosome cycle. ..................................................................................................................Figure 8 : The γ-tubulin template model. ....................................................................................................Figure 9 : Condensin architecture. ..............................................................................................................Figure 10 : Loop extrusion induced by a single condensin complex. ........................................................Figure 11 : Effects of condensin I and II on mitotic chromosome condensation. ....................................Figure 12 : Structure of the cohesin ring. ....................................................................................................Figure 13 : Models proposed for chromatin entrapment by cohesin. .......................................................Figure 14 : The cohesin cycle in human cells. .............................................................................................Figure 15 : Schematic kinetochore structure. .............................................................................................Figure 16 : The end-on conversion process. ................................................................................................Figure 17 : kinetochore orientation in mitosis and meiosis I. ....................................................................Figure 18 : Types of kinetochore-microtubule attachments. .....................................................................Figure 19 : SAC principles. ...........................................................................................................................Figure 20 : Domain organisation of human MPS1. ....................................................................................Figure 21 : Regulation of Mps1 during mitosis in humans. .......................................................................Figure 22 : Spindle Assembly Checkpoint activation. ................................................................................Figure 23 : The Mad2 template model. ........................................................................................................Figure 24 : The Chromosomal Passenger Complex and its recruitment to centromeres. ......................Figure 25 : The tension-related model. ........................................................................................................Figure 26 : The "dog leash" model for CPC function. ...............................................................................Figure 27 : Regulation of PP1 kinetochore recruitment. ...........................................................................Figure 28 : Mechanisms of Spindle Assembly Checkpoint silencing. .......................................................Figure 29 : grr1 mutations suppress the temperature-sensitivity of mps1-3 cells. .................................Figure 30 : grr1 mutations restore SAC signaling and proper chromosome segregation. ....................Figure 31 : Grr1 is degraded in the grr1 suppressors. .............................................................................Figure 32 : Grr1 localizes at kinetochores of wild type but not of mps1-3 cells. ....................................Figure 33 : Mps1 is present at similar levels in wild type and grr1-20 mutant cells. .............................Figure 34 : The Mps1-3 protein does not localize at kinetochores in the grr1-20 suppressor. .............Figure 35 : Ypi1 and Sds22 protein levels are not controlled by the SCF Grr1 complex. ........................Figure 36 : A second mutation in the mps1-3 allele suppresses the temperature-sensitivity of mps1-3 cells. ...............................................................................................................................................................Figure 37 : A second mutation in MPS1 leads to suppression of the chromosome segregation and SAC defects of the mps1-3 mutant. .....................................................................................................................Figure 38 : A 3HA tag at the C-terminus of Mps1 decreases suppressive effects of the intragenic suppressors. ..................................................................................................................................................Figure 39 : The intragenic suppressors restore Mps1 kinetochore localization. ...................................Figure 40 : The Mps1-sup proteins bear somewhat decreased kinase activity. .....................................Figure 41 : Recombinant Mps1-sup kinases might bear reduced kinase activity on Spc105 compared to Mps1-3. .........................................................................................................................................................Figure 42 : The intragenic suppressing mutations lower Mps1 kinase activity on Spc105. .................Figure 43 : Cells containing suppressing mutations proliferate normally, except for mps1-G449S. ...

Figure 13 :

 13 Figure 1 : Budding yeast mitosis. .................................................................................................................Figure 2 : A diagram of the main mitotic events in mammalian cells. .....................................................Figure 3 : The γ-tubulin small complex (γ-TuSC). .....................................................................................Figure 4 : The spindle pole body in budding yeast. ....................................................................................Figure 5 : SPB duplication. ...........................................................................................................................Figure 6 : The centrosome. ...........................................................................................................................Figure 7 : The centrosome cycle. ..................................................................................................................Figure 8 : The γ-tubulin template model. ....................................................................................................Figure 9 : Condensin architecture. ..............................................................................................................Figure 10 : Loop extrusion induced by a single condensin complex. ........................................................Figure 11 : Effects of condensin I and II on mitotic chromosome condensation. ....................................Figure 12 : Structure of the cohesin ring. ....................................................................................................Figure 13 : Models proposed for chromatin entrapment by cohesin. .......................................................Figure 14 : The cohesin cycle in human cells. .............................................................................................Figure 15 : Schematic kinetochore structure. .............................................................................................Figure 16 : The end-on conversion process. ................................................................................................Figure 17 : kinetochore orientation in mitosis and meiosis I. ....................................................................Figure 18 : Types of kinetochore-microtubule attachments. .....................................................................Figure 19 : SAC principles. ...........................................................................................................................Figure 20 : Domain organisation of human MPS1. ....................................................................................Figure 21 : Regulation of Mps1 during mitosis in humans. .......................................................................Figure 22 : Spindle Assembly Checkpoint activation. ................................................................................Figure 23 : The Mad2 template model. ........................................................................................................Figure 24 : The Chromosomal Passenger Complex and its recruitment to centromeres. ......................Figure 25 : The tension-related model. ........................................................................................................Figure 26 : The "dog leash" model for CPC function. ...............................................................................Figure 27 : Regulation of PP1 kinetochore recruitment. ...........................................................................Figure 28 : Mechanisms of Spindle Assembly Checkpoint silencing. .......................................................Figure 29 : grr1 mutations suppress the temperature-sensitivity of mps1-3 cells. .................................Figure 30 : grr1 mutations restore SAC signaling and proper chromosome segregation. ....................Figure 31 : Grr1 is degraded in the grr1 suppressors. .............................................................................Figure 32 : Grr1 localizes at kinetochores of wild type but not of mps1-3 cells. ....................................Figure 33 : Mps1 is present at similar levels in wild type and grr1-20 mutant cells. .............................Figure 34 : The Mps1-3 protein does not localize at kinetochores in the grr1-20 suppressor. .............Figure 35 : Ypi1 and Sds22 protein levels are not controlled by the SCF Grr1 complex. ........................Figure 36 : A second mutation in the mps1-3 allele suppresses the temperature-sensitivity of mps1-3 cells. ...............................................................................................................................................................Figure 37 : A second mutation in MPS1 leads to suppression of the chromosome segregation and SAC defects of the mps1-3 mutant. .....................................................................................................................Figure 38 : A 3HA tag at the C-terminus of Mps1 decreases suppressive effects of the intragenic suppressors. ..................................................................................................................................................Figure 39 : The intragenic suppressors restore Mps1 kinetochore localization. ...................................Figure 40 : The Mps1-sup proteins bear somewhat decreased kinase activity. .....................................Figure 41 : Recombinant Mps1-sup kinases might bear reduced kinase activity on Spc105 compared to Mps1-3. .........................................................................................................................................................Figure 42 : The intragenic suppressing mutations lower Mps1 kinase activity on Spc105. .................Figure 43 : Cells containing suppressing mutations proliferate normally, except for mps1-G449S. ...

Figure 14 :

 14 Figure 1 : Budding yeast mitosis. .................................................................................................................Figure 2 : A diagram of the main mitotic events in mammalian cells. .....................................................Figure 3 : The γ-tubulin small complex (γ-TuSC). .....................................................................................Figure 4 : The spindle pole body in budding yeast. ....................................................................................Figure 5 : SPB duplication. ...........................................................................................................................Figure 6 : The centrosome. ...........................................................................................................................Figure 7 : The centrosome cycle. ..................................................................................................................Figure 8 : The γ-tubulin template model. ....................................................................................................Figure 9 : Condensin architecture. ..............................................................................................................Figure 10 : Loop extrusion induced by a single condensin complex. ........................................................Figure 11 : Effects of condensin I and II on mitotic chromosome condensation. ....................................Figure 12 : Structure of the cohesin ring. ....................................................................................................Figure 13 : Models proposed for chromatin entrapment by cohesin. .......................................................Figure 14 : The cohesin cycle in human cells. .............................................................................................Figure 15 : Schematic kinetochore structure. .............................................................................................Figure 16 : The end-on conversion process. ................................................................................................Figure 17 : kinetochore orientation in mitosis and meiosis I. ....................................................................Figure 18 : Types of kinetochore-microtubule attachments. .....................................................................Figure 19 : SAC principles. ...........................................................................................................................Figure 20 : Domain organisation of human MPS1. ....................................................................................Figure 21 : Regulation of Mps1 during mitosis in humans. .......................................................................Figure 22 : Spindle Assembly Checkpoint activation. ................................................................................Figure 23 : The Mad2 template model. ........................................................................................................Figure 24 : The Chromosomal Passenger Complex and its recruitment to centromeres. ......................Figure 25 : The tension-related model. ........................................................................................................Figure 26 : The "dog leash" model for CPC function. ...............................................................................Figure 27 : Regulation of PP1 kinetochore recruitment. ...........................................................................Figure 28 : Mechanisms of Spindle Assembly Checkpoint silencing. .......................................................Figure 29 : grr1 mutations suppress the temperature-sensitivity of mps1-3 cells. .................................Figure 30 : grr1 mutations restore SAC signaling and proper chromosome segregation. ....................Figure 31 : Grr1 is degraded in the grr1 suppressors. .............................................................................Figure 32 : Grr1 localizes at kinetochores of wild type but not of mps1-3 cells. ....................................Figure 33 : Mps1 is present at similar levels in wild type and grr1-20 mutant cells. .............................Figure 34 : The Mps1-3 protein does not localize at kinetochores in the grr1-20 suppressor. .............Figure 35 : Ypi1 and Sds22 protein levels are not controlled by the SCF Grr1 complex. ........................Figure 36 : A second mutation in the mps1-3 allele suppresses the temperature-sensitivity of mps1-3 cells. ...............................................................................................................................................................Figure 37 : A second mutation in MPS1 leads to suppression of the chromosome segregation and SAC defects of the mps1-3 mutant. .....................................................................................................................Figure 38 : A 3HA tag at the C-terminus of Mps1 decreases suppressive effects of the intragenic suppressors. ..................................................................................................................................................Figure 39 : The intragenic suppressors restore Mps1 kinetochore localization. ...................................Figure 40 : The Mps1-sup proteins bear somewhat decreased kinase activity. .....................................Figure 41 : Recombinant Mps1-sup kinases might bear reduced kinase activity on Spc105 compared to Mps1-3. .........................................................................................................................................................Figure 42 : The intragenic suppressing mutations lower Mps1 kinase activity on Spc105. .................Figure 43 : Cells containing suppressing mutations proliferate normally, except for mps1-G449S. ...

Figure 15 :

 15 Figure 1 : Budding yeast mitosis. .................................................................................................................Figure 2 : A diagram of the main mitotic events in mammalian cells. .....................................................Figure 3 : The γ-tubulin small complex (γ-TuSC). .....................................................................................Figure 4 : The spindle pole body in budding yeast. ....................................................................................Figure 5 : SPB duplication. ...........................................................................................................................Figure 6 : The centrosome. ...........................................................................................................................Figure 7 : The centrosome cycle. ..................................................................................................................Figure 8 : The γ-tubulin template model. ....................................................................................................Figure 9 : Condensin architecture. ..............................................................................................................Figure 10 : Loop extrusion induced by a single condensin complex. ........................................................Figure 11 : Effects of condensin I and II on mitotic chromosome condensation. ....................................Figure 12 : Structure of the cohesin ring. ....................................................................................................Figure 13 : Models proposed for chromatin entrapment by cohesin. .......................................................Figure 14 : The cohesin cycle in human cells. .............................................................................................Figure 15 : Schematic kinetochore structure. .............................................................................................Figure 16 : The end-on conversion process. ................................................................................................Figure 17 : kinetochore orientation in mitosis and meiosis I. ....................................................................Figure 18 : Types of kinetochore-microtubule attachments. .....................................................................Figure 19 : SAC principles. ...........................................................................................................................Figure 20 : Domain organisation of human MPS1. ....................................................................................Figure 21 : Regulation of Mps1 during mitosis in humans. .......................................................................Figure 22 : Spindle Assembly Checkpoint activation. ................................................................................Figure 23 : The Mad2 template model. ........................................................................................................Figure 24 : The Chromosomal Passenger Complex and its recruitment to centromeres. ......................Figure 25 : The tension-related model. ........................................................................................................Figure 26 : The "dog leash" model for CPC function. ...............................................................................Figure 27 : Regulation of PP1 kinetochore recruitment. ...........................................................................Figure 28 : Mechanisms of Spindle Assembly Checkpoint silencing. .......................................................Figure 29 : grr1 mutations suppress the temperature-sensitivity of mps1-3 cells. .................................Figure 30 : grr1 mutations restore SAC signaling and proper chromosome segregation. ....................Figure 31 : Grr1 is degraded in the grr1 suppressors. .............................................................................Figure 32 : Grr1 localizes at kinetochores of wild type but not of mps1-3 cells. ....................................Figure 33 : Mps1 is present at similar levels in wild type and grr1-20 mutant cells. .............................Figure 34 : The Mps1-3 protein does not localize at kinetochores in the grr1-20 suppressor. .............Figure 35 : Ypi1 and Sds22 protein levels are not controlled by the SCF Grr1 complex. ........................Figure 36 : A second mutation in the mps1-3 allele suppresses the temperature-sensitivity of mps1-3 cells. ...............................................................................................................................................................Figure 37 : A second mutation in MPS1 leads to suppression of the chromosome segregation and SAC defects of the mps1-3 mutant. .....................................................................................................................Figure 38 : A 3HA tag at the C-terminus of Mps1 decreases suppressive effects of the intragenic suppressors. ..................................................................................................................................................Figure 39 : The intragenic suppressors restore Mps1 kinetochore localization. ...................................Figure 40 : The Mps1-sup proteins bear somewhat decreased kinase activity. .....................................Figure 41 : Recombinant Mps1-sup kinases might bear reduced kinase activity on Spc105 compared to Mps1-3. .........................................................................................................................................................Figure 42 : The intragenic suppressing mutations lower Mps1 kinase activity on Spc105. .................Figure 43 : Cells containing suppressing mutations proliferate normally, except for mps1-G449S. ...

Figure 16 :

 16 Figure 1 : Budding yeast mitosis. .................................................................................................................Figure 2 : A diagram of the main mitotic events in mammalian cells. .....................................................Figure 3 : The γ-tubulin small complex (γ-TuSC). .....................................................................................Figure 4 : The spindle pole body in budding yeast. ....................................................................................Figure 5 : SPB duplication. ...........................................................................................................................Figure 6 : The centrosome. ...........................................................................................................................Figure 7 : The centrosome cycle. ..................................................................................................................Figure 8 : The γ-tubulin template model. ....................................................................................................Figure 9 : Condensin architecture. ..............................................................................................................Figure 10 : Loop extrusion induced by a single condensin complex. ........................................................Figure 11 : Effects of condensin I and II on mitotic chromosome condensation. ....................................Figure 12 : Structure of the cohesin ring. ....................................................................................................Figure 13 : Models proposed for chromatin entrapment by cohesin. .......................................................Figure 14 : The cohesin cycle in human cells. .............................................................................................Figure 15 : Schematic kinetochore structure. .............................................................................................Figure 16 : The end-on conversion process. ................................................................................................Figure 17 : kinetochore orientation in mitosis and meiosis I. ....................................................................Figure 18 : Types of kinetochore-microtubule attachments. .....................................................................Figure 19 : SAC principles. ...........................................................................................................................Figure 20 : Domain organisation of human MPS1. ....................................................................................Figure 21 : Regulation of Mps1 during mitosis in humans. .......................................................................Figure 22 : Spindle Assembly Checkpoint activation. ................................................................................Figure 23 : The Mad2 template model. ........................................................................................................Figure 24 : The Chromosomal Passenger Complex and its recruitment to centromeres. ......................Figure 25 : The tension-related model. ........................................................................................................Figure 26 : The "dog leash" model for CPC function. ...............................................................................Figure 27 : Regulation of PP1 kinetochore recruitment. ...........................................................................Figure 28 : Mechanisms of Spindle Assembly Checkpoint silencing. .......................................................Figure 29 : grr1 mutations suppress the temperature-sensitivity of mps1-3 cells. .................................Figure 30 : grr1 mutations restore SAC signaling and proper chromosome segregation. ....................Figure 31 : Grr1 is degraded in the grr1 suppressors. .............................................................................Figure 32 : Grr1 localizes at kinetochores of wild type but not of mps1-3 cells. ....................................Figure 33 : Mps1 is present at similar levels in wild type and grr1-20 mutant cells. .............................Figure 34 : The Mps1-3 protein does not localize at kinetochores in the grr1-20 suppressor. .............Figure 35 : Ypi1 and Sds22 protein levels are not controlled by the SCF Grr1 complex. ........................Figure 36 : A second mutation in the mps1-3 allele suppresses the temperature-sensitivity of mps1-3 cells. ...............................................................................................................................................................Figure 37 : A second mutation in MPS1 leads to suppression of the chromosome segregation and SAC defects of the mps1-3 mutant. .....................................................................................................................Figure 38 : A 3HA tag at the C-terminus of Mps1 decreases suppressive effects of the intragenic suppressors. ..................................................................................................................................................Figure 39 : The intragenic suppressors restore Mps1 kinetochore localization. ...................................Figure 40 : The Mps1-sup proteins bear somewhat decreased kinase activity. .....................................Figure 41 : Recombinant Mps1-sup kinases might bear reduced kinase activity on Spc105 compared to Mps1-3. .........................................................................................................................................................Figure 42 : The intragenic suppressing mutations lower Mps1 kinase activity on Spc105. .................Figure 43 : Cells containing suppressing mutations proliferate normally, except for mps1-G449S. ...

Figure 17 :

 17 Figure 1 : Budding yeast mitosis. .................................................................................................................Figure 2 : A diagram of the main mitotic events in mammalian cells. .....................................................Figure 3 : The γ-tubulin small complex (γ-TuSC). .....................................................................................Figure 4 : The spindle pole body in budding yeast. ....................................................................................Figure 5 : SPB duplication. ...........................................................................................................................Figure 6 : The centrosome. ...........................................................................................................................Figure 7 : The centrosome cycle. ..................................................................................................................Figure 8 : The γ-tubulin template model. ....................................................................................................Figure 9 : Condensin architecture. ..............................................................................................................Figure 10 : Loop extrusion induced by a single condensin complex. ........................................................Figure 11 : Effects of condensin I and II on mitotic chromosome condensation. ....................................Figure 12 : Structure of the cohesin ring. ....................................................................................................Figure 13 : Models proposed for chromatin entrapment by cohesin. .......................................................Figure 14 : The cohesin cycle in human cells. .............................................................................................Figure 15 : Schematic kinetochore structure. .............................................................................................Figure 16 : The end-on conversion process. ................................................................................................Figure 17 : kinetochore orientation in mitosis and meiosis I. ....................................................................Figure 18 : Types of kinetochore-microtubule attachments. .....................................................................Figure 19 : SAC principles. ...........................................................................................................................Figure 20 : Domain organisation of human MPS1. ....................................................................................Figure 21 : Regulation of Mps1 during mitosis in humans. .......................................................................Figure 22 : Spindle Assembly Checkpoint activation. ................................................................................Figure 23 : The Mad2 template model. ........................................................................................................Figure 24 : The Chromosomal Passenger Complex and its recruitment to centromeres. ......................Figure 25 : The tension-related model. ........................................................................................................Figure 26 : The "dog leash" model for CPC function. ...............................................................................Figure 27 : Regulation of PP1 kinetochore recruitment. ...........................................................................Figure 28 : Mechanisms of Spindle Assembly Checkpoint silencing. .......................................................Figure 29 : grr1 mutations suppress the temperature-sensitivity of mps1-3 cells. .................................Figure 30 : grr1 mutations restore SAC signaling and proper chromosome segregation. ....................Figure 31 : Grr1 is degraded in the grr1 suppressors. .............................................................................Figure 32 : Grr1 localizes at kinetochores of wild type but not of mps1-3 cells. ....................................Figure 33 : Mps1 is present at similar levels in wild type and grr1-20 mutant cells. .............................Figure 34 : The Mps1-3 protein does not localize at kinetochores in the grr1-20 suppressor. .............Figure 35 : Ypi1 and Sds22 protein levels are not controlled by the SCF Grr1 complex. ........................Figure 36 : A second mutation in the mps1-3 allele suppresses the temperature-sensitivity of mps1-3 cells. ...............................................................................................................................................................Figure 37 : A second mutation in MPS1 leads to suppression of the chromosome segregation and SAC defects of the mps1-3 mutant. .....................................................................................................................Figure 38 : A 3HA tag at the C-terminus of Mps1 decreases suppressive effects of the intragenic suppressors. ..................................................................................................................................................Figure 39 : The intragenic suppressors restore Mps1 kinetochore localization. ...................................Figure 40 : The Mps1-sup proteins bear somewhat decreased kinase activity. .....................................Figure 41 : Recombinant Mps1-sup kinases might bear reduced kinase activity on Spc105 compared to Mps1-3. .........................................................................................................................................................Figure 42 : The intragenic suppressing mutations lower Mps1 kinase activity on Spc105. .................Figure 43 : Cells containing suppressing mutations proliferate normally, except for mps1-G449S. ...

Figure 18 :

 18 Figure 1 : Budding yeast mitosis. .................................................................................................................Figure 2 : A diagram of the main mitotic events in mammalian cells. .....................................................Figure 3 : The γ-tubulin small complex (γ-TuSC). .....................................................................................Figure 4 : The spindle pole body in budding yeast. ....................................................................................Figure 5 : SPB duplication. ...........................................................................................................................Figure 6 : The centrosome. ...........................................................................................................................Figure 7 : The centrosome cycle. ..................................................................................................................Figure 8 : The γ-tubulin template model. ....................................................................................................Figure 9 : Condensin architecture. ..............................................................................................................Figure 10 : Loop extrusion induced by a single condensin complex. ........................................................Figure 11 : Effects of condensin I and II on mitotic chromosome condensation. ....................................Figure 12 : Structure of the cohesin ring. ....................................................................................................Figure 13 : Models proposed for chromatin entrapment by cohesin. .......................................................Figure 14 : The cohesin cycle in human cells. .............................................................................................Figure 15 : Schematic kinetochore structure. .............................................................................................Figure 16 : The end-on conversion process. ................................................................................................Figure 17 : kinetochore orientation in mitosis and meiosis I. ....................................................................Figure 18 : Types of kinetochore-microtubule attachments. .....................................................................Figure 19 : SAC principles. ...........................................................................................................................Figure 20 : Domain organisation of human MPS1. ....................................................................................Figure 21 : Regulation of Mps1 during mitosis in humans. .......................................................................Figure 22 : Spindle Assembly Checkpoint activation. ................................................................................Figure 23 : The Mad2 template model. ........................................................................................................Figure 24 : The Chromosomal Passenger Complex and its recruitment to centromeres. ......................Figure 25 : The tension-related model. ........................................................................................................Figure 26 : The "dog leash" model for CPC function. ...............................................................................Figure 27 : Regulation of PP1 kinetochore recruitment. ...........................................................................Figure 28 : Mechanisms of Spindle Assembly Checkpoint silencing. .......................................................Figure 29 : grr1 mutations suppress the temperature-sensitivity of mps1-3 cells. .................................Figure 30 : grr1 mutations restore SAC signaling and proper chromosome segregation. ....................Figure 31 : Grr1 is degraded in the grr1 suppressors. .............................................................................Figure 32 : Grr1 localizes at kinetochores of wild type but not of mps1-3 cells. ....................................Figure 33 : Mps1 is present at similar levels in wild type and grr1-20 mutant cells. .............................Figure 34 : The Mps1-3 protein does not localize at kinetochores in the grr1-20 suppressor. .............Figure 35 : Ypi1 and Sds22 protein levels are not controlled by the SCF Grr1 complex. ........................Figure 36 : A second mutation in the mps1-3 allele suppresses the temperature-sensitivity of mps1-3 cells. ...............................................................................................................................................................Figure 37 : A second mutation in MPS1 leads to suppression of the chromosome segregation and SAC defects of the mps1-3 mutant. .....................................................................................................................Figure 38 : A 3HA tag at the C-terminus of Mps1 decreases suppressive effects of the intragenic suppressors. ..................................................................................................................................................Figure 39 : The intragenic suppressors restore Mps1 kinetochore localization. ...................................Figure 40 : The Mps1-sup proteins bear somewhat decreased kinase activity. .....................................Figure 41 : Recombinant Mps1-sup kinases might bear reduced kinase activity on Spc105 compared to Mps1-3. .........................................................................................................................................................Figure 42 : The intragenic suppressing mutations lower Mps1 kinase activity on Spc105. .................Figure 43 : Cells containing suppressing mutations proliferate normally, except for mps1-G449S. ...

Figure 19 :

 19 Figure 1 : Budding yeast mitosis. .................................................................................................................Figure 2 : A diagram of the main mitotic events in mammalian cells. .....................................................Figure 3 : The γ-tubulin small complex (γ-TuSC). .....................................................................................Figure 4 : The spindle pole body in budding yeast. ....................................................................................Figure 5 : SPB duplication. ...........................................................................................................................Figure 6 : The centrosome. ...........................................................................................................................Figure 7 : The centrosome cycle. ..................................................................................................................Figure 8 : The γ-tubulin template model. ....................................................................................................Figure 9 : Condensin architecture. ..............................................................................................................Figure 10 : Loop extrusion induced by a single condensin complex. ........................................................Figure 11 : Effects of condensin I and II on mitotic chromosome condensation. ....................................Figure 12 : Structure of the cohesin ring. ....................................................................................................Figure 13 : Models proposed for chromatin entrapment by cohesin. .......................................................Figure 14 : The cohesin cycle in human cells. .............................................................................................Figure 15 : Schematic kinetochore structure. .............................................................................................Figure 16 : The end-on conversion process. ................................................................................................Figure 17 : kinetochore orientation in mitosis and meiosis I. ....................................................................Figure 18 : Types of kinetochore-microtubule attachments. .....................................................................Figure 19 : SAC principles. ...........................................................................................................................Figure 20 : Domain organisation of human MPS1. ....................................................................................Figure 21 : Regulation of Mps1 during mitosis in humans. .......................................................................Figure 22 : Spindle Assembly Checkpoint activation. ................................................................................Figure 23 : The Mad2 template model. ........................................................................................................Figure 24 : The Chromosomal Passenger Complex and its recruitment to centromeres. ......................Figure 25 : The tension-related model. ........................................................................................................Figure 26 : The "dog leash" model for CPC function. ...............................................................................Figure 27 : Regulation of PP1 kinetochore recruitment. ...........................................................................Figure 28 : Mechanisms of Spindle Assembly Checkpoint silencing. .......................................................Figure 29 : grr1 mutations suppress the temperature-sensitivity of mps1-3 cells. .................................Figure 30 : grr1 mutations restore SAC signaling and proper chromosome segregation. ....................Figure 31 : Grr1 is degraded in the grr1 suppressors. .............................................................................Figure 32 : Grr1 localizes at kinetochores of wild type but not of mps1-3 cells. ....................................Figure 33 : Mps1 is present at similar levels in wild type and grr1-20 mutant cells. .............................Figure 34 : The Mps1-3 protein does not localize at kinetochores in the grr1-20 suppressor. .............Figure 35 : Ypi1 and Sds22 protein levels are not controlled by the SCF Grr1 complex. ........................Figure 36 : A second mutation in the mps1-3 allele suppresses the temperature-sensitivity of mps1-3 cells. ...............................................................................................................................................................Figure 37 : A second mutation in MPS1 leads to suppression of the chromosome segregation and SAC defects of the mps1-3 mutant. .....................................................................................................................Figure 38 : A 3HA tag at the C-terminus of Mps1 decreases suppressive effects of the intragenic suppressors. ..................................................................................................................................................Figure 39 : The intragenic suppressors restore Mps1 kinetochore localization. ...................................Figure 40 : The Mps1-sup proteins bear somewhat decreased kinase activity. .....................................Figure 41 : Recombinant Mps1-sup kinases might bear reduced kinase activity on Spc105 compared to Mps1-3. .........................................................................................................................................................Figure 42 : The intragenic suppressing mutations lower Mps1 kinase activity on Spc105. .................Figure 43 : Cells containing suppressing mutations proliferate normally, except for mps1-G449S. ...

Figure 20 :

 20 Figure 1 : Budding yeast mitosis. .................................................................................................................Figure 2 : A diagram of the main mitotic events in mammalian cells. .....................................................Figure 3 : The γ-tubulin small complex (γ-TuSC). .....................................................................................Figure 4 : The spindle pole body in budding yeast. ....................................................................................Figure 5 : SPB duplication. ...........................................................................................................................Figure 6 : The centrosome. ...........................................................................................................................Figure 7 : The centrosome cycle. ..................................................................................................................Figure 8 : The γ-tubulin template model. ....................................................................................................Figure 9 : Condensin architecture. ..............................................................................................................Figure 10 : Loop extrusion induced by a single condensin complex. ........................................................Figure 11 : Effects of condensin I and II on mitotic chromosome condensation. ....................................Figure 12 : Structure of the cohesin ring. ....................................................................................................Figure 13 : Models proposed for chromatin entrapment by cohesin. .......................................................Figure 14 : The cohesin cycle in human cells. .............................................................................................Figure 15 : Schematic kinetochore structure. .............................................................................................Figure 16 : The end-on conversion process. ................................................................................................Figure 17 : kinetochore orientation in mitosis and meiosis I. ....................................................................Figure 18 : Types of kinetochore-microtubule attachments. .....................................................................Figure 19 : SAC principles. ...........................................................................................................................Figure 20 : Domain organisation of human MPS1. ....................................................................................Figure 21 : Regulation of Mps1 during mitosis in humans. .......................................................................Figure 22 : Spindle Assembly Checkpoint activation. ................................................................................Figure 23 : The Mad2 template model. ........................................................................................................Figure 24 : The Chromosomal Passenger Complex and its recruitment to centromeres. ......................Figure 25 : The tension-related model. ........................................................................................................Figure 26 : The "dog leash" model for CPC function. ...............................................................................Figure 27 : Regulation of PP1 kinetochore recruitment. ...........................................................................Figure 28 : Mechanisms of Spindle Assembly Checkpoint silencing. .......................................................Figure 29 : grr1 mutations suppress the temperature-sensitivity of mps1-3 cells. .................................Figure 30 : grr1 mutations restore SAC signaling and proper chromosome segregation. ....................Figure 31 : Grr1 is degraded in the grr1 suppressors. .............................................................................Figure 32 : Grr1 localizes at kinetochores of wild type but not of mps1-3 cells. ....................................Figure 33 : Mps1 is present at similar levels in wild type and grr1-20 mutant cells. .............................Figure 34 : The Mps1-3 protein does not localize at kinetochores in the grr1-20 suppressor. .............Figure 35 : Ypi1 and Sds22 protein levels are not controlled by the SCF Grr1 complex. ........................Figure 36 : A second mutation in the mps1-3 allele suppresses the temperature-sensitivity of mps1-3 cells. ...............................................................................................................................................................Figure 37 : A second mutation in MPS1 leads to suppression of the chromosome segregation and SAC defects of the mps1-3 mutant. .....................................................................................................................Figure 38 : A 3HA tag at the C-terminus of Mps1 decreases suppressive effects of the intragenic suppressors. ..................................................................................................................................................Figure 39 : The intragenic suppressors restore Mps1 kinetochore localization. ...................................Figure 40 : The Mps1-sup proteins bear somewhat decreased kinase activity. .....................................Figure 41 : Recombinant Mps1-sup kinases might bear reduced kinase activity on Spc105 compared to Mps1-3. .........................................................................................................................................................Figure 42 : The intragenic suppressing mutations lower Mps1 kinase activity on Spc105. .................Figure 43 : Cells containing suppressing mutations proliferate normally, except for mps1-G449S. ...

Figure 21 :

 21 Figure 1 : Budding yeast mitosis. .................................................................................................................Figure 2 : A diagram of the main mitotic events in mammalian cells. .....................................................Figure 3 : The γ-tubulin small complex (γ-TuSC). .....................................................................................Figure 4 : The spindle pole body in budding yeast. ....................................................................................Figure 5 : SPB duplication. ...........................................................................................................................Figure 6 : The centrosome. ...........................................................................................................................Figure 7 : The centrosome cycle. ..................................................................................................................Figure 8 : The γ-tubulin template model. ....................................................................................................Figure 9 : Condensin architecture. ..............................................................................................................Figure 10 : Loop extrusion induced by a single condensin complex. ........................................................Figure 11 : Effects of condensin I and II on mitotic chromosome condensation. ....................................Figure 12 : Structure of the cohesin ring. ....................................................................................................Figure 13 : Models proposed for chromatin entrapment by cohesin. .......................................................Figure 14 : The cohesin cycle in human cells. .............................................................................................Figure 15 : Schematic kinetochore structure. .............................................................................................Figure 16 : The end-on conversion process. ................................................................................................Figure 17 : kinetochore orientation in mitosis and meiosis I. ....................................................................Figure 18 : Types of kinetochore-microtubule attachments. .....................................................................Figure 19 : SAC principles. ...........................................................................................................................Figure 20 : Domain organisation of human MPS1. ....................................................................................Figure 21 : Regulation of Mps1 during mitosis in humans. .......................................................................Figure 22 : Spindle Assembly Checkpoint activation. ................................................................................Figure 23 : The Mad2 template model. ........................................................................................................Figure 24 : The Chromosomal Passenger Complex and its recruitment to centromeres. ......................Figure 25 : The tension-related model. ........................................................................................................Figure 26 : The "dog leash" model for CPC function. ...............................................................................Figure 27 : Regulation of PP1 kinetochore recruitment. ...........................................................................Figure 28 : Mechanisms of Spindle Assembly Checkpoint silencing. .......................................................Figure 29 : grr1 mutations suppress the temperature-sensitivity of mps1-3 cells. .................................Figure 30 : grr1 mutations restore SAC signaling and proper chromosome segregation. ....................Figure 31 : Grr1 is degraded in the grr1 suppressors. .............................................................................Figure 32 : Grr1 localizes at kinetochores of wild type but not of mps1-3 cells. ....................................Figure 33 : Mps1 is present at similar levels in wild type and grr1-20 mutant cells. .............................Figure 34 : The Mps1-3 protein does not localize at kinetochores in the grr1-20 suppressor. .............Figure 35 : Ypi1 and Sds22 protein levels are not controlled by the SCF Grr1 complex. ........................Figure 36 : A second mutation in the mps1-3 allele suppresses the temperature-sensitivity of mps1-3 cells. ...............................................................................................................................................................Figure 37 : A second mutation in MPS1 leads to suppression of the chromosome segregation and SAC defects of the mps1-3 mutant. .....................................................................................................................Figure 38 : A 3HA tag at the C-terminus of Mps1 decreases suppressive effects of the intragenic suppressors. ..................................................................................................................................................Figure 39 : The intragenic suppressors restore Mps1 kinetochore localization. ...................................Figure 40 : The Mps1-sup proteins bear somewhat decreased kinase activity. .....................................Figure 41 : Recombinant Mps1-sup kinases might bear reduced kinase activity on Spc105 compared to Mps1-3. .........................................................................................................................................................Figure 42 : The intragenic suppressing mutations lower Mps1 kinase activity on Spc105. .................Figure 43 : Cells containing suppressing mutations proliferate normally, except for mps1-G449S. ...

Figure 22 :

 22 Figure 1 : Budding yeast mitosis. .................................................................................................................Figure 2 : A diagram of the main mitotic events in mammalian cells. .....................................................Figure 3 : The γ-tubulin small complex (γ-TuSC). .....................................................................................Figure 4 : The spindle pole body in budding yeast. ....................................................................................Figure 5 : SPB duplication. ...........................................................................................................................Figure 6 : The centrosome. ...........................................................................................................................Figure 7 : The centrosome cycle. ..................................................................................................................Figure 8 : The γ-tubulin template model. ....................................................................................................Figure 9 : Condensin architecture. ..............................................................................................................Figure 10 : Loop extrusion induced by a single condensin complex. ........................................................Figure 11 : Effects of condensin I and II on mitotic chromosome condensation. ....................................Figure 12 : Structure of the cohesin ring. ....................................................................................................Figure 13 : Models proposed for chromatin entrapment by cohesin. .......................................................Figure 14 : The cohesin cycle in human cells. .............................................................................................Figure 15 : Schematic kinetochore structure. .............................................................................................Figure 16 : The end-on conversion process. ................................................................................................Figure 17 : kinetochore orientation in mitosis and meiosis I. ....................................................................Figure 18 : Types of kinetochore-microtubule attachments. .....................................................................Figure 19 : SAC principles. ...........................................................................................................................Figure 20 : Domain organisation of human MPS1. ....................................................................................Figure 21 : Regulation of Mps1 during mitosis in humans. .......................................................................Figure 22 : Spindle Assembly Checkpoint activation. ................................................................................Figure 23 : The Mad2 template model. ........................................................................................................Figure 24 : The Chromosomal Passenger Complex and its recruitment to centromeres. ......................Figure 25 : The tension-related model. ........................................................................................................Figure 26 : The "dog leash" model for CPC function. ...............................................................................Figure 27 : Regulation of PP1 kinetochore recruitment. ...........................................................................Figure 28 : Mechanisms of Spindle Assembly Checkpoint silencing. .......................................................Figure 29 : grr1 mutations suppress the temperature-sensitivity of mps1-3 cells. .................................Figure 30 : grr1 mutations restore SAC signaling and proper chromosome segregation. ....................Figure 31 : Grr1 is degraded in the grr1 suppressors. .............................................................................Figure 32 : Grr1 localizes at kinetochores of wild type but not of mps1-3 cells. ....................................Figure 33 : Mps1 is present at similar levels in wild type and grr1-20 mutant cells. .............................Figure 34 : The Mps1-3 protein does not localize at kinetochores in the grr1-20 suppressor. .............Figure 35 : Ypi1 and Sds22 protein levels are not controlled by the SCF Grr1 complex. ........................Figure 36 : A second mutation in the mps1-3 allele suppresses the temperature-sensitivity of mps1-3 cells. ...............................................................................................................................................................Figure 37 : A second mutation in MPS1 leads to suppression of the chromosome segregation and SAC defects of the mps1-3 mutant. .....................................................................................................................Figure 38 : A 3HA tag at the C-terminus of Mps1 decreases suppressive effects of the intragenic suppressors. ..................................................................................................................................................Figure 39 : The intragenic suppressors restore Mps1 kinetochore localization. ...................................Figure 40 : The Mps1-sup proteins bear somewhat decreased kinase activity. .....................................Figure 41 : Recombinant Mps1-sup kinases might bear reduced kinase activity on Spc105 compared to Mps1-3. .........................................................................................................................................................Figure 42 : The intragenic suppressing mutations lower Mps1 kinase activity on Spc105. .................Figure 43 : Cells containing suppressing mutations proliferate normally, except for mps1-G449S. ...

Figure 23 :

 23 Figure 1 : Budding yeast mitosis. .................................................................................................................Figure 2 : A diagram of the main mitotic events in mammalian cells. .....................................................Figure 3 : The γ-tubulin small complex (γ-TuSC). .....................................................................................Figure 4 : The spindle pole body in budding yeast. ....................................................................................Figure 5 : SPB duplication. ...........................................................................................................................Figure 6 : The centrosome. ...........................................................................................................................Figure 7 : The centrosome cycle. ..................................................................................................................Figure 8 : The γ-tubulin template model. ....................................................................................................Figure 9 : Condensin architecture. ..............................................................................................................Figure 10 : Loop extrusion induced by a single condensin complex. ........................................................Figure 11 : Effects of condensin I and II on mitotic chromosome condensation. ....................................Figure 12 : Structure of the cohesin ring. ....................................................................................................Figure 13 : Models proposed for chromatin entrapment by cohesin. .......................................................Figure 14 : The cohesin cycle in human cells. .............................................................................................Figure 15 : Schematic kinetochore structure. .............................................................................................Figure 16 : The end-on conversion process. ................................................................................................Figure 17 : kinetochore orientation in mitosis and meiosis I. ....................................................................Figure 18 : Types of kinetochore-microtubule attachments. .....................................................................Figure 19 : SAC principles. ...........................................................................................................................Figure 20 : Domain organisation of human MPS1. ....................................................................................Figure 21 : Regulation of Mps1 during mitosis in humans. .......................................................................Figure 22 : Spindle Assembly Checkpoint activation. ................................................................................Figure 23 : The Mad2 template model. ........................................................................................................Figure 24 : The Chromosomal Passenger Complex and its recruitment to centromeres. ......................Figure 25 : The tension-related model. ........................................................................................................Figure 26 : The "dog leash" model for CPC function. ...............................................................................Figure 27 : Regulation of PP1 kinetochore recruitment. ...........................................................................Figure 28 : Mechanisms of Spindle Assembly Checkpoint silencing. .......................................................Figure 29 : grr1 mutations suppress the temperature-sensitivity of mps1-3 cells. .................................Figure 30 : grr1 mutations restore SAC signaling and proper chromosome segregation. ....................Figure 31 : Grr1 is degraded in the grr1 suppressors. .............................................................................Figure 32 : Grr1 localizes at kinetochores of wild type but not of mps1-3 cells. ....................................Figure 33 : Mps1 is present at similar levels in wild type and grr1-20 mutant cells. .............................Figure 34 : The Mps1-3 protein does not localize at kinetochores in the grr1-20 suppressor. .............Figure 35 : Ypi1 and Sds22 protein levels are not controlled by the SCF Grr1 complex. ........................Figure 36 : A second mutation in the mps1-3 allele suppresses the temperature-sensitivity of mps1-3 cells. ...............................................................................................................................................................Figure 37 : A second mutation in MPS1 leads to suppression of the chromosome segregation and SAC defects of the mps1-3 mutant. .....................................................................................................................Figure 38 : A 3HA tag at the C-terminus of Mps1 decreases suppressive effects of the intragenic suppressors. ..................................................................................................................................................Figure 39 : The intragenic suppressors restore Mps1 kinetochore localization. ...................................Figure 40 : The Mps1-sup proteins bear somewhat decreased kinase activity. .....................................Figure 41 : Recombinant Mps1-sup kinases might bear reduced kinase activity on Spc105 compared to Mps1-3. .........................................................................................................................................................Figure 42 : The intragenic suppressing mutations lower Mps1 kinase activity on Spc105. .................Figure 43 : Cells containing suppressing mutations proliferate normally, except for mps1-G449S. ...

Figure 24 :

 24 Figure 1 : Budding yeast mitosis. .................................................................................................................Figure 2 : A diagram of the main mitotic events in mammalian cells. .....................................................Figure 3 : The γ-tubulin small complex (γ-TuSC). .....................................................................................Figure 4 : The spindle pole body in budding yeast. ....................................................................................Figure 5 : SPB duplication. ...........................................................................................................................Figure 6 : The centrosome. ...........................................................................................................................Figure 7 : The centrosome cycle. ..................................................................................................................Figure 8 : The γ-tubulin template model. ....................................................................................................Figure 9 : Condensin architecture. ..............................................................................................................Figure 10 : Loop extrusion induced by a single condensin complex. ........................................................Figure 11 : Effects of condensin I and II on mitotic chromosome condensation. ....................................Figure 12 : Structure of the cohesin ring. ....................................................................................................Figure 13 : Models proposed for chromatin entrapment by cohesin. .......................................................Figure 14 : The cohesin cycle in human cells. .............................................................................................Figure 15 : Schematic kinetochore structure. .............................................................................................Figure 16 : The end-on conversion process. ................................................................................................Figure 17 : kinetochore orientation in mitosis and meiosis I. ....................................................................Figure 18 : Types of kinetochore-microtubule attachments. .....................................................................Figure 19 : SAC principles. ...........................................................................................................................Figure 20 : Domain organisation of human MPS1. ....................................................................................Figure 21 : Regulation of Mps1 during mitosis in humans. .......................................................................Figure 22 : Spindle Assembly Checkpoint activation. ................................................................................Figure 23 : The Mad2 template model. ........................................................................................................Figure 24 : The Chromosomal Passenger Complex and its recruitment to centromeres. ......................Figure 25 : The tension-related model. ........................................................................................................Figure 26 : The "dog leash" model for CPC function. ...............................................................................Figure 27 : Regulation of PP1 kinetochore recruitment. ...........................................................................Figure 28 : Mechanisms of Spindle Assembly Checkpoint silencing. .......................................................Figure 29 : grr1 mutations suppress the temperature-sensitivity of mps1-3 cells. .................................Figure 30 : grr1 mutations restore SAC signaling and proper chromosome segregation. ....................Figure 31 : Grr1 is degraded in the grr1 suppressors. .............................................................................Figure 32 : Grr1 localizes at kinetochores of wild type but not of mps1-3 cells. ....................................Figure 33 : Mps1 is present at similar levels in wild type and grr1-20 mutant cells. .............................Figure 34 : The Mps1-3 protein does not localize at kinetochores in the grr1-20 suppressor. .............Figure 35 : Ypi1 and Sds22 protein levels are not controlled by the SCF Grr1 complex. ........................Figure 36 : A second mutation in the mps1-3 allele suppresses the temperature-sensitivity of mps1-3 cells. ...............................................................................................................................................................Figure 37 : A second mutation in MPS1 leads to suppression of the chromosome segregation and SAC defects of the mps1-3 mutant. .....................................................................................................................Figure 38 : A 3HA tag at the C-terminus of Mps1 decreases suppressive effects of the intragenic suppressors. ..................................................................................................................................................Figure 39 : The intragenic suppressors restore Mps1 kinetochore localization. ...................................Figure 40 : The Mps1-sup proteins bear somewhat decreased kinase activity. .....................................Figure 41 : Recombinant Mps1-sup kinases might bear reduced kinase activity on Spc105 compared to Mps1-3. .........................................................................................................................................................Figure 42 : The intragenic suppressing mutations lower Mps1 kinase activity on Spc105. .................Figure 43 : Cells containing suppressing mutations proliferate normally, except for mps1-G449S. ...

Figure 25 :

 25 Figure 1 : Budding yeast mitosis. .................................................................................................................Figure 2 : A diagram of the main mitotic events in mammalian cells. .....................................................Figure 3 : The γ-tubulin small complex (γ-TuSC). .....................................................................................Figure 4 : The spindle pole body in budding yeast. ....................................................................................Figure 5 : SPB duplication. ...........................................................................................................................Figure 6 : The centrosome. ...........................................................................................................................Figure 7 : The centrosome cycle. ..................................................................................................................Figure 8 : The γ-tubulin template model. ....................................................................................................Figure 9 : Condensin architecture. ..............................................................................................................Figure 10 : Loop extrusion induced by a single condensin complex. ........................................................Figure 11 : Effects of condensin I and II on mitotic chromosome condensation. ....................................Figure 12 : Structure of the cohesin ring. ....................................................................................................Figure 13 : Models proposed for chromatin entrapment by cohesin. .......................................................Figure 14 : The cohesin cycle in human cells. .............................................................................................Figure 15 : Schematic kinetochore structure. .............................................................................................Figure 16 : The end-on conversion process. ................................................................................................Figure 17 : kinetochore orientation in mitosis and meiosis I. ....................................................................Figure 18 : Types of kinetochore-microtubule attachments. .....................................................................Figure 19 : SAC principles. ...........................................................................................................................Figure 20 : Domain organisation of human MPS1. ....................................................................................Figure 21 : Regulation of Mps1 during mitosis in humans. .......................................................................Figure 22 : Spindle Assembly Checkpoint activation. ................................................................................Figure 23 : The Mad2 template model. ........................................................................................................Figure 24 : The Chromosomal Passenger Complex and its recruitment to centromeres. ......................Figure 25 : The tension-related model. ........................................................................................................Figure 26 : The "dog leash" model for CPC function. ...............................................................................Figure 27 : Regulation of PP1 kinetochore recruitment. ...........................................................................Figure 28 : Mechanisms of Spindle Assembly Checkpoint silencing. .......................................................Figure 29 : grr1 mutations suppress the temperature-sensitivity of mps1-3 cells. .................................Figure 30 : grr1 mutations restore SAC signaling and proper chromosome segregation. ....................Figure 31 : Grr1 is degraded in the grr1 suppressors. .............................................................................Figure 32 : Grr1 localizes at kinetochores of wild type but not of mps1-3 cells. ....................................Figure 33 : Mps1 is present at similar levels in wild type and grr1-20 mutant cells. .............................Figure 34 : The Mps1-3 protein does not localize at kinetochores in the grr1-20 suppressor. .............Figure 35 : Ypi1 and Sds22 protein levels are not controlled by the SCF Grr1 complex. ........................Figure 36 : A second mutation in the mps1-3 allele suppresses the temperature-sensitivity of mps1-3 cells. ...............................................................................................................................................................Figure 37 : A second mutation in MPS1 leads to suppression of the chromosome segregation and SAC defects of the mps1-3 mutant. .....................................................................................................................Figure 38 : A 3HA tag at the C-terminus of Mps1 decreases suppressive effects of the intragenic suppressors. ..................................................................................................................................................Figure 39 : The intragenic suppressors restore Mps1 kinetochore localization. ...................................Figure 40 : The Mps1-sup proteins bear somewhat decreased kinase activity. .....................................Figure 41 : Recombinant Mps1-sup kinases might bear reduced kinase activity on Spc105 compared to Mps1-3. .........................................................................................................................................................Figure 42 : The intragenic suppressing mutations lower Mps1 kinase activity on Spc105. .................Figure 43 : Cells containing suppressing mutations proliferate normally, except for mps1-G449S. ...

Figure 26 :

 26 Figure 1 : Budding yeast mitosis. .................................................................................................................Figure 2 : A diagram of the main mitotic events in mammalian cells. .....................................................Figure 3 : The γ-tubulin small complex (γ-TuSC). .....................................................................................Figure 4 : The spindle pole body in budding yeast. ....................................................................................Figure 5 : SPB duplication. ...........................................................................................................................Figure 6 : The centrosome. ...........................................................................................................................Figure 7 : The centrosome cycle. ..................................................................................................................Figure 8 : The γ-tubulin template model. ....................................................................................................Figure 9 : Condensin architecture. ..............................................................................................................Figure 10 : Loop extrusion induced by a single condensin complex. ........................................................Figure 11 : Effects of condensin I and II on mitotic chromosome condensation. ....................................Figure 12 : Structure of the cohesin ring. ....................................................................................................Figure 13 : Models proposed for chromatin entrapment by cohesin. .......................................................Figure 14 : The cohesin cycle in human cells. .............................................................................................Figure 15 : Schematic kinetochore structure. .............................................................................................Figure 16 : The end-on conversion process. ................................................................................................Figure 17 : kinetochore orientation in mitosis and meiosis I. ....................................................................Figure 18 : Types of kinetochore-microtubule attachments. .....................................................................Figure 19 : SAC principles. ...........................................................................................................................Figure 20 : Domain organisation of human MPS1. ....................................................................................Figure 21 : Regulation of Mps1 during mitosis in humans. .......................................................................Figure 22 : Spindle Assembly Checkpoint activation. ................................................................................Figure 23 : The Mad2 template model. ........................................................................................................Figure 24 : The Chromosomal Passenger Complex and its recruitment to centromeres. ......................Figure 25 : The tension-related model. ........................................................................................................Figure 26 : The "dog leash" model for CPC function. ...............................................................................Figure 27 : Regulation of PP1 kinetochore recruitment. ...........................................................................Figure 28 : Mechanisms of Spindle Assembly Checkpoint silencing. .......................................................Figure 29 : grr1 mutations suppress the temperature-sensitivity of mps1-3 cells. .................................Figure 30 : grr1 mutations restore SAC signaling and proper chromosome segregation. ....................Figure 31 : Grr1 is degraded in the grr1 suppressors. .............................................................................Figure 32 : Grr1 localizes at kinetochores of wild type but not of mps1-3 cells. ....................................Figure 33 : Mps1 is present at similar levels in wild type and grr1-20 mutant cells. .............................Figure 34 : The Mps1-3 protein does not localize at kinetochores in the grr1-20 suppressor. .............Figure 35 : Ypi1 and Sds22 protein levels are not controlled by the SCF Grr1 complex. ........................Figure 36 : A second mutation in the mps1-3 allele suppresses the temperature-sensitivity of mps1-3 cells. ...............................................................................................................................................................Figure 37 : A second mutation in MPS1 leads to suppression of the chromosome segregation and SAC defects of the mps1-3 mutant. .....................................................................................................................Figure 38 : A 3HA tag at the C-terminus of Mps1 decreases suppressive effects of the intragenic suppressors. ..................................................................................................................................................Figure 39 : The intragenic suppressors restore Mps1 kinetochore localization. ...................................Figure 40 : The Mps1-sup proteins bear somewhat decreased kinase activity. .....................................Figure 41 : Recombinant Mps1-sup kinases might bear reduced kinase activity on Spc105 compared to Mps1-3. .........................................................................................................................................................Figure 42 : The intragenic suppressing mutations lower Mps1 kinase activity on Spc105. .................Figure 43 : Cells containing suppressing mutations proliferate normally, except for mps1-G449S. ...

Figure 27 :

 27 Figure 1 : Budding yeast mitosis. .................................................................................................................Figure 2 : A diagram of the main mitotic events in mammalian cells. .....................................................Figure 3 : The γ-tubulin small complex (γ-TuSC). .....................................................................................Figure 4 : The spindle pole body in budding yeast. ....................................................................................Figure 5 : SPB duplication. ...........................................................................................................................Figure 6 : The centrosome. ...........................................................................................................................Figure 7 : The centrosome cycle. ..................................................................................................................Figure 8 : The γ-tubulin template model. ....................................................................................................Figure 9 : Condensin architecture. ..............................................................................................................Figure 10 : Loop extrusion induced by a single condensin complex. ........................................................Figure 11 : Effects of condensin I and II on mitotic chromosome condensation. ....................................Figure 12 : Structure of the cohesin ring. ....................................................................................................Figure 13 : Models proposed for chromatin entrapment by cohesin. .......................................................Figure 14 : The cohesin cycle in human cells. .............................................................................................Figure 15 : Schematic kinetochore structure. .............................................................................................Figure 16 : The end-on conversion process. ................................................................................................Figure 17 : kinetochore orientation in mitosis and meiosis I. ....................................................................Figure 18 : Types of kinetochore-microtubule attachments. .....................................................................Figure 19 : SAC principles. ...........................................................................................................................Figure 20 : Domain organisation of human MPS1. ....................................................................................Figure 21 : Regulation of Mps1 during mitosis in humans. .......................................................................Figure 22 : Spindle Assembly Checkpoint activation. ................................................................................Figure 23 : The Mad2 template model. ........................................................................................................Figure 24 : The Chromosomal Passenger Complex and its recruitment to centromeres. ......................Figure 25 : The tension-related model. ........................................................................................................Figure 26 : The "dog leash" model for CPC function. ...............................................................................Figure 27 : Regulation of PP1 kinetochore recruitment. ...........................................................................Figure 28 : Mechanisms of Spindle Assembly Checkpoint silencing. .......................................................Figure 29 : grr1 mutations suppress the temperature-sensitivity of mps1-3 cells. .................................Figure 30 : grr1 mutations restore SAC signaling and proper chromosome segregation. ....................Figure 31 : Grr1 is degraded in the grr1 suppressors. .............................................................................Figure 32 : Grr1 localizes at kinetochores of wild type but not of mps1-3 cells. ....................................Figure 33 : Mps1 is present at similar levels in wild type and grr1-20 mutant cells. .............................Figure 34 : The Mps1-3 protein does not localize at kinetochores in the grr1-20 suppressor. .............Figure 35 : Ypi1 and Sds22 protein levels are not controlled by the SCF Grr1 complex. ........................Figure 36 : A second mutation in the mps1-3 allele suppresses the temperature-sensitivity of mps1-3 cells. ...............................................................................................................................................................Figure 37 : A second mutation in MPS1 leads to suppression of the chromosome segregation and SAC defects of the mps1-3 mutant. .....................................................................................................................Figure 38 : A 3HA tag at the C-terminus of Mps1 decreases suppressive effects of the intragenic suppressors. ..................................................................................................................................................Figure 39 : The intragenic suppressors restore Mps1 kinetochore localization. ...................................Figure 40 : The Mps1-sup proteins bear somewhat decreased kinase activity. .....................................Figure 41 : Recombinant Mps1-sup kinases might bear reduced kinase activity on Spc105 compared to Mps1-3. .........................................................................................................................................................Figure 42 : The intragenic suppressing mutations lower Mps1 kinase activity on Spc105. .................Figure 43 : Cells containing suppressing mutations proliferate normally, except for mps1-G449S. ...

Figure 28 :

 28 Figure 1 : Budding yeast mitosis. .................................................................................................................Figure 2 : A diagram of the main mitotic events in mammalian cells. .....................................................Figure 3 : The γ-tubulin small complex (γ-TuSC). .....................................................................................Figure 4 : The spindle pole body in budding yeast. ....................................................................................Figure 5 : SPB duplication. ...........................................................................................................................Figure 6 : The centrosome. ...........................................................................................................................Figure 7 : The centrosome cycle. ..................................................................................................................Figure 8 : The γ-tubulin template model. ....................................................................................................Figure 9 : Condensin architecture. ..............................................................................................................Figure 10 : Loop extrusion induced by a single condensin complex. ........................................................Figure 11 : Effects of condensin I and II on mitotic chromosome condensation. ....................................Figure 12 : Structure of the cohesin ring. ....................................................................................................Figure 13 : Models proposed for chromatin entrapment by cohesin. .......................................................Figure 14 : The cohesin cycle in human cells. .............................................................................................Figure 15 : Schematic kinetochore structure. .............................................................................................Figure 16 : The end-on conversion process. ................................................................................................Figure 17 : kinetochore orientation in mitosis and meiosis I. ....................................................................Figure 18 : Types of kinetochore-microtubule attachments. .....................................................................Figure 19 : SAC principles. ...........................................................................................................................Figure 20 : Domain organisation of human MPS1. ....................................................................................Figure 21 : Regulation of Mps1 during mitosis in humans. .......................................................................Figure 22 : Spindle Assembly Checkpoint activation. ................................................................................Figure 23 : The Mad2 template model. ........................................................................................................Figure 24 : The Chromosomal Passenger Complex and its recruitment to centromeres. ......................Figure 25 : The tension-related model. ........................................................................................................Figure 26 : The "dog leash" model for CPC function. ...............................................................................Figure 27 : Regulation of PP1 kinetochore recruitment. ...........................................................................Figure 28 : Mechanisms of Spindle Assembly Checkpoint silencing. .......................................................Figure 29 : grr1 mutations suppress the temperature-sensitivity of mps1-3 cells. .................................Figure 30 : grr1 mutations restore SAC signaling and proper chromosome segregation. ....................Figure 31 : Grr1 is degraded in the grr1 suppressors. .............................................................................Figure 32 : Grr1 localizes at kinetochores of wild type but not of mps1-3 cells. ....................................Figure 33 : Mps1 is present at similar levels in wild type and grr1-20 mutant cells. .............................Figure 34 : The Mps1-3 protein does not localize at kinetochores in the grr1-20 suppressor. .............Figure 35 : Ypi1 and Sds22 protein levels are not controlled by the SCF Grr1 complex. ........................Figure 36 : A second mutation in the mps1-3 allele suppresses the temperature-sensitivity of mps1-3 cells. ...............................................................................................................................................................Figure 37 : A second mutation in MPS1 leads to suppression of the chromosome segregation and SAC defects of the mps1-3 mutant. .....................................................................................................................Figure 38 : A 3HA tag at the C-terminus of Mps1 decreases suppressive effects of the intragenic suppressors. ..................................................................................................................................................Figure 39 : The intragenic suppressors restore Mps1 kinetochore localization. ...................................Figure 40 : The Mps1-sup proteins bear somewhat decreased kinase activity. .....................................Figure 41 : Recombinant Mps1-sup kinases might bear reduced kinase activity on Spc105 compared to Mps1-3. .........................................................................................................................................................Figure 42 : The intragenic suppressing mutations lower Mps1 kinase activity on Spc105. .................Figure 43 : Cells containing suppressing mutations proliferate normally, except for mps1-G449S. ...

Figure 29 :

 29 Figure 1 : Budding yeast mitosis. .................................................................................................................Figure 2 : A diagram of the main mitotic events in mammalian cells. .....................................................Figure 3 : The γ-tubulin small complex (γ-TuSC). .....................................................................................Figure 4 : The spindle pole body in budding yeast. ....................................................................................Figure 5 : SPB duplication. ...........................................................................................................................Figure 6 : The centrosome. ...........................................................................................................................Figure 7 : The centrosome cycle. ..................................................................................................................Figure 8 : The γ-tubulin template model. ....................................................................................................Figure 9 : Condensin architecture. ..............................................................................................................Figure 10 : Loop extrusion induced by a single condensin complex. ........................................................Figure 11 : Effects of condensin I and II on mitotic chromosome condensation. ....................................Figure 12 : Structure of the cohesin ring. ....................................................................................................Figure 13 : Models proposed for chromatin entrapment by cohesin. .......................................................Figure 14 : The cohesin cycle in human cells. .............................................................................................Figure 15 : Schematic kinetochore structure. .............................................................................................Figure 16 : The end-on conversion process. ................................................................................................Figure 17 : kinetochore orientation in mitosis and meiosis I. ....................................................................Figure 18 : Types of kinetochore-microtubule attachments. .....................................................................Figure 19 : SAC principles. ...........................................................................................................................Figure 20 : Domain organisation of human MPS1. ....................................................................................Figure 21 : Regulation of Mps1 during mitosis in humans. .......................................................................Figure 22 : Spindle Assembly Checkpoint activation. ................................................................................Figure 23 : The Mad2 template model. ........................................................................................................Figure 24 : The Chromosomal Passenger Complex and its recruitment to centromeres. ......................Figure 25 : The tension-related model. ........................................................................................................Figure 26 : The "dog leash" model for CPC function. ...............................................................................Figure 27 : Regulation of PP1 kinetochore recruitment. ...........................................................................Figure 28 : Mechanisms of Spindle Assembly Checkpoint silencing. .......................................................Figure 29 : grr1 mutations suppress the temperature-sensitivity of mps1-3 cells. .................................Figure 30 : grr1 mutations restore SAC signaling and proper chromosome segregation. ....................Figure 31 : Grr1 is degraded in the grr1 suppressors. .............................................................................Figure 32 : Grr1 localizes at kinetochores of wild type but not of mps1-3 cells. ....................................Figure 33 : Mps1 is present at similar levels in wild type and grr1-20 mutant cells. .............................Figure 34 : The Mps1-3 protein does not localize at kinetochores in the grr1-20 suppressor. .............Figure 35 : Ypi1 and Sds22 protein levels are not controlled by the SCF Grr1 complex. ........................Figure 36 : A second mutation in the mps1-3 allele suppresses the temperature-sensitivity of mps1-3 cells. ...............................................................................................................................................................Figure 37 : A second mutation in MPS1 leads to suppression of the chromosome segregation and SAC defects of the mps1-3 mutant. .....................................................................................................................Figure 38 : A 3HA tag at the C-terminus of Mps1 decreases suppressive effects of the intragenic suppressors. ..................................................................................................................................................Figure 39 : The intragenic suppressors restore Mps1 kinetochore localization. ...................................Figure 40 : The Mps1-sup proteins bear somewhat decreased kinase activity. .....................................Figure 41 : Recombinant Mps1-sup kinases might bear reduced kinase activity on Spc105 compared to Mps1-3. .........................................................................................................................................................Figure 42 : The intragenic suppressing mutations lower Mps1 kinase activity on Spc105. .................Figure 43 : Cells containing suppressing mutations proliferate normally, except for mps1-G449S. ...

Figure 30 :

 30 Figure 1 : Budding yeast mitosis. .................................................................................................................Figure 2 : A diagram of the main mitotic events in mammalian cells. .....................................................Figure 3 : The γ-tubulin small complex (γ-TuSC). .....................................................................................Figure 4 : The spindle pole body in budding yeast. ....................................................................................Figure 5 : SPB duplication. ...........................................................................................................................Figure 6 : The centrosome. ...........................................................................................................................Figure 7 : The centrosome cycle. ..................................................................................................................Figure 8 : The γ-tubulin template model. ....................................................................................................Figure 9 : Condensin architecture. ..............................................................................................................Figure 10 : Loop extrusion induced by a single condensin complex. ........................................................Figure 11 : Effects of condensin I and II on mitotic chromosome condensation. ....................................Figure 12 : Structure of the cohesin ring. ....................................................................................................Figure 13 : Models proposed for chromatin entrapment by cohesin. .......................................................Figure 14 : The cohesin cycle in human cells. .............................................................................................Figure 15 : Schematic kinetochore structure. .............................................................................................Figure 16 : The end-on conversion process. ................................................................................................Figure 17 : kinetochore orientation in mitosis and meiosis I. ....................................................................Figure 18 : Types of kinetochore-microtubule attachments. .....................................................................Figure 19 : SAC principles. ...........................................................................................................................Figure 20 : Domain organisation of human MPS1. ....................................................................................Figure 21 : Regulation of Mps1 during mitosis in humans. .......................................................................Figure 22 : Spindle Assembly Checkpoint activation. ................................................................................Figure 23 : The Mad2 template model. ........................................................................................................Figure 24 : The Chromosomal Passenger Complex and its recruitment to centromeres. ......................Figure 25 : The tension-related model. ........................................................................................................Figure 26 : The "dog leash" model for CPC function. ...............................................................................Figure 27 : Regulation of PP1 kinetochore recruitment. ...........................................................................Figure 28 : Mechanisms of Spindle Assembly Checkpoint silencing. .......................................................Figure 29 : grr1 mutations suppress the temperature-sensitivity of mps1-3 cells. .................................Figure 30 : grr1 mutations restore SAC signaling and proper chromosome segregation. ....................Figure 31 : Grr1 is degraded in the grr1 suppressors. .............................................................................Figure 32 : Grr1 localizes at kinetochores of wild type but not of mps1-3 cells. ....................................Figure 33 : Mps1 is present at similar levels in wild type and grr1-20 mutant cells. .............................Figure 34 : The Mps1-3 protein does not localize at kinetochores in the grr1-20 suppressor. .............Figure 35 : Ypi1 and Sds22 protein levels are not controlled by the SCF Grr1 complex. ........................Figure 36 : A second mutation in the mps1-3 allele suppresses the temperature-sensitivity of mps1-3 cells. ...............................................................................................................................................................Figure 37 : A second mutation in MPS1 leads to suppression of the chromosome segregation and SAC defects of the mps1-3 mutant. .....................................................................................................................Figure 38 : A 3HA tag at the C-terminus of Mps1 decreases suppressive effects of the intragenic suppressors. ..................................................................................................................................................Figure 39 : The intragenic suppressors restore Mps1 kinetochore localization. ...................................Figure 40 : The Mps1-sup proteins bear somewhat decreased kinase activity. .....................................Figure 41 : Recombinant Mps1-sup kinases might bear reduced kinase activity on Spc105 compared to Mps1-3. .........................................................................................................................................................Figure 42 : The intragenic suppressing mutations lower Mps1 kinase activity on Spc105. .................Figure 43 : Cells containing suppressing mutations proliferate normally, except for mps1-G449S. ...

Figure 31 :

 31 Figure 1 : Budding yeast mitosis. .................................................................................................................Figure 2 : A diagram of the main mitotic events in mammalian cells. .....................................................Figure 3 : The γ-tubulin small complex (γ-TuSC). .....................................................................................Figure 4 : The spindle pole body in budding yeast. ....................................................................................Figure 5 : SPB duplication. ...........................................................................................................................Figure 6 : The centrosome. ...........................................................................................................................Figure 7 : The centrosome cycle. ..................................................................................................................Figure 8 : The γ-tubulin template model. ....................................................................................................Figure 9 : Condensin architecture. ..............................................................................................................Figure 10 : Loop extrusion induced by a single condensin complex. ........................................................Figure 11 : Effects of condensin I and II on mitotic chromosome condensation. ....................................Figure 12 : Structure of the cohesin ring. ....................................................................................................Figure 13 : Models proposed for chromatin entrapment by cohesin. .......................................................Figure 14 : The cohesin cycle in human cells. .............................................................................................Figure 15 : Schematic kinetochore structure. .............................................................................................Figure 16 : The end-on conversion process. ................................................................................................Figure 17 : kinetochore orientation in mitosis and meiosis I. ....................................................................Figure 18 : Types of kinetochore-microtubule attachments. .....................................................................Figure 19 : SAC principles. ...........................................................................................................................Figure 20 : Domain organisation of human MPS1. ....................................................................................Figure 21 : Regulation of Mps1 during mitosis in humans. .......................................................................Figure 22 : Spindle Assembly Checkpoint activation. ................................................................................Figure 23 : The Mad2 template model. ........................................................................................................Figure 24 : The Chromosomal Passenger Complex and its recruitment to centromeres. ......................Figure 25 : The tension-related model. ........................................................................................................Figure 26 : The "dog leash" model for CPC function. ...............................................................................Figure 27 : Regulation of PP1 kinetochore recruitment. ...........................................................................Figure 28 : Mechanisms of Spindle Assembly Checkpoint silencing. .......................................................Figure 29 : grr1 mutations suppress the temperature-sensitivity of mps1-3 cells. .................................Figure 30 : grr1 mutations restore SAC signaling and proper chromosome segregation. ....................Figure 31 : Grr1 is degraded in the grr1 suppressors. .............................................................................Figure 32 : Grr1 localizes at kinetochores of wild type but not of mps1-3 cells. ....................................Figure 33 : Mps1 is present at similar levels in wild type and grr1-20 mutant cells. .............................Figure 34 : The Mps1-3 protein does not localize at kinetochores in the grr1-20 suppressor. .............Figure 35 : Ypi1 and Sds22 protein levels are not controlled by the SCF Grr1 complex. ........................Figure 36 : A second mutation in the mps1-3 allele suppresses the temperature-sensitivity of mps1-3 cells. ...............................................................................................................................................................Figure 37 : A second mutation in MPS1 leads to suppression of the chromosome segregation and SAC defects of the mps1-3 mutant. .....................................................................................................................Figure 38 : A 3HA tag at the C-terminus of Mps1 decreases suppressive effects of the intragenic suppressors. ..................................................................................................................................................Figure 39 : The intragenic suppressors restore Mps1 kinetochore localization. ...................................Figure 40 : The Mps1-sup proteins bear somewhat decreased kinase activity. .....................................Figure 41 : Recombinant Mps1-sup kinases might bear reduced kinase activity on Spc105 compared to Mps1-3. .........................................................................................................................................................Figure 42 : The intragenic suppressing mutations lower Mps1 kinase activity on Spc105. .................Figure 43 : Cells containing suppressing mutations proliferate normally, except for mps1-G449S. ...

Figure 32 :

 32 Figure 1 : Budding yeast mitosis. .................................................................................................................Figure 2 : A diagram of the main mitotic events in mammalian cells. .....................................................Figure 3 : The γ-tubulin small complex (γ-TuSC). .....................................................................................Figure 4 : The spindle pole body in budding yeast. ....................................................................................Figure 5 : SPB duplication. ...........................................................................................................................Figure 6 : The centrosome. ...........................................................................................................................Figure 7 : The centrosome cycle. ..................................................................................................................Figure 8 : The γ-tubulin template model. ....................................................................................................Figure 9 : Condensin architecture. ..............................................................................................................Figure 10 : Loop extrusion induced by a single condensin complex. ........................................................Figure 11 : Effects of condensin I and II on mitotic chromosome condensation. ....................................Figure 12 : Structure of the cohesin ring. ....................................................................................................Figure 13 : Models proposed for chromatin entrapment by cohesin. .......................................................Figure 14 : The cohesin cycle in human cells. .............................................................................................Figure 15 : Schematic kinetochore structure. .............................................................................................Figure 16 : The end-on conversion process. ................................................................................................Figure 17 : kinetochore orientation in mitosis and meiosis I. ....................................................................Figure 18 : Types of kinetochore-microtubule attachments. .....................................................................Figure 19 : SAC principles. ...........................................................................................................................Figure 20 : Domain organisation of human MPS1. ....................................................................................Figure 21 : Regulation of Mps1 during mitosis in humans. .......................................................................Figure 22 : Spindle Assembly Checkpoint activation. ................................................................................Figure 23 : The Mad2 template model. ........................................................................................................Figure 24 : The Chromosomal Passenger Complex and its recruitment to centromeres. ......................Figure 25 : The tension-related model. ........................................................................................................Figure 26 : The "dog leash" model for CPC function. ...............................................................................Figure 27 : Regulation of PP1 kinetochore recruitment. ...........................................................................Figure 28 : Mechanisms of Spindle Assembly Checkpoint silencing. .......................................................Figure 29 : grr1 mutations suppress the temperature-sensitivity of mps1-3 cells. .................................Figure 30 : grr1 mutations restore SAC signaling and proper chromosome segregation. ....................Figure 31 : Grr1 is degraded in the grr1 suppressors. .............................................................................Figure 32 : Grr1 localizes at kinetochores of wild type but not of mps1-3 cells. ....................................Figure 33 : Mps1 is present at similar levels in wild type and grr1-20 mutant cells. .............................Figure 34 : The Mps1-3 protein does not localize at kinetochores in the grr1-20 suppressor. .............Figure 35 : Ypi1 and Sds22 protein levels are not controlled by the SCF Grr1 complex. ........................Figure 36 : A second mutation in the mps1-3 allele suppresses the temperature-sensitivity of mps1-3 cells. ...............................................................................................................................................................Figure 37 : A second mutation in MPS1 leads to suppression of the chromosome segregation and SAC defects of the mps1-3 mutant. .....................................................................................................................Figure 38 : A 3HA tag at the C-terminus of Mps1 decreases suppressive effects of the intragenic suppressors. ..................................................................................................................................................Figure 39 : The intragenic suppressors restore Mps1 kinetochore localization. ...................................Figure 40 : The Mps1-sup proteins bear somewhat decreased kinase activity. .....................................Figure 41 : Recombinant Mps1-sup kinases might bear reduced kinase activity on Spc105 compared to Mps1-3. .........................................................................................................................................................Figure 42 : The intragenic suppressing mutations lower Mps1 kinase activity on Spc105. .................Figure 43 : Cells containing suppressing mutations proliferate normally, except for mps1-G449S. ...

Figure 33 :

 33 Figure 1 : Budding yeast mitosis. .................................................................................................................Figure 2 : A diagram of the main mitotic events in mammalian cells. .....................................................Figure 3 : The γ-tubulin small complex (γ-TuSC). .....................................................................................Figure 4 : The spindle pole body in budding yeast. ....................................................................................Figure 5 : SPB duplication. ...........................................................................................................................Figure 6 : The centrosome. ...........................................................................................................................Figure 7 : The centrosome cycle. ..................................................................................................................Figure 8 : The γ-tubulin template model. ....................................................................................................Figure 9 : Condensin architecture. ..............................................................................................................Figure 10 : Loop extrusion induced by a single condensin complex. ........................................................Figure 11 : Effects of condensin I and II on mitotic chromosome condensation. ....................................Figure 12 : Structure of the cohesin ring. ....................................................................................................Figure 13 : Models proposed for chromatin entrapment by cohesin. .......................................................Figure 14 : The cohesin cycle in human cells. .............................................................................................Figure 15 : Schematic kinetochore structure. .............................................................................................Figure 16 : The end-on conversion process. ................................................................................................Figure 17 : kinetochore orientation in mitosis and meiosis I. ....................................................................Figure 18 : Types of kinetochore-microtubule attachments. .....................................................................Figure 19 : SAC principles. ...........................................................................................................................Figure 20 : Domain organisation of human MPS1. ....................................................................................Figure 21 : Regulation of Mps1 during mitosis in humans. .......................................................................Figure 22 : Spindle Assembly Checkpoint activation. ................................................................................Figure 23 : The Mad2 template model. ........................................................................................................Figure 24 : The Chromosomal Passenger Complex and its recruitment to centromeres. ......................Figure 25 : The tension-related model. ........................................................................................................Figure 26 : The "dog leash" model for CPC function. ...............................................................................Figure 27 : Regulation of PP1 kinetochore recruitment. ...........................................................................Figure 28 : Mechanisms of Spindle Assembly Checkpoint silencing. .......................................................Figure 29 : grr1 mutations suppress the temperature-sensitivity of mps1-3 cells. .................................Figure 30 : grr1 mutations restore SAC signaling and proper chromosome segregation. ....................Figure 31 : Grr1 is degraded in the grr1 suppressors. .............................................................................Figure 32 : Grr1 localizes at kinetochores of wild type but not of mps1-3 cells. ....................................Figure 33 : Mps1 is present at similar levels in wild type and grr1-20 mutant cells. .............................Figure 34 : The Mps1-3 protein does not localize at kinetochores in the grr1-20 suppressor. .............Figure 35 : Ypi1 and Sds22 protein levels are not controlled by the SCF Grr1 complex. ........................Figure 36 : A second mutation in the mps1-3 allele suppresses the temperature-sensitivity of mps1-3 cells. ...............................................................................................................................................................Figure 37 : A second mutation in MPS1 leads to suppression of the chromosome segregation and SAC defects of the mps1-3 mutant. .....................................................................................................................Figure 38 : A 3HA tag at the C-terminus of Mps1 decreases suppressive effects of the intragenic suppressors. ..................................................................................................................................................Figure 39 : The intragenic suppressors restore Mps1 kinetochore localization. ...................................Figure 40 : The Mps1-sup proteins bear somewhat decreased kinase activity. .....................................Figure 41 : Recombinant Mps1-sup kinases might bear reduced kinase activity on Spc105 compared to Mps1-3. .........................................................................................................................................................Figure 42 : The intragenic suppressing mutations lower Mps1 kinase activity on Spc105. .................Figure 43 : Cells containing suppressing mutations proliferate normally, except for mps1-G449S. ...

Figure 34 :

 34 Figure 1 : Budding yeast mitosis. .................................................................................................................Figure 2 : A diagram of the main mitotic events in mammalian cells. .....................................................Figure 3 : The γ-tubulin small complex (γ-TuSC). .....................................................................................Figure 4 : The spindle pole body in budding yeast. ....................................................................................Figure 5 : SPB duplication. ...........................................................................................................................Figure 6 : The centrosome. ...........................................................................................................................Figure 7 : The centrosome cycle. ..................................................................................................................Figure 8 : The γ-tubulin template model. ....................................................................................................Figure 9 : Condensin architecture. ..............................................................................................................Figure 10 : Loop extrusion induced by a single condensin complex. ........................................................Figure 11 : Effects of condensin I and II on mitotic chromosome condensation. ....................................Figure 12 : Structure of the cohesin ring. ....................................................................................................Figure 13 : Models proposed for chromatin entrapment by cohesin. .......................................................Figure 14 : The cohesin cycle in human cells. .............................................................................................Figure 15 : Schematic kinetochore structure. .............................................................................................Figure 16 : The end-on conversion process. ................................................................................................Figure 17 : kinetochore orientation in mitosis and meiosis I. ....................................................................Figure 18 : Types of kinetochore-microtubule attachments. .....................................................................Figure 19 : SAC principles. ...........................................................................................................................Figure 20 : Domain organisation of human MPS1. ....................................................................................Figure 21 : Regulation of Mps1 during mitosis in humans. .......................................................................Figure 22 : Spindle Assembly Checkpoint activation. ................................................................................Figure 23 : The Mad2 template model. ........................................................................................................Figure 24 : The Chromosomal Passenger Complex and its recruitment to centromeres. ......................Figure 25 : The tension-related model. ........................................................................................................Figure 26 : The "dog leash" model for CPC function. ...............................................................................Figure 27 : Regulation of PP1 kinetochore recruitment. ...........................................................................Figure 28 : Mechanisms of Spindle Assembly Checkpoint silencing. .......................................................Figure 29 : grr1 mutations suppress the temperature-sensitivity of mps1-3 cells. .................................Figure 30 : grr1 mutations restore SAC signaling and proper chromosome segregation. ....................Figure 31 : Grr1 is degraded in the grr1 suppressors. .............................................................................Figure 32 : Grr1 localizes at kinetochores of wild type but not of mps1-3 cells. ....................................Figure 33 : Mps1 is present at similar levels in wild type and grr1-20 mutant cells. .............................Figure 34 : The Mps1-3 protein does not localize at kinetochores in the grr1-20 suppressor. .............Figure 35 : Ypi1 and Sds22 protein levels are not controlled by the SCF Grr1 complex. ........................Figure 36 : A second mutation in the mps1-3 allele suppresses the temperature-sensitivity of mps1-3 cells. ...............................................................................................................................................................Figure 37 : A second mutation in MPS1 leads to suppression of the chromosome segregation and SAC defects of the mps1-3 mutant. .....................................................................................................................Figure 38 : A 3HA tag at the C-terminus of Mps1 decreases suppressive effects of the intragenic suppressors. ..................................................................................................................................................Figure 39 : The intragenic suppressors restore Mps1 kinetochore localization. ...................................Figure 40 : The Mps1-sup proteins bear somewhat decreased kinase activity. .....................................Figure 41 : Recombinant Mps1-sup kinases might bear reduced kinase activity on Spc105 compared to Mps1-3. .........................................................................................................................................................Figure 42 : The intragenic suppressing mutations lower Mps1 kinase activity on Spc105. .................Figure 43 : Cells containing suppressing mutations proliferate normally, except for mps1-G449S. ...

Figure 35 :

 35 Figure 1 : Budding yeast mitosis. .................................................................................................................Figure 2 : A diagram of the main mitotic events in mammalian cells. .....................................................Figure 3 : The γ-tubulin small complex (γ-TuSC). .....................................................................................Figure 4 : The spindle pole body in budding yeast. ....................................................................................Figure 5 : SPB duplication. ...........................................................................................................................Figure 6 : The centrosome. ...........................................................................................................................Figure 7 : The centrosome cycle. ..................................................................................................................Figure 8 : The γ-tubulin template model. ....................................................................................................Figure 9 : Condensin architecture. ..............................................................................................................Figure 10 : Loop extrusion induced by a single condensin complex. ........................................................Figure 11 : Effects of condensin I and II on mitotic chromosome condensation. ....................................Figure 12 : Structure of the cohesin ring. ....................................................................................................Figure 13 : Models proposed for chromatin entrapment by cohesin. .......................................................Figure 14 : The cohesin cycle in human cells. .............................................................................................Figure 15 : Schematic kinetochore structure. .............................................................................................Figure 16 : The end-on conversion process. ................................................................................................Figure 17 : kinetochore orientation in mitosis and meiosis I. ....................................................................Figure 18 : Types of kinetochore-microtubule attachments. .....................................................................Figure 19 : SAC principles. ...........................................................................................................................Figure 20 : Domain organisation of human MPS1. ....................................................................................Figure 21 : Regulation of Mps1 during mitosis in humans. .......................................................................Figure 22 : Spindle Assembly Checkpoint activation. ................................................................................Figure 23 : The Mad2 template model. ........................................................................................................Figure 24 : The Chromosomal Passenger Complex and its recruitment to centromeres. ......................Figure 25 : The tension-related model. ........................................................................................................Figure 26 : The "dog leash" model for CPC function. ...............................................................................Figure 27 : Regulation of PP1 kinetochore recruitment. ...........................................................................Figure 28 : Mechanisms of Spindle Assembly Checkpoint silencing. .......................................................Figure 29 : grr1 mutations suppress the temperature-sensitivity of mps1-3 cells. .................................Figure 30 : grr1 mutations restore SAC signaling and proper chromosome segregation. ....................Figure 31 : Grr1 is degraded in the grr1 suppressors. .............................................................................Figure 32 : Grr1 localizes at kinetochores of wild type but not of mps1-3 cells. ....................................Figure 33 : Mps1 is present at similar levels in wild type and grr1-20 mutant cells. .............................Figure 34 : The Mps1-3 protein does not localize at kinetochores in the grr1-20 suppressor. .............Figure 35 : Ypi1 and Sds22 protein levels are not controlled by the SCF Grr1 complex. ........................Figure 36 : A second mutation in the mps1-3 allele suppresses the temperature-sensitivity of mps1-3 cells. ...............................................................................................................................................................Figure 37 : A second mutation in MPS1 leads to suppression of the chromosome segregation and SAC defects of the mps1-3 mutant. .....................................................................................................................Figure 38 : A 3HA tag at the C-terminus of Mps1 decreases suppressive effects of the intragenic suppressors. ..................................................................................................................................................Figure 39 : The intragenic suppressors restore Mps1 kinetochore localization. ...................................Figure 40 : The Mps1-sup proteins bear somewhat decreased kinase activity. .....................................Figure 41 : Recombinant Mps1-sup kinases might bear reduced kinase activity on Spc105 compared to Mps1-3. .........................................................................................................................................................Figure 42 : The intragenic suppressing mutations lower Mps1 kinase activity on Spc105. .................Figure 43 : Cells containing suppressing mutations proliferate normally, except for mps1-G449S. ...

Figure 36 :

 36 Figure 1 : Budding yeast mitosis. .................................................................................................................Figure 2 : A diagram of the main mitotic events in mammalian cells. .....................................................Figure 3 : The γ-tubulin small complex (γ-TuSC). .....................................................................................Figure 4 : The spindle pole body in budding yeast. ....................................................................................Figure 5 : SPB duplication. ...........................................................................................................................Figure 6 : The centrosome. ...........................................................................................................................Figure 7 : The centrosome cycle. ..................................................................................................................Figure 8 : The γ-tubulin template model. ....................................................................................................Figure 9 : Condensin architecture. ..............................................................................................................Figure 10 : Loop extrusion induced by a single condensin complex. ........................................................Figure 11 : Effects of condensin I and II on mitotic chromosome condensation. ....................................Figure 12 : Structure of the cohesin ring. ....................................................................................................Figure 13 : Models proposed for chromatin entrapment by cohesin. .......................................................Figure 14 : The cohesin cycle in human cells. .............................................................................................Figure 15 : Schematic kinetochore structure. .............................................................................................Figure 16 : The end-on conversion process. ................................................................................................Figure 17 : kinetochore orientation in mitosis and meiosis I. ....................................................................Figure 18 : Types of kinetochore-microtubule attachments. .....................................................................Figure 19 : SAC principles. ...........................................................................................................................Figure 20 : Domain organisation of human MPS1. ....................................................................................Figure 21 : Regulation of Mps1 during mitosis in humans. .......................................................................Figure 22 : Spindle Assembly Checkpoint activation. ................................................................................Figure 23 : The Mad2 template model. ........................................................................................................Figure 24 : The Chromosomal Passenger Complex and its recruitment to centromeres. ......................Figure 25 : The tension-related model. ........................................................................................................Figure 26 : The "dog leash" model for CPC function. ...............................................................................Figure 27 : Regulation of PP1 kinetochore recruitment. ...........................................................................Figure 28 : Mechanisms of Spindle Assembly Checkpoint silencing. .......................................................Figure 29 : grr1 mutations suppress the temperature-sensitivity of mps1-3 cells. .................................Figure 30 : grr1 mutations restore SAC signaling and proper chromosome segregation. ....................Figure 31 : Grr1 is degraded in the grr1 suppressors. .............................................................................Figure 32 : Grr1 localizes at kinetochores of wild type but not of mps1-3 cells. ....................................Figure 33 : Mps1 is present at similar levels in wild type and grr1-20 mutant cells. .............................Figure 34 : The Mps1-3 protein does not localize at kinetochores in the grr1-20 suppressor. .............Figure 35 : Ypi1 and Sds22 protein levels are not controlled by the SCF Grr1 complex. ........................Figure 36 : A second mutation in the mps1-3 allele suppresses the temperature-sensitivity of mps1-3 cells. ...............................................................................................................................................................Figure 37 : A second mutation in MPS1 leads to suppression of the chromosome segregation and SAC defects of the mps1-3 mutant. .....................................................................................................................Figure 38 : A 3HA tag at the C-terminus of Mps1 decreases suppressive effects of the intragenic suppressors. ..................................................................................................................................................Figure 39 : The intragenic suppressors restore Mps1 kinetochore localization. ...................................Figure 40 : The Mps1-sup proteins bear somewhat decreased kinase activity. .....................................Figure 41 : Recombinant Mps1-sup kinases might bear reduced kinase activity on Spc105 compared to Mps1-3. .........................................................................................................................................................Figure 42 : The intragenic suppressing mutations lower Mps1 kinase activity on Spc105. .................Figure 43 : Cells containing suppressing mutations proliferate normally, except for mps1-G449S. ...

Figure 37 :

 37 Figure 1 : Budding yeast mitosis. .................................................................................................................Figure 2 : A diagram of the main mitotic events in mammalian cells. .....................................................Figure 3 : The γ-tubulin small complex (γ-TuSC). .....................................................................................Figure 4 : The spindle pole body in budding yeast. ....................................................................................Figure 5 : SPB duplication. ...........................................................................................................................Figure 6 : The centrosome. ...........................................................................................................................Figure 7 : The centrosome cycle. ..................................................................................................................Figure 8 : The γ-tubulin template model. ....................................................................................................Figure 9 : Condensin architecture. ..............................................................................................................Figure 10 : Loop extrusion induced by a single condensin complex. ........................................................Figure 11 : Effects of condensin I and II on mitotic chromosome condensation. ....................................Figure 12 : Structure of the cohesin ring. ....................................................................................................Figure 13 : Models proposed for chromatin entrapment by cohesin. .......................................................Figure 14 : The cohesin cycle in human cells. .............................................................................................Figure 15 : Schematic kinetochore structure. .............................................................................................Figure 16 : The end-on conversion process. ................................................................................................Figure 17 : kinetochore orientation in mitosis and meiosis I. ....................................................................Figure 18 : Types of kinetochore-microtubule attachments. .....................................................................Figure 19 : SAC principles. ...........................................................................................................................Figure 20 : Domain organisation of human MPS1. ....................................................................................Figure 21 : Regulation of Mps1 during mitosis in humans. .......................................................................Figure 22 : Spindle Assembly Checkpoint activation. ................................................................................Figure 23 : The Mad2 template model. ........................................................................................................Figure 24 : The Chromosomal Passenger Complex and its recruitment to centromeres. ......................Figure 25 : The tension-related model. ........................................................................................................Figure 26 : The "dog leash" model for CPC function. ...............................................................................Figure 27 : Regulation of PP1 kinetochore recruitment. ...........................................................................Figure 28 : Mechanisms of Spindle Assembly Checkpoint silencing. .......................................................Figure 29 : grr1 mutations suppress the temperature-sensitivity of mps1-3 cells. .................................Figure 30 : grr1 mutations restore SAC signaling and proper chromosome segregation. ....................Figure 31 : Grr1 is degraded in the grr1 suppressors. .............................................................................Figure 32 : Grr1 localizes at kinetochores of wild type but not of mps1-3 cells. ....................................Figure 33 : Mps1 is present at similar levels in wild type and grr1-20 mutant cells. .............................Figure 34 : The Mps1-3 protein does not localize at kinetochores in the grr1-20 suppressor. .............Figure 35 : Ypi1 and Sds22 protein levels are not controlled by the SCF Grr1 complex. ........................Figure 36 : A second mutation in the mps1-3 allele suppresses the temperature-sensitivity of mps1-3 cells. ...............................................................................................................................................................Figure 37 : A second mutation in MPS1 leads to suppression of the chromosome segregation and SAC defects of the mps1-3 mutant. .....................................................................................................................Figure 38 : A 3HA tag at the C-terminus of Mps1 decreases suppressive effects of the intragenic suppressors. ..................................................................................................................................................Figure 39 : The intragenic suppressors restore Mps1 kinetochore localization. ...................................Figure 40 : The Mps1-sup proteins bear somewhat decreased kinase activity. .....................................Figure 41 : Recombinant Mps1-sup kinases might bear reduced kinase activity on Spc105 compared to Mps1-3. .........................................................................................................................................................Figure 42 : The intragenic suppressing mutations lower Mps1 kinase activity on Spc105. .................Figure 43 : Cells containing suppressing mutations proliferate normally, except for mps1-G449S. ...

  Figure 1 : Budding yeast mitosis. .................................................................................................................Figure 2 : A diagram of the main mitotic events in mammalian cells. .....................................................Figure 3 : The γ-tubulin small complex (γ-TuSC). .....................................................................................Figure 4 : The spindle pole body in budding yeast. ....................................................................................Figure 5 : SPB duplication. ...........................................................................................................................Figure 6 : The centrosome. ...........................................................................................................................Figure 7 : The centrosome cycle. ..................................................................................................................Figure 8 : The γ-tubulin template model. ....................................................................................................Figure 9 : Condensin architecture. ..............................................................................................................Figure 10 : Loop extrusion induced by a single condensin complex. ........................................................Figure 11 : Effects of condensin I and II on mitotic chromosome condensation. ....................................Figure 12 : Structure of the cohesin ring. ....................................................................................................Figure 13 : Models proposed for chromatin entrapment by cohesin. .......................................................Figure 14 : The cohesin cycle in human cells. .............................................................................................Figure 15 : Schematic kinetochore structure. .............................................................................................Figure 16 : The end-on conversion process. ................................................................................................Figure 17 : kinetochore orientation in mitosis and meiosis I. ....................................................................Figure 18 : Types of kinetochore-microtubule attachments. .....................................................................Figure 19 : SAC principles. ...........................................................................................................................Figure 20 : Domain organisation of human MPS1. ....................................................................................Figure 21 : Regulation of Mps1 during mitosis in humans. .......................................................................Figure 22 : Spindle Assembly Checkpoint activation. ................................................................................Figure 23 : The Mad2 template model. ........................................................................................................Figure 24 : The Chromosomal Passenger Complex and its recruitment to centromeres. ......................Figure 25 : The tension-related model. ........................................................................................................Figure 26 : The "dog leash" model for CPC function. ...............................................................................Figure 27 : Regulation of PP1 kinetochore recruitment. ...........................................................................Figure 28 : Mechanisms of Spindle Assembly Checkpoint silencing. .......................................................Figure 29 : grr1 mutations suppress the temperature-sensitivity of mps1-3 cells. .................................Figure 30 : grr1 mutations restore SAC signaling and proper chromosome segregation. ....................Figure 31 : Grr1 is degraded in the grr1 suppressors. .............................................................................Figure 32 : Grr1 localizes at kinetochores of wild type but not of mps1-3 cells. ....................................Figure 33 : Mps1 is present at similar levels in wild type and grr1-20 mutant cells. .............................Figure 34 : The Mps1-3 protein does not localize at kinetochores in the grr1-20 suppressor. .............Figure 35 : Ypi1 and Sds22 protein levels are not controlled by the SCF Grr1 complex. ........................Figure 36 : A second mutation in the mps1-3 allele suppresses the temperature-sensitivity of mps1-3 cells. ...............................................................................................................................................................Figure 37 : A second mutation in MPS1 leads to suppression of the chromosome segregation and SAC defects of the mps1-3 mutant. .....................................................................................................................Figure 38 : A 3HA tag at the C-terminus of Mps1 decreases suppressive effects of the intragenic suppressors. ..................................................................................................................................................Figure 39 : The intragenic suppressors restore Mps1 kinetochore localization. ...................................Figure 40 : The Mps1-sup proteins bear somewhat decreased kinase activity. .....................................Figure 41 : Recombinant Mps1-sup kinases might bear reduced kinase activity on Spc105 compared to Mps1-3. .........................................................................................................................................................Figure 42 : The intragenic suppressing mutations lower Mps1 kinase activity on Spc105. .................Figure 43 : Cells containing suppressing mutations proliferate normally, except for mps1-G449S. ...

Figure 39 :

 39 Figure 1 : Budding yeast mitosis. .................................................................................................................Figure 2 : A diagram of the main mitotic events in mammalian cells. .....................................................Figure 3 : The γ-tubulin small complex (γ-TuSC). .....................................................................................Figure 4 : The spindle pole body in budding yeast. ....................................................................................Figure 5 : SPB duplication. ...........................................................................................................................Figure 6 : The centrosome. ...........................................................................................................................Figure 7 : The centrosome cycle. ..................................................................................................................Figure 8 : The γ-tubulin template model. ....................................................................................................Figure 9 : Condensin architecture. ..............................................................................................................Figure 10 : Loop extrusion induced by a single condensin complex. ........................................................Figure 11 : Effects of condensin I and II on mitotic chromosome condensation. ....................................Figure 12 : Structure of the cohesin ring. ....................................................................................................Figure 13 : Models proposed for chromatin entrapment by cohesin. .......................................................Figure 14 : The cohesin cycle in human cells. .............................................................................................Figure 15 : Schematic kinetochore structure. .............................................................................................Figure 16 : The end-on conversion process. ................................................................................................Figure 17 : kinetochore orientation in mitosis and meiosis I. ....................................................................Figure 18 : Types of kinetochore-microtubule attachments. .....................................................................Figure 19 : SAC principles. ...........................................................................................................................Figure 20 : Domain organisation of human MPS1. ....................................................................................Figure 21 : Regulation of Mps1 during mitosis in humans. .......................................................................Figure 22 : Spindle Assembly Checkpoint activation. ................................................................................Figure 23 : The Mad2 template model. ........................................................................................................Figure 24 : The Chromosomal Passenger Complex and its recruitment to centromeres. ......................Figure 25 : The tension-related model. ........................................................................................................Figure 26 : The "dog leash" model for CPC function. ...............................................................................Figure 27 : Regulation of PP1 kinetochore recruitment. ...........................................................................Figure 28 : Mechanisms of Spindle Assembly Checkpoint silencing. .......................................................Figure 29 : grr1 mutations suppress the temperature-sensitivity of mps1-3 cells. .................................Figure 30 : grr1 mutations restore SAC signaling and proper chromosome segregation. ....................Figure 31 : Grr1 is degraded in the grr1 suppressors. .............................................................................Figure 32 : Grr1 localizes at kinetochores of wild type but not of mps1-3 cells. ....................................Figure 33 : Mps1 is present at similar levels in wild type and grr1-20 mutant cells. .............................Figure 34 : The Mps1-3 protein does not localize at kinetochores in the grr1-20 suppressor. .............Figure 35 : Ypi1 and Sds22 protein levels are not controlled by the SCF Grr1 complex. ........................Figure 36 : A second mutation in the mps1-3 allele suppresses the temperature-sensitivity of mps1-3 cells. ...............................................................................................................................................................Figure 37 : A second mutation in MPS1 leads to suppression of the chromosome segregation and SAC defects of the mps1-3 mutant. .....................................................................................................................Figure 38 : A 3HA tag at the C-terminus of Mps1 decreases suppressive effects of the intragenic suppressors. ..................................................................................................................................................Figure 39 : The intragenic suppressors restore Mps1 kinetochore localization. ...................................Figure 40 : The Mps1-sup proteins bear somewhat decreased kinase activity. .....................................Figure 41 : Recombinant Mps1-sup kinases might bear reduced kinase activity on Spc105 compared to Mps1-3. .........................................................................................................................................................Figure 42 : The intragenic suppressing mutations lower Mps1 kinase activity on Spc105. .................Figure 43 : Cells containing suppressing mutations proliferate normally, except for mps1-G449S. ...

Figure 40 :

 40 Figure 1 : Budding yeast mitosis. .................................................................................................................Figure 2 : A diagram of the main mitotic events in mammalian cells. .....................................................Figure 3 : The γ-tubulin small complex (γ-TuSC). .....................................................................................Figure 4 : The spindle pole body in budding yeast. ....................................................................................Figure 5 : SPB duplication. ...........................................................................................................................Figure 6 : The centrosome. ...........................................................................................................................Figure 7 : The centrosome cycle. ..................................................................................................................Figure 8 : The γ-tubulin template model. ....................................................................................................Figure 9 : Condensin architecture. ..............................................................................................................Figure 10 : Loop extrusion induced by a single condensin complex. ........................................................Figure 11 : Effects of condensin I and II on mitotic chromosome condensation. ....................................Figure 12 : Structure of the cohesin ring. ....................................................................................................Figure 13 : Models proposed for chromatin entrapment by cohesin. .......................................................Figure 14 : The cohesin cycle in human cells. .............................................................................................Figure 15 : Schematic kinetochore structure. .............................................................................................Figure 16 : The end-on conversion process. ................................................................................................Figure 17 : kinetochore orientation in mitosis and meiosis I. ....................................................................Figure 18 : Types of kinetochore-microtubule attachments. .....................................................................Figure 19 : SAC principles. ...........................................................................................................................Figure 20 : Domain organisation of human MPS1. ....................................................................................Figure 21 : Regulation of Mps1 during mitosis in humans. .......................................................................Figure 22 : Spindle Assembly Checkpoint activation. ................................................................................Figure 23 : The Mad2 template model. ........................................................................................................Figure 24 : The Chromosomal Passenger Complex and its recruitment to centromeres. ......................Figure 25 : The tension-related model. ........................................................................................................Figure 26 : The "dog leash" model for CPC function. ...............................................................................Figure 27 : Regulation of PP1 kinetochore recruitment. ...........................................................................Figure 28 : Mechanisms of Spindle Assembly Checkpoint silencing. .......................................................Figure 29 : grr1 mutations suppress the temperature-sensitivity of mps1-3 cells. .................................Figure 30 : grr1 mutations restore SAC signaling and proper chromosome segregation. ....................Figure 31 : Grr1 is degraded in the grr1 suppressors. .............................................................................Figure 32 : Grr1 localizes at kinetochores of wild type but not of mps1-3 cells. ....................................Figure 33 : Mps1 is present at similar levels in wild type and grr1-20 mutant cells. .............................Figure 34 : The Mps1-3 protein does not localize at kinetochores in the grr1-20 suppressor. .............Figure 35 : Ypi1 and Sds22 protein levels are not controlled by the SCF Grr1 complex. ........................Figure 36 : A second mutation in the mps1-3 allele suppresses the temperature-sensitivity of mps1-3 cells. ...............................................................................................................................................................Figure 37 : A second mutation in MPS1 leads to suppression of the chromosome segregation and SAC defects of the mps1-3 mutant. .....................................................................................................................Figure 38 : A 3HA tag at the C-terminus of Mps1 decreases suppressive effects of the intragenic suppressors. ..................................................................................................................................................Figure 39 : The intragenic suppressors restore Mps1 kinetochore localization. ...................................Figure 40 : The Mps1-sup proteins bear somewhat decreased kinase activity. .....................................Figure 41 : Recombinant Mps1-sup kinases might bear reduced kinase activity on Spc105 compared to Mps1-3. .........................................................................................................................................................Figure 42 : The intragenic suppressing mutations lower Mps1 kinase activity on Spc105. .................Figure 43 : Cells containing suppressing mutations proliferate normally, except for mps1-G449S. ...

Figure 41 :

 41 Figure 1 : Budding yeast mitosis. .................................................................................................................Figure 2 : A diagram of the main mitotic events in mammalian cells. .....................................................Figure 3 : The γ-tubulin small complex (γ-TuSC). .....................................................................................Figure 4 : The spindle pole body in budding yeast. ....................................................................................Figure 5 : SPB duplication. ...........................................................................................................................Figure 6 : The centrosome. ...........................................................................................................................Figure 7 : The centrosome cycle. ..................................................................................................................Figure 8 : The γ-tubulin template model. ....................................................................................................Figure 9 : Condensin architecture. ..............................................................................................................Figure 10 : Loop extrusion induced by a single condensin complex. ........................................................Figure 11 : Effects of condensin I and II on mitotic chromosome condensation. ....................................Figure 12 : Structure of the cohesin ring. ....................................................................................................Figure 13 : Models proposed for chromatin entrapment by cohesin. .......................................................Figure 14 : The cohesin cycle in human cells. .............................................................................................Figure 15 : Schematic kinetochore structure. .............................................................................................Figure 16 : The end-on conversion process. ................................................................................................Figure 17 : kinetochore orientation in mitosis and meiosis I. ....................................................................Figure 18 : Types of kinetochore-microtubule attachments. .....................................................................Figure 19 : SAC principles. ...........................................................................................................................Figure 20 : Domain organisation of human MPS1. ....................................................................................Figure 21 : Regulation of Mps1 during mitosis in humans. .......................................................................Figure 22 : Spindle Assembly Checkpoint activation. ................................................................................Figure 23 : The Mad2 template model. ........................................................................................................Figure 24 : The Chromosomal Passenger Complex and its recruitment to centromeres. ......................Figure 25 : The tension-related model. ........................................................................................................Figure 26 : The "dog leash" model for CPC function. ...............................................................................Figure 27 : Regulation of PP1 kinetochore recruitment. ...........................................................................Figure 28 : Mechanisms of Spindle Assembly Checkpoint silencing. .......................................................Figure 29 : grr1 mutations suppress the temperature-sensitivity of mps1-3 cells. .................................Figure 30 : grr1 mutations restore SAC signaling and proper chromosome segregation. ....................Figure 31 : Grr1 is degraded in the grr1 suppressors. .............................................................................Figure 32 : Grr1 localizes at kinetochores of wild type but not of mps1-3 cells. ....................................Figure 33 : Mps1 is present at similar levels in wild type and grr1-20 mutant cells. .............................Figure 34 : The Mps1-3 protein does not localize at kinetochores in the grr1-20 suppressor. .............Figure 35 : Ypi1 and Sds22 protein levels are not controlled by the SCF Grr1 complex. ........................Figure 36 : A second mutation in the mps1-3 allele suppresses the temperature-sensitivity of mps1-3 cells. ...............................................................................................................................................................Figure 37 : A second mutation in MPS1 leads to suppression of the chromosome segregation and SAC defects of the mps1-3 mutant. .....................................................................................................................Figure 38 : A 3HA tag at the C-terminus of Mps1 decreases suppressive effects of the intragenic suppressors. ..................................................................................................................................................Figure 39 : The intragenic suppressors restore Mps1 kinetochore localization. ...................................Figure 40 : The Mps1-sup proteins bear somewhat decreased kinase activity. .....................................Figure 41 : Recombinant Mps1-sup kinases might bear reduced kinase activity on Spc105 compared to Mps1-3. .........................................................................................................................................................Figure 42 : The intragenic suppressing mutations lower Mps1 kinase activity on Spc105. .................Figure 43 : Cells containing suppressing mutations proliferate normally, except for mps1-G449S. ...

Figure 42 :

 42 Figure 1 : Budding yeast mitosis. .................................................................................................................Figure 2 : A diagram of the main mitotic events in mammalian cells. .....................................................Figure 3 : The γ-tubulin small complex (γ-TuSC). .....................................................................................Figure 4 : The spindle pole body in budding yeast. ....................................................................................Figure 5 : SPB duplication. ...........................................................................................................................Figure 6 : The centrosome. ...........................................................................................................................Figure 7 : The centrosome cycle. ..................................................................................................................Figure 8 : The γ-tubulin template model. ....................................................................................................Figure 9 : Condensin architecture. ..............................................................................................................Figure 10 : Loop extrusion induced by a single condensin complex. ........................................................Figure 11 : Effects of condensin I and II on mitotic chromosome condensation. ....................................Figure 12 : Structure of the cohesin ring. ....................................................................................................Figure 13 : Models proposed for chromatin entrapment by cohesin. .......................................................Figure 14 : The cohesin cycle in human cells. .............................................................................................Figure 15 : Schematic kinetochore structure. .............................................................................................Figure 16 : The end-on conversion process. ................................................................................................Figure 17 : kinetochore orientation in mitosis and meiosis I. ....................................................................Figure 18 : Types of kinetochore-microtubule attachments. .....................................................................Figure 19 : SAC principles. ...........................................................................................................................Figure 20 : Domain organisation of human MPS1. ....................................................................................Figure 21 : Regulation of Mps1 during mitosis in humans. .......................................................................Figure 22 : Spindle Assembly Checkpoint activation. ................................................................................Figure 23 : The Mad2 template model. ........................................................................................................Figure 24 : The Chromosomal Passenger Complex and its recruitment to centromeres. ......................Figure 25 : The tension-related model. ........................................................................................................Figure 26 : The "dog leash" model for CPC function. ...............................................................................Figure 27 : Regulation of PP1 kinetochore recruitment. ...........................................................................Figure 28 : Mechanisms of Spindle Assembly Checkpoint silencing. .......................................................Figure 29 : grr1 mutations suppress the temperature-sensitivity of mps1-3 cells. .................................Figure 30 : grr1 mutations restore SAC signaling and proper chromosome segregation. ....................Figure 31 : Grr1 is degraded in the grr1 suppressors. .............................................................................Figure 32 : Grr1 localizes at kinetochores of wild type but not of mps1-3 cells. ....................................Figure 33 : Mps1 is present at similar levels in wild type and grr1-20 mutant cells. .............................Figure 34 : The Mps1-3 protein does not localize at kinetochores in the grr1-20 suppressor. .............Figure 35 : Ypi1 and Sds22 protein levels are not controlled by the SCF Grr1 complex. ........................Figure 36 : A second mutation in the mps1-3 allele suppresses the temperature-sensitivity of mps1-3 cells. ...............................................................................................................................................................Figure 37 : A second mutation in MPS1 leads to suppression of the chromosome segregation and SAC defects of the mps1-3 mutant. .....................................................................................................................Figure 38 : A 3HA tag at the C-terminus of Mps1 decreases suppressive effects of the intragenic suppressors. ..................................................................................................................................................Figure 39 : The intragenic suppressors restore Mps1 kinetochore localization. ...................................Figure 40 : The Mps1-sup proteins bear somewhat decreased kinase activity. .....................................Figure 41 : Recombinant Mps1-sup kinases might bear reduced kinase activity on Spc105 compared to Mps1-3. .........................................................................................................................................................Figure 42 : The intragenic suppressing mutations lower Mps1 kinase activity on Spc105. .................Figure 43 : Cells containing suppressing mutations proliferate normally, except for mps1-G449S. ...

  Syndromes caused by erroneous chromosome segregations in meiosis. ................................... Table 2 : Mutations in grr1 and rsc30 found in the suppressor screen. ................................................. : γ-Tubulin ring complex MT : microtubule NEBD : Nuclear Envelope Breakdown ATP : Adenosine Triphosphate ABC : ATP-binding cassette HEAT : Huntingtin, Elongation factor 3, Protein Phosphatase 2A, TOR1 MELT : Met-Glu-Leu-Thr TADs : Topologically Associating Domain GTP : Guanosine Triphosphate GDP : Guanosine Diphosphate MCC : Mitotic Checkpoint Complex APC : Anaphase Promoting Complex NTE : N-Terminal Extension TPR : N-terminal Tetratricopeptide Repeat domain CH : Calponin Homology MR : Middle Region GEF Guanine-nucleotide Exchange Factor CPC : Chromosomal Passenger Complex RVxF : Arginine, Valine, any aminoacid, Phenylalanine SCF : Skp1-Cullin-F-box protein RSC : Chromatin structure Remodeling Complex CYC : cycling cells aa : amino acid supp : suppressor kDa : kilodalton Kb : kilobase

Figure 1 :

 1 Figure 1 : Budding yeast mitosis.Budding yeast mitosis is closed, meaning that the nuclear membrane never breaks down. Therefore chromosome segregation takes place inside the nucleus. During G1, yeast cells grow and start to duplicate the SPBs (spindle pole bodies, the microtubule organising centers); here first monopolar attachments take place. At the G1/S transition bud emergence occurs, SPBs duplicate and chromosomal DNA starts being replicated, leading to the temporary dissociation of kinetochores from microtubules. In G2 the spindle becomes bipolar and in metaphase sister chromatids bi-orient on the mitotic spindle, meaning that they attach to microtubules emanating from the two opposite spindle poles. During anaphase the spindle elongates and the two sets of sister chromatids segregate to spindle poles. Finally during telophase the nuclear membrane separates into two separated nuclei along with the two newformed cells, which separate through cytokinesis.

Figure 2 :

 2 Figure 2 : A diagram of the main mitotic events in mammalian cells. Interphase (G2): in this stage the cell prepares for division and synthesizes essential cell cycle proteins (e.g. mitotic cyclins). Prophase: in this stage chromosomes condense and the centrosomes separate and start forming a mitotic spindle. Prometaphase: the nuclear envelope breaks down and microtubules start attaching to kinetochores. Metaphase: chromosomes are connected to spindle fibers in a bipolar fashion and become aligned on the metaphase plate. Anaphase: sister chromatids separate and are pulled apart towards the spindle poles. Telophase and cytokinesis: chromosomes decondense and are again surrounded by the nuclear envelope that reforms. The two daughter cells physically separate. From Campbell Biology (10 th edition) by J. B. Reece & S. A. Wasserman.

Figure 3 :

 3 Figure 3 : The γ-tubulin small complex (γ-TuSC). The γ-TuSC is composed of two molecules of Tub4 (γ-tubulin) and one molecule of each γ-tubulin complex proteins (GCP) Spc97 and Spc98. Adapted from Tovey and Conduit, 2018.

Figure 4 :

 4 Figure4: The spindle pole body in budding yeast. i) Organization of the budding yeast SPB. The SPBs (brown spheres) are inserted into the nuclear membrane (light circle); green = microtubules. ii) SPB structure and protein composition. For details see main text. From[START_REF] Cavanaugh | Big Lessons from Little Yeast: Budding and Fission Yeast Centrosome Structure, Duplication, and Function[END_REF]

Figure 5 :

 5 Figure 5 : SPB duplication.SPB duplication can be divided into three steps: 1) half-bridge elongation and deposition of the satellite; 2) expansion of the satellite into a duplication plaque; 3) insertion of the duplication plaque into the nuclear envelope and assembly of the inner plaque; 4) after cleavage of the bridge connecting the two SPBs, the SPBs move to opposite sides of the nuclear envelope. From[START_REF] Jaspersen | THE BUDDING YEAST SPINDLE POLE BODY: Structure, Duplication, and Function[END_REF]

Figure 6 :

 6 Figure 6 : The centrosome.Centrosomes contains a mother and a daughter centriole connected by fibers embedded in the Pericentriolar Material (PCM). PCM nucleates microtubules (MT), which, after release, will become anchored to the mother centriole by subdistal appendages of the mother centriole. Microtubule minus (-) ends are stabilized at the centrosome, and dynamic plus (+) ends are directed outside the PCM. FromKloc et al., 2013.

Figure 7 :

 7 Figure 7 : The centrosome cycle.At the beginning of G1, cells contain a single centrosome with two perpendicular centrioles. During G1 the link between the centrioles (purple bar) is dissolved (centriole disengagement) and centrioles are only connected by a fibrous structure (purple strands). In S phase, the duplicating centrosomes assemble two new centrioles perpendicular to the two existing ones. Then, the daughter centriole finally acquires the same features of the mother centriole and the fibrous linker between daughter and mother centrioles is cleaved. Due to the link between the old and the newly formed centriole for each couple (purple bars), the two centrosomes are now engaged and prevented from further replication. In late G2, the two centrosomes mature by recruiting additional PCM components (grey shadow) in order to form the mitotic spindle. The centrosomes then separate and move to the opposite sides of the nucleus. This is simultaneous with nuclear envelope breakdown (NEBD). FromBarr and Gergely, 2007.

Figure 8 :

 8 Figure 8 : The γ-tubulin template model. Microtubule polymerization involves longitudinal interactions of αβ-tubulin heterodimers with γ-tubulin in the γTuRC (template nucleation model).From[START_REF] Sulimenko | Regulation of microtubule nucleation mediated by γ-tubulin complexes[END_REF]

Figure 9 :

 9 Figure 9 : Condensin architecture.The eukaryotic condensin complexes (I and II) share the same SMC proteins core subunits (SMC2/4). In contrast, they have distinct sets of non-SMC regulatory subunits, which comprise a kleisin-subunit (CAP-H or CAP-H2) and two HEAT repeats containing proteins (CAP-D2/G and CAP-D3/G2). From[START_REF] Hirano | Condensin-Based Chromosome Organization from Bacteria to Vertebrates[END_REF]

Figure 10 :

 10 Figure 10 : Loop extrusion induced by a single condensin complex.One strand of DNA is anchored by the kleisin and HEAT-repeated subunits (in yellow) of the condensin complex, which extrudes a loop of DNA. From[START_REF] Ganji | Real-time imaging of DNA loop extrusion by condensin[END_REF]

Figure 11 :

 11 Figure 11 : Effects of condensin I and II on mitotic chromosome condensation. CAP-H siRNA cells lack of condensin I and CAP-D3 siRNA cells lack of condensin II.In the first case it is possible to notice shorter and larger chromosomes, whereas in the second case they are longer and less rigid. From[START_REF] Kalitsis | Condensin, master organizer of the genome[END_REF]

Figure 12 :

 12 Figure 12 : Structure of the cohesin ring.The cohesin ring is formed by two rod-shaped SMC proteins, Smc1 (in red) and Smc3 (in light blue), that hold a dimerization domain on one end and an ATPase domain on the other. The interaction between the dimerization domains creates a V-structure. The ring is closed by an αkleisin subunit (in yellow), which binds to Smc3 and Smc1 and also, with its central domain, to Scc3/SA and Pds5. Pds5 in turn binds to Wapl1. Adapted from Nasmyth, 2011.

Figure 13 :

 13 Figure 13 : Models proposed for chromatin entrapment by cohesin. A: one ring (embrace) model: the two sister chromatids (10nm each) are entrapped by one cohesin ring (40nm). B: two rings (handcuff) model: each cohesin ring embraces a single chromatid.C: bracelet model: cohesin rings oligomerize in a hand-to-hand fashion to hold the sister chromatids together. D: rod model: cohesin rings associate with one another by the SMC coiled coils to make a rod-like structure. From[START_REF] Mehta | Cohesin: A guardian of genome integrity[END_REF]

Figure 14 :

 14 Figure 14 : The cohesin cycle in human cells.The Scc2/Scc4 complex promotes cohesin loading in telophase and early G1. During DNA replication, Esco1/2 and Sororin are required to establish chromosome cohesion. In prophase, cohesin is removed from the chromosome arms by the action of mitotic kinases and the Wapl1/Pds5 complex, whereas cohesin at centromeres is protected by the Sgo1/PP2A complex until anaphase. After sister chromatid bi-orientation and SAC silencing, Separase is activated and cleaves centromeric cohesin, enabling sister chromatid separation in anaphase. From[START_REF] Zheng | Regulation of sister chromatid cohesion during the mitotic cell cycle[END_REF]

Figure 15 :

 15 Figure 15 : Schematic kinetochore structure.The kinetochore is assembled on the centromere to form an interface for the interaction with microtubules. Major components are the Constitutive Centromere Associated Network (CCAN) and the KMN (Knl1, Mis12 and the Ndc80 complex) network. The CCAN binds to chromatin through the CENP-A-containing nucleosomes. The KMNnetwork binds to the CCAN through interaction with CENP-C or CENP-T. Finally, the Ndc80 complex directly contacts microtubules. FromHara and Fukagawa, 2018.

Figure 16 :

 16 Figure16: The end-on conversion process. Kinetochores are first captured along lateral walls of microtubules, then are tethered to the ends of microtubules, guided by the kinetochore and microtubule-associated proteins. The end-on conversion process enables the formation of mature end-on tethered kinetochores. FromConti et al., 2017.

Figure 17 :

 17 Figure 17 : kinetochore orientation in mitosis and meiosis I.Left: in mitosis sister chromatids are attached from microtubules emanated from opposite spindle poles, leading to chromatids biorientation. Right: during meiosis I homologous chromosomes are attached to microtubules emanating from opposite spindle poles, leading to bi-orientation of homologs, while sister chromatids are attached to microtubules emanating from the same pole, leading to sister chromatids monoorientation. From[START_REF] Duro | From equator to pole: splitting chromosomes in mitosis and meiosis[END_REF]

Figure 18 :

 18 Figure 18 : Types of kinetochore-microtubule attachments.Monotelic attachment: one of the sister kinetochores attaches to microtubules, whereas the other does not. Syntelic attachment: both sister kinetochores attach to microtubules emanating from the same spindle pole. Amphitelic attachment: sister kinetochores attach to microtubules emanating from opposite spindle poles. Merotelic attachment: one sister kinetochore attaches simultaneously to microtubules extending from both spindle poles. FromTanaka et al., 2005.

Figure 19 :

 19 Figure 19 : SAC principles. During prometaphase, unattached kinetochores promote the formation of the MCC (Mitotic Checkpoint Complex), composed of BubR1, Bub3, Mad2 and Cdc20, and inhibiting the APC/C bound to its activator Cdc20. Once all chromosomes are bi-oriented in metaphase, the MCC is no longer generated, thus leading to APC/C activation by Cdc20, allowing ubiquitylation and degradation of Securin and cyclin B1. Degradation of Securin causes disruption of cohesin rings through liberation of Separase and subsequent cleavage of the cohesion subunit Scc1. This leads to sister chromatid separation. Moreover, cyclin B1 degradation inactivates Cdk1, causing mitotic exit. From Lara-Gonzales et al., 2011.

Figure 20 :

 20 Figure 20 : Domain organisation of human MPS1. The kinase domain is located at the Cterminus. The other functionally characterised sequences are involved in Mps1 activation and kinetochore localisation. The NTE and MR domains are necessary for the interaction with the NDC80 complex. The TPR domain is involved in the regulation of Mps1 kinetochore recruitment. From Pachis and Kops, 2018.

Figure 21 :

 21 Figure 21 : Regulation of Mps1 during mitosis in humans.In the absence of Aurora B activity (a), the TPR domain inhibits the NTE (A) and HEC1 interacts with NUF2, blocking the MR-NUF2 interaction (B). When Aurora B is active (b), the NTE is released from the TPR inhibition. ARHGEF17 interacts with inactive Mps1 (D) and localises it to kinetochores where it binds to the NDC80 complex via the NTE and MR regions (E). The phosphorylation of the HEC1 tail by Aurora B prevents the binding to NUF2 and allows the MR to bind there. Binding of inactive Mps1 to the NDC80 with both subunits causes tight binding and long kinetochore residence time. The slow turnover allows Mps1 autoactivation, which will change conformation and will bind only HEC1 (F). Preventing MR binding to NUF2 lowers the affinity of Mps1 for the NDC80 complex, causing a higher turnover rate at kinetochores. From[START_REF] Pachis | Leader of the SAC: molecular mechanisms of Mps1/TTK regulation in mitosis[END_REF]

Figure 22 :

 22 Figure 22 : Spindle Assembly Checkpoint activation. The SAC is activated in presence of unattached kinetochores. The kinase Mps1 phosphorylates Knl1 on its MELT repeats, leading to recruitment of the dimer Bub3-Bub1. BubR1 is also recruited, binding to Bub1. Mps1 also phosphorylates Bub1 and Mad1 in order to allow their interaction and the interaction of Mad1 with Cdc20. Once Mad2 binds to Mad1, it is activated and incorporated into the MCC, thus inhibiting the APC/C. Adapted from Henriques et al., 2019.

Figure 23 :

 23 Figure 23 : The Mad2 template model. The Mad1-Mad2 complex recruits O-Mad2 to kinetochores, and O-Mad2 is converted into C-Mad2 bound to Cdc20. The C-Mad2-Cdc20 complex dissociates from the Mad1-Mad2 core complex. In the cytosol, Mad2-Cdc20 acts as a structural equivalent of Mad1-Mad2 and converts more O-Mad2 into Cdc20-bound C-Mad2. Signal amplification occurs because Mad2-Cdc20 converts O-Mad2 to Cdc20 bound C-Mad2. Dashed arrows represent hypothetical steps related to the amplification of the SAC signal by Mad2-Cdc20. From De Antoni et al., 2005.

  During the metaphase arrest induced by SAC, cells need to catch up and correct improper kinetochore-microtubule attachments to achieve chromatids bi-orientation. The main player in this process is the Chromosomal Passenger Complex (CPC). The CPC is composed by different proteins that form two main modules linked by a central region: the localization module is composed of the N-terminal part of INCENP (INner CENtromere Protein), Survivin and Borealin, that all together form a three-helix bundle; the kinase module is composed of the C-terminal part of INCENP together with the kinase Aurora B. Aurora B is part of the conserved family of serine-threonine kinases, which includes Aurora A, Aurora B and Aurora C, three protein kinases that plays essential roles during mitosis and meiosis (reviewed inCarmena et al. 2012). CPC subunits are conserved in budding yeast:

Figure 26 :

 26 Figure 26 : The "dog leash" model for CPC function.A. The idea behind the dog leash model is that the localisation model of the CPC (the owner) is tethered at the base of centromeres. INCENP acts as a "dog leash" allowing "the dog" (Aurora B) to phosphorylates substrates only in the limit defined by the length of the leash. B-C. Application of the leash model. B. Under low tension Aurora B can reach the kinetochore and phosphorylate its substrates. C. Under high tension substrates have crossed the limit defined by the leash and can no longer be phosphorylated by the kinase. FromKrenn and Musacchio, 2015.

Figure 27 :

 27 Figure 27 : Regulation of PP1 kinetochore recruitment. When kinetochores are not attached, Aurora B inhibits PP1 recruitment to kinetochores by phosphorylating its binding motif on KNL1. Dephosphorylation of this site promotes recruitment of PP1, thus allowing the phosphatase to silence the SAC. From Musacchio, 2015.

Figure 28 :

 28 Figure 28 : Mechanisms of Spindle Assembly Checkpoint silencing. (A) Mad2 inhibition by p31 comet and TRIP13, which convert active C-Mad2 to the unbound O-Mad2 conformation, thus promoting MCC dissociation. (B) Release of Mps1 from kinetochores and dephosphorylation of KNL1 by PP2A and PP1. (C) Dynein-mediated stripping of SAC components, from kinetochores towards spindle poles. (D) Ubiquitination and degradation of Cdc20, mediated by the Apc15 subunit of the APC/C. (E) Consequences of APC/C reactivation: ubiquitylation and degradation of cyclin B and Securin. Adapted from Henriques et al., 2019.

  Figure 1.

FFigure 2 .

 2 Figure 2.

  Fig S2). Y-axis shows enrichment values (linear scale, range is 0-10). Values below 1.5 are shown in grey, and values above 1.5 (i.e. sequences enriched in ChIP samples) are red coloured.E Cells with the indicated genotypes were synchronised in G1 with a-factor at 25°C and then released in fresh medium at 34°C (t = 0). Cells were collected at the indicated time points for FACS analysis of DNA contents.

  Figure 3.

2 or 4

 4 min by time-lapse fluorescence microscopy. Chromosome V segregation errors are reported in the table (H). Representative cells are shown as examples in the montages (G). Representative montages for wild-type and mps1-3 cells are shown in Fig 1F. DIC: differential interference contrast. Scale bar: 5 lm. I Wild-type, mps1-3, mps1-3 spc105-18 and mps1-3 GLC7-24 cells were synchronised in G1 with a-factor at 25°C and then released at 32°C in the presence of nocodazole (note that the presence of 3HA tags at the C-terminus of Mps1-3 slightly decreases the maximal temperature of suppression). Cells were collected after 90 min and fixed with formaldehyde for ChIP-seq analysis. ChIP sequence reads were normalised against sequence reads from corresponding input samples, and relative enrichment is plotted for chromosome III around the centromere (see the centromeric regions of all 16 yeast chromosomes in Appendix Fig S5). Y-axis shows enrichment values (linear scale, range is 0-10). Values below 1.5 are shown in grey, and values above 1.5 (i.e. sequences enriched in ChIP samples) are red coloured. 60-150 min after release from G1, Fig 4A). Phosphorylation was impaired in mps1-3 cells under the same conditions, in agreement with the notion that Spc105 is a critical Mps1 substrate during SAC activation [15]. Importantly, Spc105 phosphorylation was efficiently restored, if not accentuated, in the mps1-3 spc105-18 and mps1-3 GLC7-24 mutants (Fig 4A), consistent with defective PP1 activity at kinetochores that counteracts Mps1-dependent phosphorylation of Spc105 during SAC signalling. Interestingly, we could detect a slight electrophoretic mobility shift of Spc105 also in wild-type cells undergoing a normal cell cycle in the absence of nocodazole (Fig 4B, times 30 0 -40 0 after release from G1, which corresponds to cells in S phase), suggesting that Mps1 might phosphorylate a low number of MELT repeats in unperturbed conditions. Consistent with this hypothesis, an unphosphorylatable MELT mutant (spc105-6A) is unable to recruit Bub1 to kinetochores also during unperturbed conditions and displays chromosome segregation defects [15]. Spc105 phosphorylation was not apparent in mps1-3 cells at 34°C and was restored in mps1-3 spc105-18 cells (Fig 4B). It is worth noting that the mobility shift of Spc105 was even more conspicuous and present for a larger cell cycle window in mps1-3 spc105-18 cells

Figure 4 .

 4 Figure 4. Spc105 phosphorylation and Bub1 kinetochore recruitment are impaired in mps1-3 cells.

  Montages show representative cells (C). Arrowheads indicate Bub1-GFP signals at kinetochores. DIC: differential interference contrast. Scale bar: 5 lm. GFP signals (Bub1) co-localising with Tomato signals (kinetochores) were scored on the movies (D). E-H Wild-type and mps1-3 cells were synchronised in G1 with a-factor at 25°C and then released at 34°C in the presence (E, F) or absence (G, H) of nocodazole (t = 0). Cells were collected at the indicated time points for Western blot analysis of the indicated proteins (E, G) and for FACS analysis of DNA contents (F, H). Equal amounts of protein extracts were loaded on two different gels, for Western blot of Bub1-3HA/Cdc5 and Clb2/Pgk1, respectively. A white asterisk indicates a hyperphosphorylated isoform of Bub1 that in wild-type cells correlates with lack of chromosome biorientation. Cdc5 and Clb2 were used as mitotic markers and Pgk1 as loading control. Cyc: cycling cells. ª 2020 The Authors EMBO reports e50257 | 2020 than in the wild type (Fig 4B), in line with the impaired PP1 activity at Spc105 in this mutant. Moreover, it was prominent in mps1-3 spc105-18 cells at 25°C, but less so at 34°C (Appendix Fig S6A), confirming that it depends on Mps1 activity at kinetochores.

  e. during S phase and early mitosis, in 100% of the cells (n = 260, Fig4C and D). In mps1-3 cells, we detected low levels of Bub1 at kinetochores only in 20% of the cells at 34°C (n = 170, Fig4C and D), while the protein co-localised normally with Mtw1 in small-and medium-budded cells at 25°C(Appendix Fig S6B). Therefore, lack of Spc105 phosphorylation in mps1-3 cells under unperturbed conditions correlates with loss of Bub1 at kinetochores. Remarkably, Bub1 kinetochore localisation was restored to nearly wild-type levels in mps1-3 spc105-18 and mps1-3 GLC7-24 cells (Fig4C and D), further strengthening the correlation between Spc105 phosphorylation and Bub1 residence at kinetochores. It is worth noting that while in wild-type cells Bub1 dissociated from the kinetochores at the time of chromosome biorientation, it persisted at kinetochores up to anaphase in the majority of mps1-3 spc105-18, in good agreement with Spc105 hyperphosphorylation in these cells.

  ). While this hyperphosphorylation was transient in the absence of nocodazole, disappearing by 60 0 (Fig 4G), it accumulated upon microtubule depolymerisation in wild-type cells (Fig 4E). Under both conditions, this isoform was not apparent in mps1-3 cells, suggesting that it is phosphorylated by kinetochore-bound Mps1 (Fig 4E and G). Furthermore, Bub1 hyperphosphorylation was not restored in the mps1-3 spc105-18 and mps1-3 GLC7-24 suppressors undergoing cell cycle progression in the absence (Appendix Fig S6C) or in the presence of nocodazole (Appendix Fig S6D), although a modest Bub1 hyperphosphorylation could be observed in the mps1-3 GLC7-24 suppressor treated with nocodazole at late time points (Appendix Fig S6D).

Figure 5 .

 5 Figure 5. Artificial recruitment of Bub1 to Spc105 is sufficient to suppress the chromosome biorientation defects of mps1-3 cells.A SPC105-GBD cells expressing Bub1-GFP were grown at 30°C and filmed every 4 min at 30°C by time-lapse fluorescence microscopy. DIC: differential interference contrast. Scale bar: 5 lm. B Serial dilutions of stationary phase cultures of the indicated strains were spotted on YEPD and incubated at the indicated temperatures. C Cells with the indicated genotypes were grown at 25°C and then shifted at 34°C (t = 0). Every hour after the temperature shift, the same number of cells was plated on YEPD for each strain and incubated at 25°C to determine the number of colony-forming units. Percentages of viable cells have been calculated for each strain relative to t = 0. Error bars: SD. N = 3. D Cells with the indicated genotypes were synchronised in G1 with a-factor at 25°C and then released in fresh medium at 34°C. Cells were collected at the indicated time points for FACS analysis of DNA contents.

  5 lm (Figs 1G, 3G, 4C, and 5A, Movie EV1), 0.3 lm (Appendix Fig S8A) or 0.67 lm (Appendix Fig S1A). Images of Sgo1-GFP (Appendix Fig S8A) have been deconvolved using the software Huygens using the following settings: background = 200; ratio signal/noise = 4; and iterations = 5.

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Hardwick KG, Weiss E, Luca FC, Winey M, Murray AW (1996) Activation of the budding yeast spindle assembly checkpoint without mitotic spindle disruption. Science 273: 953 -956 63. Hardwick KG, Murray AW (1995) Mad1p, a phosphoprotein component of the spindle assembly checkpoint in budding yeast. J Cell Biol 131: 709 -720 microtubule attachments by the aurora kinase Ipl1p. Cell 111: 163 -172 82. Krenn V, Musacchio A (2015) The Aurora B kinase in chromosome biorientation and spindle checkpoint signaling. Front Oncol 5: 225 83. Araki Y, Gombos L, Migueleti SPS, Sivashanmugam L, Antony C, Schiebel E (2010) N-terminal regions of Mps1 kinase determine functional bifurcation. J Cell Biol 189: 41 -56 84. Chu MLH, Chavas LMG, Douglas KT, Eyers PA, Tabernero L (2008) Crystal structure of the catalytic domain of the mitotic checkpoint kinase Mps1 in complex with SP600125. J Biol Chem 283: 21495 -21500 85. 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Tsukahara T, Tanno Y, Watanabe Y (2010) Phosphorylation of the CPC by Cdk1 promotes chromosome bi-orientation. Nature 467: 719 -723 95. Musacchio A (2011) Spindle assembly checkpoint: the third decade.

  Top: GST-tagged Mps1, Mps1-3 and Mps1-1 expressed from the GAL1 promoter[START_REF] Lauzé | Yeast spindle pole body duplication gene MPS1 encodes an essential dual specificity protein kinase[END_REF] were affinity purified and incubated in vitro with radioactive ATP and myelin basic protein (MBP) for the indicated times. Bottom left: 0.2, 0.1 and 0.05 OD280 of each extract were loaded on SDS page for western blot analysis with an anti-GST antibody to quantify Mps1 protein levels.Note that Mps1 overexpression from the GAL1 promoter leads to accumulation of Mps1 phosphorylated isoforms. Right: autophosphorylation and MBP kinase activity was quantified at each time point on the autoradiographs by Image J and normalized to the protein levels quantified by Image J in the lowest OD280 (0.05). A.U.: Arbitrary units. B: Cells expressing 3HA-tagged Mps1 (wild type) or Mps1-3 cells were grown at 25°C, synchronised in G1 with α-factor and released into the cell cycle at 34°C to monitor Mps1 protein levels at the indicated time points by western blot. αfactor was re-added after 60' to re-accumulate cells in the next G1 phase (note, however, that wild type cells re-entered into a new cell cycle at 135'). Clb2 was used as mitotic marker, while Pgk1 was used as loading control. Mps1 protein levels were quantified by ImageJ and normalised to Pgk1 levels. A.U.: Arbitrary units.

w

  il d t y p e m p s 1 -3 ip l1 -3 2 1 w il d t y p e m p s 1 -3 ip l1 -3 2 1 w il d t y p e m p s 1 -3 ip l1 -3 2 1 w il d t y p e m p s 1 -3 ip l1 -3 2 1 w il d t y p e m p s 1 -3 ip l1 -3 2 1 w il d t y p e m p s 1 -3 ip l1 -3

Figure 29 :

 29 Figure 29 : grr1 mutations suppress the temperature-sensitivity of mps1-3 cells. Serial dilutions of stationary phase cultures of the indicated strains were spotted on YEPD and incubated at the indicated temperatures.

Figure 31 :

 31 Figure31: Grr1 is degraded in the grr1 suppressors. Cells with the indicated genotypes were synchronized in G1 with α-factor at 25°C and then released in fresh medium at 34°C (t = 0). Cells were collected at the indicated time points for western blot analysis of Grr1-3PK and Pgk1. Pgk1 has been used as loading control. Cyc: cycling cells.

Figure 32 :

 32 Figure 32 : Grr1 localizes at kinetochores of wild type but not of mps1-3 cells.

Figure 35 :

 35 Figure 35 : Ypi1 and Sds22 protein levels are not controlled by the SCF Grr1 complex.A-B: Cells with the indicated genotypes were synchronized in G1 with α-factor at 25°C and then released in fresh medium at 34°C in presence of nocodazole (t = 0). Cells were collected at the indicated time points for western blot analysis of Ypi1-3PK or Sds22-3PK, Clb2 and Pgk1. Clb2 has been used as mitotic marker and Pgk1 has been used as loading control. Cyc: cycling cells.

Figure 36 :

 36 Figure 36 : A second mutation in the mps1-3 allele suppresses the temperature-sensitivity of mps1-3 cells. A: Position of the MPS1 suppressing mutations found in the genetic screen (sequence position is relative to the ATG). B: Serial dilutions of stationary phase cultures of the indicated strains were spotted on YEPD and incubated at the indicated temperatures.

Figure 38 :

 38 Figure 38 : A 3HA tag at the C-terminus of Mps1 decreases suppressive effects of the intragenic suppressors. Serial dilutions of stationary phase cultures of the indicated strains were spotted on YEPD and incubated at the indicated temperatures.

Figure 39 :

 39 Figure39: The intragenic suppressors restore Mps1 kinetochore localization. Wild type, mps1-3 and mps1-3-sup cells were synchronized in G1 with α-factor at 25°C and then released at 34°C (wild type, mps1-3, mps1-sup1 and mps1-sup5) or at 32°C (mps1-sup22) in presence of nocodazole. Cells were collected after 90 minutes and processed for ChIP-seq analysis. ChIP sequence reads were normalized against sequence reads from corresponding input samples, and relative enrichment is plotted for chromosome II around the centromere. Y-axis shows enrichment values (linear scale, range is 0-8). Values below 1.5 are shown in grey, and values above 1.5 (i.e. sequences enriched in ChIP samples) are red coloured.

Figure 40 :

 40 Figure 40 : The Mps1-sup proteins bear somewhat decreased kinase activity.GST-tagged Mps1, Mps1-3, Mps1-1 and Mps1-sup proteins expressed in yeast cells from the GAL1 promoter[START_REF] Lauzé | Yeast spindle pole body duplication gene MPS1 encodes an essential dual specificity protein kinase[END_REF] were affinity-purified and incubated in vitro with radioactive ATP and myelin basic protein (MBP) for 4 minutes.

Figure 41 :

 41 Figure 41 : Recombinant Mps1-sup kinases might bear reduced kinase activity on Spc105 compared to Mps1-3.In vitro kinase assays with recombinant Mps1, Mps1-3 and Mps1-sup proteins purified from E. coli and incubated at 34°C for 1 or 5 minutes in the presence of radioactive gP 33 ATP and recombinant Spc105 Nterminal fragment (aa 1-320) as substrate.

Figure 42 :

 42 Figure 42 : The intragenic suppressing mutations lower Mps1 kinase activity on Spc105.In vitro kinase assays with recombinant Mps1, Mps1-3 and various Mps1 mutant proteins incubated at 34°C for 1 or 5 minutes in the presence of γP 33 ATP and a recombinant Spc105 N-terminal fragment (aa 1-320) as substrate.

Figure 43 :

 43 Figure 43 : Cells containing suppressing mutations proliferate normally, except for mps1-G449S. Serial dilutions of stationary phase cultures of the indicated strains were spotted on YEPD or YEPD containing Benomyl 12.5 µg/mL and incubated at the indicated temperatures.

Fig. 1

 1 Fig. 1 Budding yeast Mps1 in the control of chromosome biorientation. In wild type cells Mps1 promotes the error correction pathway through Spc105 phosphorylation and Bub1 recruitment, which at

  LD: 1% bactotryptone, 0.5% yeast extract, 0.5% NaCl pH 7.25 LD agar: LD + 1% agar LD ampicillin: LD + 50 µg/mL ampicillin LD chloramphenicol: LD + 34 µg/mL chloramphenicol Media for S. cerevisiae YEP: 1% yeast extract, 2% bactopeptone, 50mg/L adenine YEPD: YEP + 2% glucose YEPG: YEP + 2% galactose YEPR: YEP + 2% raffinose YEPRG: YEP + 2% galactose, 1% raffinose Sporulation plates (VB): 1.36% CH3CooNa3H2O, 0.19% KCl, 0.0035% MgSO4, 0.12% NaCl, 2% agar SD medium/plates: 0.7% yeast nitrogen base (YNB), 50 µg/mL aminoacids (excluding the amino acid that want to be used for selection), 2% agar, appropriate sugar bearing YIplac211 using primers MP1171-MP1172 or MP1173-MP1174, respectively. Plasmids were integrated into URA3 using StuI. Single integrations were checked by Southern Blot.Primers used in this study for gene tagging: Sequences in bold anneal to the tagbearing cassette SP84 (tagging BUB1 with 3HA::K.l.URA3; fwd) ACGTAATTCTAAGCATTGAAGAGGAGTTATCACATTTTCAATATAAGGGGAAACCGTC AAGGAGATTTTCCGGTTCTGCTGCTAG SP85 (tagging BUB1 with 3HA::K.l.URA3; rev) GTCATTGCTATGGAATCTGGCAGGACACCAAAAAGTCACCTATGCGGGAGATGAAGGC ATATTTATTCACCTCGAGGCCAGAAGAC SP96 (tagging MPS1 with 3HA::K.l.URA3; fwd) CATATATCACAAGATGATCTCAATGATGTGGTAGACACTGTTTTAAGGAAATTTGCAG ATTACAAAATTTCCGGTTCTGCTGCTAG SP97 (tagging MPS1 with 3HA::K.l.URA3; rev) CATGCATGGCAAACTCTAAATGTATTTATGTTCATAACTGGCACATGCTTTTCTTCCTTA TGCGGCTCTTCCTCGAGGCCAGAAGAC SP134 (tagging MAD1 with 3HA::K.l.URA3; fwd) GATAGAGGTCAACTTCCGTGCTTTTTGGCAACAATAACATTGCGTCTGTGGGAACAGC GACAAGCCAAATCCGGTTCTGCTGCTAG SP135 (tagging MAD1 with 3HA::K.l.URA3; rev) ATGTCAGCGGATAGGAGTTTATCATATTATAAAACCGATTACTATTATCTATTAGAAAT GTATATACACCCTCGAGGCCAGAAGAC MP560 (tagging SPC97 with mCherry::HPHMX; fwd) GTTCTTTACTCTATAGTACCTCCTCGCTCAGCATCTGCTTCTTCCCAAAGAGGTCGACG GATCCCCGGG MP561 (tagging SPC97 with mCherry::HPHMX; rev) GATTCCACTTTCCGCAAGTTGGTGCACGTCGTTAGTGACATAACGCCGCGTATCGATG AATTCGAGCTCG MP629 (tagging mps1-3 with 3HA::K.l.URA3; fwd) CTCAATGATGTGGTAGACACTGTTTTAAGGAAATTTGCAGATTACAAAATTTCCGGTT CTGCTGCTAG MP630 (tagging mps1-3 with 3HA::K.l.URA3; rev) ATGTATTTATGTTCATAACTGGCACATGCTTTTCTTCCTTATGCGGCTCTTCCTCGAGG CCAGAAGAC MP761 (tagging MPS1 with 6Gly-eGFP::kanMX ; fwd) CTCAATGATGTGGTAGACACTGTTTTAAGGAAATTTGCAGATTACAAAATTGGGGGAG GCGGGGGTGGAGGTGACGGTGCTGGTTTA MP762 (tagging MPS1 with 6Gly-eGFP::kanMX ; rev) ATGTATTTATGTTCATAACTGGCACATGCTTTTCTTCCTTATGCGGCTCTTTCGATGAAT TCGAGCTCG MP763 (tagging SPC105 with 3PK::K.l.HIS3; fwd) AGTGGAGTTCTTCCTTCATTTACGAAAAGTAGAATACATTTAGAGTTTACGTCCGGTT CTGCTGCTAG MP764 (tagging SPC105 with 3PK::K.l.HIS3; rev) AAAAAAAAGTGATGAGATATTACTAGTCATCGTTGTCCTATTATAAACACTCCTCGAG GCCAGAAGAC MP807 (tagging SDS22 with 3PK::K.l.HIS3; fwd) ATGAACTTACCTCCATCCCTACAGAAGATTGATGCGACATATATAAGAGGCTCCGGTT CTGCTGCTAG MP808 (tagging SDS22 with 3PK::K.l.HIS3; rev) ATATATATGTTTGTGTGTGTATATAAAAAAAAAATCATTTCTTGATCAAGACCTCGAG GCCAGAAGAC MP811 (tagging GRR1 with 3PK::K.l.HIS3; fwd) ATTGATTCTCAAATGGATGAAGCATCAGGAACGCCCGATGAAGATATGTTATCCGGTT CTGCTGCTAG MP812 (tagging GRR1 with 3PK::K.l.HIS3; rev) GATTTTGAAAATAAAGGTGTAGTAGGACAGTAAGTATTCAATGAAATACAACCTCGA GGCCAGAAGAC MP848 (tagging YPI1 with 3PK::K.l.HIS3; fwd) ATCCAACCAGATTATTCTGAATACAGGCGAAAACAGCAGGAAAAGAAGGACTCCGGT TCTGCTGCTAG MP849 (tagging YPI1 with 3PK::K.l.HIS3; rev) AAAAGCCATTTGCTGCTTCATCGAATATTTTGGCTTTGCTTGTACAAAGCCCCTCGAGG CCAGAAGAC MP894 (tagging DAM1 with 3PK::K.l.HIS3; fwd) TCTGTTGCAAAGAAAACTGAAAAAAAAATAAATACAAGGCCCCCCTTCAGATCCGGT TCTGCTGCTAG MP895 (tagging DAM1 with 3PK::K.l.HIS3; rev) CTAGCGATATATTTTGTGAGGAGGATAATTCTTTGGTTGGGTTGGGCGTAGCCTCGAG GCCAGAAGAC MP900 (tagging SNF1 with 3PK::K.l.HIS3; fwd) CATTTAACAACAAAACTAATTATGGAATTAGCCGTTAACAGTCAAAGCAATTCCGGTT CTGCTGCTAG MP901 (tagging SNF1 with 3PK::K.l.HIS3; rev) ACGATACATAAAAAAAAGGGAACTTCCATATCATTCTTTTACGTTCCACCACCTCGAG GCCAGAAGAC MP940 (tagging SLI15 with 3HA::K.l.URA3; fwd) AATCCTAGGCTAAACAGGTTGAAACCGCGTCAAATTGTGCCCAAAAGGTCTTCCGGTT CTGCTGCTAG MP941 (tagging SLI15 with 3HA::K.l.URA3; rev) CTTGATTTAATGTTAACCAGTTTGAATTTTTCTTTTCTGGGGTAATCGAATCCTCGAGG CCAGAAGAC MP1024 (tagging BUB3 with eGFP::kanMX; fwd) ACTATTGAACTAAACGCAAGTTCAATATACATAATATTTGACTATGAGAACGGGGGAG GCGGGGGTGGAGGTGACGGTGCTGGTTTA MP1025 (tagging BUB3 with eGFP::kanMX; rev) AATTTTTTTTCTGGAATGTTCTATCATACTACACGAATCTTCACGAAGATATCGATGAA TTCGAGCTCG MP1128 (tagging MTW1 with GBD::kanMX; fwd) GTTAGTATAGATATTGAAGAGCCTCAATTGGATTTACTTGATGATGTGTTACGTACGC TGCAGGTCGAC MP1129 (tagging MTW1 with GBD::kanMX; rev) ATTCGTGAATACATACATCATATCATAGCACATACTTTTTCCCACTTTATAATCGATGA ATTCGAGCTCG Primers used in this study for cloning: sequences in bold indicate the restriction sites SP127 (to clone MPS1, EcoRI) CGGAATTCCTGCAAGAACTGGATGATCTC SP129 (to clone MPS1, XhoI) CCGCTCGAGCGGTGTAAAAAAGAGTTGATC SP238 (cloning of GAL1-GST-MPS1; fwd) TCCCCCGGGATCAACAAACTCATTCCATGATTAG SP239 (cloning of GAL1-GST-MPS1; rev) CCCAAGCTTAAGTATTCAGTGTCTGTGTCG MP774 (cloning of GAL1-GST-mps1-sup; rev) AGTACATGCATGGCAAACTC MP948 (cloning of GAL1-GST-mps1-sup; fwd) GGATGATGCAATAACGAATG MP1124 (cloning of MPS1, EcoRI; fwd) ACTTAGAATTCTCAACAAACTCATTCCATGATTATG MP1125 (cloning of MPS1, HindIII; rev) ACTTAAAGCTTCTAAATTTTGTAATCTGCAAATTTC MP1126 (cloning N-ter SPC105, BamHI; fwd) ACTTAGGATCCAATGTGGATGAAAGAAGCCG MP1127 (cloning N-ter SPC105, PstI; rev) ACTTACTGCAGCTAGTCAAAGTTTGTCTGTATTTGCG Primers used in this study for mutagenesis: sequences in bold indicate the mutated codon MP1171 (to mutagenize MPS1 C1807>A (Pro603Thr); fwd) GTGAAACTCAAATTGGGACTACAAATTATATGGCACCAGAAGC MP1172 (to mutagenize MPS1 C1807>A (Pro603Thr); rev) GCTTCTGGTGCCATATAATTTGTAGTCCCAATTTGAGTTTCAC MP1173 (to mutagenize MPS1 C1904>A (Ser635Tyr); fwd) GGAAAGTGGGGAGACCATATGATATGTGGTCATGCGG MP1174 (to mutagenize MPS1 C1904>A (Ser635Tyr); rev) CCGCATGACCACATATCATATGGTCTCCCCACTTTCC Synchronization with α-factor

0 .

 0 1M Maleic acid 0.15M NaCl 0.3% tween, one with Blocking Reagent 0.1M Maleic acid 0.15M NaCl 0.3% tween containing anti-Dig antibodies diluted 1:10000, two with 0.1M Maleic acid 0.15M NaCl 0.3% tween, the membrane was incubated 15minutes with 0.1M TrisHCl 0.1M NaCl pH 9.5 containing 1:200 CSPD (Disodium 3-(4-methoxyspiro {1,2-dioxetane-3,2′-(5′-chloro)tricyclo

[

  polymerase (Biolabs), Pfu polymerase (Stratagene), LongAmp polymerase (Biolabs), depending on the kind of DNA template and required fidelity. A typical reaction mix was: DNA amplification was performed with a PTC-100 Peltier Thermal Cycler (MJ research), with the following parameters:

Table 1 :

 1 Syndromes caused by erroneous chromosome segregations in meiosis.

	Down	21	Growth delay, intellectual disability,
			characteristic facial features
	Patau	13	Intellectual disability, heart defects,
			body malformations including sex
			organs, cysts in the kidneys
	Edwards	18	Small babies, heart defects, body
			malformations, intellectual disability
	Klinefelter (males)	X	Infertility, weaker muscles, greater
			height, poor coordination, breast
			growth, less body hair
	Turner (females)	X	Webbed neck, short stature, swollen
			hands and feet, heart defects, diabetes
	Triple X (females)	X	Learning difficulties, decreased
			muscle tone, seizures, kidney
			problems
	Jacobs (males)	Y	Tall stature, learning problems, acne,
			ADHD

Bub1 aux kinétochores et est antagonisée par la phosphatase PP1, est une fonction cruciale de Mps1 dans la correction des liaisons inappropriées kinétochores-microtubules comme pour l'activation du SAC. De façon cohérente, la phosphorylation de Spc105 et la localisation de Bub1au kinétochore étaient affectées dans les cellules mps1-3 et restaurées dans les suppresseurs susmentionnés. De plus, le recrutement artificiel de Bub1 à Spc105 supprimait les défauts de ségrégation des cellules mutantes mps1-3. Ainsi, une conclusion principale de mon travail de thèse est que le SAC et la correction d'erreurs sont déclenchés par un seul dispositif sensoriel impliquant Mps1 et antagonisé par PP1. Grâce au même crible génétique ci-dessus, j'ai également trouvé des suppresseurs portant des mutations dans la protéine F-box Grr1, qui fait partie du complexe ubiquitine-ligase SCF. Mes données préliminaires indiquent que Grr1 se localise aux kinétochores, suggérant que le complexe SCF Grr1 pourrait être un nouvel acteur dans le contrôle de la bi-orientation des chromosomes. Enfin, j'ai pu isoler des suppresseurs intragéniques, qui portent une seconde mutation dans MPS1 et restaurent les fonctions mitotiques de la protéine Mps1-3. Les données recueillies jusqu'à présent indiquent que cette classe de mutations, contrairement aux suppresseurs extragéniques ci-dessus, rétablit la localisation de Mps1 au kinétochore. Étant donné que Mps1 régule son propre turnover aux kinétochores par autophosphorylation, nous avions émis l'hypothèse que les suppresseurs puissent rétablir la localisation de Mps1-3 au kinétochore en diminuant son activité catalytique. Étonnamment, bien que la plupart des mutations suppressives réduisaient la capacité de Mps1-3 à phosphoryler une cible exogène, elles n'affectaient pas de façon significative l'autophosphorylation, ce qui suggère que la phosphorylation dépendante de Mps1 d'une protéine non identifiée au kinétochore pourrait influencer le turnover de Mps1.

Acknowledgements

We are grateful to P. Pietruszka for initial experiments on mps1-3; to A. Amon, S. Biggins, C. Campbell, F. Cross, T. Davis, A. Desai, P. De Wulf, H. Funabiki, A. Hoyt, J. Lechner, A. Marston, K. Nasmyth, E. Schwob, S. Storchova, A. Straight, T. Tanaka and W. Zachariae for sharing reagents; to K. Fujiki for analysis of ChIP-seq data; to V. Georget, S. De Rossi and O. Faklaris for their invaluable help with live cell imaging; to A. Musacchio and E. Schwob for insightful

Acknowledgements

We thank all members of Piatti's lab for useful discussions. Work in Piatti's lab is supported by Fondation pour la Recherche Médicale (DEQ20150331740 to S.P.), Agence Nationale de la Recherche (ANR-18-CE13-0015-01 to S.P.) and by Labex Epi-GenMed (PhD fellowship to G.B.).

ACKNOWLEDGMENTS

I thank the member of my thesis committee, Etienne and Andrea, who always advised me well during my PhD and the member of my thesis jury who will spend their time reading my manuscript and participating to my defense. I also thank the head of the CRBM for giving me the opportunity to have a 4 th year of thesis.

I thank the Helleday lab in Stockholm, who's giving me a great opportunity for my future, allowing me to take exactly the direction I wanted to take.

Last but not least, this PhD offered me the best I could get: Damien. These four years together have been perfect, and our life together has just started. Hoping is the beginning of a wonderful trip! The product of your PhD is not your thesis, but the person you became.

RESUME

Mps1 est une protéine kinase conservée qui, pendant la mitose, corrige les attachements inappropriés entre kinétochores et microtubules, assurant ainsi la bi-orientation des chromosomes. Cependant, ses cibles critiques dans ce processus restent largement inconnues. Mps1 contrôle également le « spindle assembly checkpoint » (SAC), qui arrête la ségrégation des chromosomes en métaphase jusqu'à ce que la bi-orientation soit atteinte. Son rôle dans l'activation du SAC est antagonisé par la phosphatase PP1 et implique la phosphorylation de la protéine d'échafaudage au kinétochore KNL1/Spc105, qui à son tour recrute la kinase Bub1 pour favoriser l'assemblage des complexes effecteurs du SAC. Une question cruciale est si la correction des erreurs et l'activation du SAC font partie d'une seule et même voie ou des voies séparables. Au cours de ma thèse, j'ai caractérisé un nouveau mutant thermosensible, mps1-3, qui est défectueux dans la bi-orientation des chromosomes et la signalisation du SAC. La mutation mps1-3 change une sérine conservée dans le domaine kinase en phénylalanine et, de façon surprenante, la protéine Mps1-3 a une activité catalytique accrue par rapport à la protéine sauvage. Inversement, la protéine ne se localise pas aux kinétochores, ce qui suggère que les défauts mitotiques du mutant mps1-3 proviennent du manque de phosphorylation des cibles critiques aux kinétochores. Grâce à un crible génétique, j'ai identifié des mutations qui supprimaient la thermosensibilité du mutant mps1-3 en affectant l'interaction entre la protéine du kinétochore Spc105/KNL1 et la phosphatase PP1, et j'ai démontré que des niveaux réduits de PP1 aux kinétochores sous-tendent la suppression. Remarquablement, les suppresseurs restauraient à la fois la signalisation du SAC et la bi-orientation des chromosomes, ce qui suggère que Mps1 contrôle les deux processus par le même mécanisme moléculaire. Nous avons proposé que la phosphorylation de Spc105/KNL1, qui conduit au recrutement de

acknowledge the imaging core facility MRI, member of the national infrastructure France-BioImaging supported by the French National Research Agency (ANR-10-INBS-04, "Investments for the future"). This work has been supported by the Fondation pour la Recherche Médicale (DEQ20150331740 to S.P.) and by LabexEpiGenMed (PhD fellowship to G.B.). The authors declare no

Data availability

The ChIP-seq data from this publication have been deposited to the GEO NCBI database (https://www.ncbi.nlm.nih.gov/geo/) and assigned the identifier GSE144741.

Expanded View for this article is available online.

Yeast strains

All yeast strains are congenic to or at least four times backcrossed to W303 (ade2-1, trp1-1, leu2-3,112, his3-11, and 15 ura3).

Name

Relevant genotype ySP642 MATa, mps1-1 ySP1578

MATa, bub1::S.p.HIS5 ySP1593

MATa, BUB1-3HA::KlURA3 ySP1791 MATa, his3::HIS3-GFP-TUB1 ySP1923

MATa MPS1-3HA::K.l.URA3 ySP2216

MATa, MAD1-3HA::K.l.URA3 ySP3846

MATa, [2m GAL1-GST-MPS1::URA3] ySP3848

MATa, [2m GAL1-GST-mps1-1::URA3] ySP5639

MATa, cdc16-1, tetO2X224::URA3 (-12,6 Kb from CEN5), leu2::tetR-GFP::LEU2 ySP7280 MATalpha, sgo1::TRP1 ySP7961 MATa, bar1::hisGURA3hisG, PDS1-myc18::LEU2 ySP11069 MATa, mps1-3 ySP11678

MATa, bar1::hisGURA3hisG, PDS1-myc18::LEU2, mps1-1 ySP11680

MATa, bar1::hisGURA3hisG, PDS1-myc18::LEU2, mps1-3 ySP11681

MATa, his3::HIS3-GFP-TUB1, mps1-3 ySP11683

MATa, his3::HIS3-GFP-TUB1, mps1-1 ySP11954

MATa, mps1-Ser635Phe-Pro603Thr (sup1)

MATa, mps1-3, spc105-3 (Ala79Thr) ySP11958

MATa, mps1-Ser635Phe-Phe483Leu (sup5) ySP11959

MATalpha, mps1-3, GLC7-6 (Phe256Ile) ySP12005

MATa, MTW1-tdTomato::NAT, SGO1-GFP::KanMX, mps1-3 ySP12007

MATa, MTW1-tdTomato::NAT, SGO1-GFP::KanMX ySP12086

MATalpha, mps1-Ser635Phe-Leu548Met (sup7) ySP12096

MATalpha, mps1-3, grr1-Ser772Pro, rsc30-Gly571Asp (sup17) ySP12098

MATalpha, mps1-3, grr1-Asn1069Lys, rsc30Gly571Asp (sup19) ySP12099

MATalpha, mps1-3, grr1-Ser772Pro (sup20) ySP12097

MATalpha, mps1-3, spc105-18 (Val76Gly) ySP12100

MATalpha, mps1-3, spc105-21 (Val82Asp) ySP12100

MATalpha, mps1-Ser635Tyr (sup22) ySP12102

MATalpha, mps1-3, spc105-23 (Val82Asp) ySP12103

MATalpha, mps1-3, GLC7-24 (Phe256Val) ySP12105

MATa, mps1-3, grr1-Cys730Trp, rsc30-Gly571Asp (sup26) ySP12106

MATalpha, mps1-Ser635Phe-Ser451Ala (sup27) ySP12117

MATa, cdc16-1, tetO2X224::URA3 (-12,6 Kb from CEN5), leu2::tetR-GFP::LEU2, mps1-1 ySP12119

MATa, CEN5::tetO2X112::HIS3, LEU2::tetR-GFP, cdc15-2, mps1-1, SPC97-mcherry::HPH ySP12120

MATa, CEN5::tetO2X112::HIS3, LEU2::tetR-GFP, cdc15-2, SPC97-mcherry::HPH ySP12157

MATa, [2m GAL1-GST-mps1-3::URA3] ySP12158

MATa, cdc16-1, tetO2X224::URA3 (-12,6 Kb from CEN5), leu2::tetR-GFP::LEU2, mps1-3 ySP12239

MATa, CEN5::tetO2X112::HIS3, LEU2::tetR-GFP, cdc15-2, mps1-3, SPC97-mcherry::HPH ySP12463

MATa, mps1-Ser635Tyr (sup22) ySP12535

MATa, mps1-3-3HA::K.l.URA3 ySP13011

MATa, mps1-Ser635Tyr-3HA::K.l.URA3 (sup22) ySP13030

MATa, mps1-3, grr1-Ser772Pro, rsc30-Gly571Asp (sup17) ySP13031

MATa, mps1-3, grr1-Asn1069Lys, rsc30Gly571Asp (sup19) ySP13032

MATa, mps1-3, grr1-Ser772Pro (sup20) ySP13324

MATa, MTW1-tdTomato::NAT, BUB1-GFP::URA3 ySP13492

MATa, MAD1-3HA::K.l.URA3, mps1-3 ySP13553

MATa, SPC105-3PK::K.l.HIS3, mps1-3 ySP13613

MATa, CEN5::tetO2X112::HIS3, LEU2::tetR-GFP, ura3::URA3::mCherry-TUB1 ySP13618

MATa, GRR1-3PK::K.l.HIS3 ySP13622 MATa, GRR1-3PK::K.l.HIS3, mps1-3 ySP13649

MATa, grr1-Ser772Pro-3PK::K.l.HIS3 (sup20), mps1-3 ySP13650

MATa, grr1-Ser772Pro-3PK::K.l.HIS3, rsc30-Gly571Asp (sup17), mps1-3 ySP13698

MATa, CEN5::tetO2X112::HIS3, LEU2::tetR-GFP, ura3::URA3::mCherry-TUB1, mps1-3 ySP13743

MATa, SPC105-3PK::K.l.HIS3 ySP13796

MATa, MAD1-3HA::K.l.URA3, mps1-3, GLC7-24 (Phe256Val) ySP13830

MATa, SDS22-3PK::K.l.HIS3, mps1-3 ySP13833

MATa, SDS22-3PK::K.l.HIS3, grr1-Ser772Pro, rsc30-Gly571Asp (sup17), mps1-3 ySP13844

MATa, grr1-Ser772Pro, rsc30-Gly571Asp (sup17), mps1-3 YPI1-3PK::K.l.HIS3 ySP13846 MATa, mps1-3 YPI1-3PK::K.l.HIS3 ySP13849

MATa, YPI1-3PK::K.l.HIS3 ySP13891

MATa, SDS22-3PK::K.l.HIS3, ySP14014

MATa, mps1-3-3HA::K.l.URA3, spc105-18 (Val76Gly) ySP14016

MATa, mps1-3-3HA::K.l.URA3, GLC7-24 (Phe256Val) ySP14028

MATa, mps1-3-3HA::K.l.URA3, grr1-Ser772Pro (sup20) ySP14150

MATa, MPS1-3HA::K.l.URA3, grr1-Ser772Pro (sup20) ySP14194

MATa, DAM1-3PK::K.l.HIS3 ySP14196 MATa, DAM1-3PK::K.l.HIS3, mps1-3 ySP14298

MATa, SNF1-3PK::K.l.HIS3, mps1-3 ySP14231

MATa, SPC105-3PK::K.l.HIS3, mps1-3, GLC7-24 (Phe256Val) 147 ySP14280 MATa, mps1-3, spc105-18-3PK::K.l.HIS3 ySP14282

MATa, mps1-Ser635Phe-Phe483Leu-3HA::K.l.URA3 (sup5) ySP14296

MATa, SNF1-3PK::K.l.HIS3 ySP14298 MATa, SNF1-3PK::K.l.HIS3, mps1-3, GLC7-24 (Phe256Val) ySP14302

MATa, SLI15-3HA::K.l.URA3 ySP14306

MATa, SNF1-3PK::K.l.HIS3, GLC7-24 (Phe256Val) ySP14310

MATa, SLI15-3HA::K.l.URA3, GLC7-24 (Phe256Val) ySP14312

MATa, mps1-Ser635Phe-Pro603Thr-3HA::K.l.URA3 (sup1) ySP14437

MATa, BUB1-3HA::K.l.URA3, mps1-3 ySP14477

MATa, [2m GAL1-GST-mps1-Ser635Phe-Pro603Thr::URA3] (sup1) ySP14479

MATa, [2m GAL1-GST-mps1-Ser635Phe-Gly449Ser::URA3] (sup2) ySP14481

MATa, [2m GAL1-GST-mps1-Ser635Phe-Phe483Leu::URA3] (sup5) ySP14483

MATa, [2m GAL1-GST-mps1-Ser635Tyr::URA3] (sup22) ySP14485

MATa, [2m GAL1-GST-mps1-Ser635Phe-Ser451Ala::URA3] (sup27) ySP14537

MATa, BUB1-HA3::K.l.URA3, mps1-3, GLC7-24 (Phe256Val) ySP14591

MATa, BUB1-3HA::K.l.URA3, mps1-3, spc105-18 (Val76Gly) ySP14592

MATa, [2m GAL1-GST-mps1-Ser635Phe-Leu548Met::URA3] (sup7) ySP14719

MATa, mps1-3, [CEN::GALs-MPS1::HIS3], MTW1-tdTomato::NAT, BUB1-GFP::URA3 ySP14817

MATa, MTW1-tdTomato::NAT, BUB1-GFP::URA3, GLC7-24 (Phe256Val), mps1-3 ySP14829

MATa, CEN5::tetO2X112::HIS3, LEU2::tetR-GFP, ura3::URA3::mCherry-TUB1, mps1-3, spc105-18 (Val76Gly) ySP14831

MATa, CEN5::tetO2X112::HIS3, LEU2::tetR-GFP, fura3::URA3::mCherry-TUB1, mps1-3, GLC7-

) ySP14854 MATa, MTW1-tdTomato::NAT, BUB1-GFP::URA3, spc105-18 (Val76Gly), mps1-3 ySP14860 MATa, mad2∆::KanMX, lys2::GAL1-HO::LYS2, his3::HIS3-TUB1-GFP, spc105-RASA::ADE2 ySP14861 MATa, lys2::GAL1-HO::LYS2, GLC7-spc105-RASA::ADE2 ySP14900 MATalpha, GLC7-spc105-RASA::ADE2, mps1-3 ySP14946 MATa, mps1-3, SLI15::SLI15(del 2-228)::URA3 ySP14947 MATa, bub1::S.p.HIS5, SLI15::SLI15(del 2-228)::URA3 ySP14950 MATa, mps1-3, SLI15::SLI15-6A (S335A,S373A,S427A,S437A,S462A,T474A)::URA3 ySP14951 MATa,bub1::S.p.HIS5,SLI15::SLI15-6A (S335A,S373A,S427A,S437A,S462A,T474A)::URA3 ySP14953 MATalpha, mad2∆::KanMX, spc105-RASA::ADE2, mps1-3 ySP14987 MATa, spc105::SPC105-GBD::TRP1 ySP15006 MATa, BUB1-GFP::URA3, spc105::SPC105-GBD::TRP1 ySP15047 MATalpha, spc105::SPC105-GBD::TRP1, mps1-3 ySP15080 MATa, BUB1-GFP::URA3, spc105::SPC105-GBD::TRP1, mps1-3 ySP15088 MATa, spc105::SPC105-GBD::TRP1, BUB3-eGFP::KANMX, mps1-3 ySP15091 MATa, spc105::SPC105-GBD::TRP1, BUB3-eGFP::KANMX ySP15282 MATa, mps1-Ser635Phe-Ser451Ala (sup27) ySP15351 MATa, DAM1-3PK::K.l.HIS3, ipl1-321 ySP15367 MATa, mps1-Ser635Phe-Leu548Met (sup7) ySP15435 MATa, spc105::SPC105-GBD::TRP1, BUB3-eGFP::KANMX, mps1-3, mad2::KanMX ySP15519 MATa, CEN5::tetO2X112::HIS3 (1,4Kb), LEU2::tetR-GFP, ura3::pRS306-mCherry-TUB1::URA3, spc105-18 (Val76Gly), mps1-3, mad2::TRP1 ySP15522 MATa, CEN5::tetO2X112::HIS3 (1,4Kb), LEU2::tetR-GFP, ura3::pRS306-mCherry-TUB1::URA3, GLC7-24 (Phe256Val), mps1-3, mad2::TRP1 ySP15597 MATa, MPS1-eGFP::KanMX, MTW1-GBD::KanMX, mad2::TRP1 ySP15598 MATa, mps1-3-eGFP::KanMX, MTW1-GBD::KanMX, mad2::TRP1 ySP15842 MATa/MATalpha, MPS1/mps1::kanMX, ura3::YIplac211-MPS1::URA3 ySP15843 MATa/MATalpha, MPS1/mps1::kanMX, ura3::YIplac211-mps1-Pro603Thr::URA3 ySP15844 MATa/MATalpha, MPS1/mps1::kanMX, ura3::YIplac211-mps1-Gly449Ser::URA3 ySP15845 MATa/MATalpha, MPS1/mps1::kanMX, ura3::YIplac211-mps1-Phe483Leu::URA3

LIST OF ABBREVIATIONS

Conflict of interest

The authors declare that they have no conflict of interest.

APPENDIX Benzi et al. -pT210 Snf1-3PK wild type mps1-3 GLC7-24 GLC7-24 Appendix Figure S4. The GLC7-24 mutation might impair PP1 activity at kinetochores A: Cells with the indicated genotypes were grown at 25°C in YEP containing 4% glucose (glu) and then shifted to YEP containing 1% glucose for 5 minutes. Cells were collected for western blot analysis in the two conditions. The phosphorylated active form of Snf1 was detected with antiphospho AMPK (Thr172 that corresponds to Thr210 of Snf1), while an anti-PK antibody was used to detect total levels of Snf1-3PK. Signals were quantified by ImageJ and the signals due to Snf1 phosphorylation were normalised for the total amounts of Snf1, in turn normalised for the amounts of Pgk1 in each lane (loading control). Error bars: SD. N=3. B: Wild type and GLC7-24 cells were synchronised with α-factor at 25°C and then released in fresh medium containing nocodazole at the same temperature (t=0). Cells were collected at the indicated time points for western blot analysis.

Table of contents

Anti-HA antibodies have been used to detect Sli15-3HA. Clb2 was used as mitotic marker. Cyc: cycling cells.

Appendix Figure S5. The Mps1-3 mutant protein fails to re-accumulate at kinetochores in the spc105-18 and GLC7-24 suppressors

Cells with the indicated genotypes were synchronised in G1 with α-factor at 25°C and then released at 32°C in the presence of nocodazole (note that the presence of 3HA tags at the C-terminus of Mps1-3 decreased the maximal permissive temperature of the suppressors from 34°C to 32°C).

Cells were collected after 90 minutes and fixed with formaldehyde for ChIP-seq analysis. ChIP sequence reads were normalised against sequence reads from corresponding input samples, and relative enrichment is plotted for all yeast chromosomes except for chromosome V (shown in Fig. 3I) around the centromere. Y!axis shows enrichment values (linear scale, range is 0-10). Values below 1.5 are shown in grey, and values above 1.5 (i.e. sequences enriched in ChIP samples) are red coloured. 1 0 ' 2 0 ' 3 0 ' 4 0 ' 5 0 ' 6 0 ' 7 0 ' c y c 0 ' 1 0 ' 2 0 ' 3 0 ' 4 0 ' 5 0 ' 6 0 ' 7 0 ' c y c 0 ' 1 0 ' 2 0 ' 3 0 ' 4 0 ' 5 0 ' 6 0 ' 7 0 ' c y c 0 ' 1 0 ' 2 0 ' 3 0 ' 4 0 ' 5 0 ' 6 0 ' 7 0 ! "#! Appendix Table S1. List of S. cerevisiae strains used in this study (plasmids are indicated in brackets)

Name

Relevant genotype effect on Mps1 protein levels). However, Mps1 did not seem to accumulate in the grr1-20 mutant (Fig. 33), suggesting that Grr1 probably has no role in Mps1 proteolysis. loses its centromeric localization in mps1-3 cells. It is however possible that in the suppressors, although the overall levels of Grr1 decrease, the protein might re-gain association to centromeres, a possibility that will be worth testing in the future. interaction partners at kinetochores cannot be excluded. In the future, it will be interesting to study the interactome of Mps1-3 versus wild type Mps1, as differences in their binding partners might help distinguishing kinetochore substrates from kinetochore receptors of Mps1 in yeast. Additionally, studying the phosphorylation pattern of the different subunits of the NDC80 complex in wild type and mps1-3 cells could reveal important insights into the molecular mechanism underlying Mps1 localization at kinetochores. Another important, unanswered question is why the Mps1-3 kinase is hyperactive. Recent data showed that the catalytic activity of Mps1 is autoinhibited by the N-terminal extension, which interacts intra-molecularly with the kinase domain at the C-terminus. Mps1-dependent phosphorylation of the N-terminal extension in mitosis relieves autoinhibition (Combes et al., 2018).

We could therefore envision that in the mps1-3 mutant interaction between the N-and the C-terminal regions of Mps1 could be impaired due to the Ser635Phe mutation, thereby leading to loss of autoinhibition and increase in the kinase activity. To test this intriguing hypothesis, I plan to check the interaction between an N-terminal and a C-terminal recombinant fragment of Mps1 and test the possible impact of the Ser635Phe mutation on their binding. is unaffected (data not shown), in contrast to high level overexpression of MPS1 that causes a SACdependent metaphase arrest [START_REF] Hardwick | Activation of the budding yeast spindle assembly checkpoint without mitotic spindle disruption[END_REF].

The GAL1-GST-MPS1 plasmid (pSP228) was constructed by amplifying MPS1 by PCR with primers SP238 and SP239 and cloning it into pEG(KT) (Pierce and Wendland, 2009) after cutting SmaI/HindIII. The GAL1-GST-mps1-3 plasmid (pSP1202) was generated by replacing a BspEI/XbaI fragment of pSP228 internal to MPS1 coding region by the same fragment containing the mps1-3 mutation from pSP1041.

The GAL1- GST-mps1-sup plasmids (pSP1438, pSP1439, pSP1440, pSP1141, pSP1442, pSP1456) were generated by replacing a BspEI/XbaI fragment of pSP228 internal to the MPS1 coding region by the same fragment containing the mps1-sup mutations amplified from yeast genomic DNA of suppressing mutants using primers MP948-MP774.

The plasmids for expression of His-tagged Mps1 and Mps1-3 in bacteria (pSP1575, pSP1576) were generated by amplifying the coding sequence of MPS1/mps1-3 by PCR with primers MP1124-MP1125, followed by cloning into EcoRI/HindIII of pPROEX-HTa.

The plasmid for expression of His-tagged Spc105 N-terminus (aa 1-320) in E. coli (pSP1571) was generated by PCR-amplifying part of the coding sequence of SPC105 with primers MP1126-1127, followed by cloning into BamHI/PstI of pPROEX-HTa.

The plasmids for expression of His-tagged mps1-sup double mutant alleles (pSP1596, pSP1597, pSP1598, pSP1599, pSP1600, pSP1601) were generated by cloning XbaI/NdeI protein fragments from GAL-GST-mps1-sup plasmids in XbaI/NdeI cut pPROEX-HTa.

The plasmids for expression of His-tagged Mps1-G449S, Y483L, L548M, S451A single mutants (pSP1623, pSP1624, pSP1625, pSP1626) were generated by cloning KpnI/SacI DNA fragments derived from the mps1-sup pPROEX-HTa plasmids in KpnI/SacI cut pPROEX-HTa already containing MPS1. Plasmids containing His-tagged Mps1-P603T and Mps1-S635T (pSP1627, pSP1628) were generated by site-directed mutagenesis of MPS1-bearing pPROEX-HTa (pSP1575) using primers MP1171-MP1172 or MP1173-MP1174.

The plasmids for integration of single suppressing mutant alleles in yeast cells (pSP1620, pSP1619, pSP1618, pSP1617) were generated by cloning an EcoRI/PstI fragment carrying the coding sequence of mutant derivatives of MPS1, along with MPS1 promoter region (480 bp) and 3' UTR (150 bp), into YIplac211 cut EcoRI/PstI and then by substituting a KpnI/SacI fragment taken from pPROEXHTa containing mps1-sup mutations. Plasmids for integration of single mutants mps1-P603T and mps1-S635T (pSP1621, pSP1622) were generated by site-directed mutagenesis of MPS1-To determine which gene was responsible for suppression in the mps1-3 grr1 rsc30 suppressors, the grr1 and rsc30 mutant alleles were marked at their 3' UTR with a NAT and LEU2 marker, respectively, and co-segregation of temperature-resistance at 34°C with either marker was assessed after crossing to a wild type strain and tetrad analysis. By this means suppression could be attributed to the grr1 mutations.

Viability test

Cells were grown at 25°C to 2x10 6 cells/mL and then serially diluted to plate 200 cells/plate in duplicate (wild type, mps1-3 SPC105-GBD BUB1-GFP and mps1-3 SPC105-GBD BUB3-GFP) or 200, 500 and 1000 cells/plate in duplicate for mps1-3 cells (t=0). Cells were then shifted to 34°C, withdrawn each hour, serially diluted to plate 200 cells/plate in duplicate (wild type, mps1-3 SPC105-GBD BUB1-GFP and mps1-3 SPC105-GBD BUB3-GFP) or 200, 500 and 1000 cells/plate in duplicate for mps1-3 cells. Plates were incubated at 25°C until colony appearance. The number of colonies at each time point (average values of the duplicates) was divided by the number of colonies at t=0 to obtain percentages of viability.

Microscopy

Scoring of GFP-tagged chromatid separation was carried out on cells fixed in 100% ethanol at -20°C.

After two washes with PBS, cells were laid between a glass slide and a coverslip and imaged by fluorescence microscopy (Epifluorescent widefield Zeiss Axioimager Z1 controlled by the Metamorph software). Z-stacks (11 planes, 0.3 µm spaced) were max-projected before scoring.

To detect spindle formation and elongation, alpha-tubulin immunostaining was performed on formaldehyde-fixed cells using the YOL34 monoclonal antibody (1:100; MCA78S AbD Serotec, Raleigh, NC), followed by indirect immunofluorescence using CY2-conjugated anti-rat antibody Kinase assays were performed as above using 2 µg of MBP as exogenous substrate.

ChIP-seq analysis 100 mL of cells (concentration 10 7 cells/mL) were fixed in 1% formaldehyde for 30' at RT with shaking and then transferred ON at 4°C. Cells were then spun down 3min at 3000 rpm at 4°C and washed three times with TBS. Cells were resuspended in 0.4 mL of 50mM Hepes-KOH pH7.5, 140mM NaCl, 1mM EDTA, 1% Triton-X100, 0.1% Na-deoxicholate containing a cocktail of protease inhibitors (Complete EDTA-free Roche) and 1mM PMSF, followed by cell breakage through a Multi-beads shaker (Yasui-Kikai) at 0°C. Lysates were eluted in 15mL Falcon tubes (centrifuged twice at 3000 rpm for 1 min at 4°C), transferred in 1.5 Eppendorf tube, centrifuged at 5000 rpm 1 min at 4°C and sonicated through a Sonifyer Branson 2508 (5-7 cycles of 15sec, tune 1.5 with centrifugation of 5000 rpm for 1 min between each cycle). Sonicated lysates were then centrifuged for 5 min at 15000 rpm at 4°C. Supernatants were recovered. 10 µL of lysates were withdrawn at this stage (=Inputs). For immunoprecipitations (IPs), 60 µL of Protein G-dynabeads pre-washed twice with PBS containing 5 mg/mL BSA and pre-adsorbed with 3 µg Babco 16B12 anti-HA antibody were added to each lysate and incubated ON at 4°C on a nutator. IPs were then washed twice with ice-cold 50mM Hepes-KOH pH7.5, 140mM NaCl, 1mM EDTA, 1% Triton-X100, 0.1% Na-deoxicholate, twice with ice-cold 50mM Hepes-KOH pH7.5, 500mM NaCl, 1mM EDTA, 1% Triton-X100, 0.1% Na-deoxicholate, twice with ice-cold 10mM Tris-Cl pH8, 250mM LiCl, 0.5% NP-40, 0.5% Na-deoxycholate, 1mM EDTA and once with ice-cold TE (50mM Tris-Cl pH8, 1mM EDTA pH8). IPs were eluted at 65°C for 15min with 50µL of 50mM Tris-Cl pH8, 10mM EDTA, 1% SDS, resuspended in 50 µL of TE/1% SDS and incubated overnight at 65°C to reverse crosslinking.

IPs were then incubated at 37°C for 1 hour with 100 µL of TE containing 10 µg of RNAseA and 2 hours at 37°C with 2 µL of ProteinaseK 50 mg/mL. DNA was then purified with the Qiagen PCR purification kit. Libraries for Illumina sequencing have been then prepared following the NEBNext Ultra II DNA Library Prep Kit for Illumina. Sequencing results have been analysed using the DROMPA software (Nakato and Shirahige, 2018).

Preparation of yeast genomic DNA

For PCR SUMMARY Mps1 is a conserved protein kinase that during mitosis corrects improper kinetochore-microtubule attachments, thereby ensuring chromosome biorientation and balanced chromosome segregation. Yet, its critical phosphorylation targets in this process remain largely elusive. Mps1 also controls the spindle assembly checkpoint (SAC), which halts chromosome segregation in metaphase until biorientation is attained. Its role in SAC activation is antagonised by the PP1 phosphatase and involves phosphorylation of the kinetochore scaffold Knl1/Spc105, which in turn recruits the Bub1 kinase to promote assembly of SAC effector complexes. A crucial question is whether error correction and SAC activation are part of a single or separable pathways. During my PhD thesis, I characterized a novel temperature sensitive mutant, mps1-3, that is defective in chromosome biorientation and SAC signaling. T he mps1-3 mutation changes a conserved serine in the kinase domain to phenylalanine, and, surprisingly, the Mps1-3 protein has enhanced catalytic activity compared to the wild type protein. Conversely, the protein does not localize at kinetochores, suggesting that the mps1-3 mitotic defects stem from the lack of phosphorylation of critical kinetochore substrates. T hrough an unbiased screen for spontaneous suppressors of the temperature-sensitivity of the mps1-3 mutant, I found suppressing mutations that affected the interaction between the kinetochore protein Spc105/KNL1 and the phosphatase PP1, suggesting that reduced levels of PP1 at kinetochores underlie the suppression of the temperature-sensitivity of mps1-3 cells. Importantly, the suppressors restored both SAC signaling and proper chromosome biorientation, suggesting that the same molecular mechanism underlies the role of Mps1 in both processes. We have proposed that phosphorylation of Spc105/KNL1, which leads to Bub1 kinetochore recruitment and is antagonized by the phosphatase PP1, is a crucial function of Mps1 in the correction of improper kinetochore-microtubule attachments as it is for SAC activation. Consistently, Spc105 phosphorylation and Bub1 kinetochore localization were affected in mps1-3 cells and restored in the aforementioned suppressors. Moreover, artificial recruitment of Bub1 to Spc105 suppressed the chromosome segregation defects of mps1-3 mutant cells. T hus, a main conclusion of my thesis work is that SAC and error correction are triggered by a single sensory device involving Mps1 and antagonized by PP1. T hrough the same genetic screen, I also found suppressors carrying mutations in the F-box protein Grr1 that is part of the SCF ubiquitin-ligase complex. My preliminary data indicate that Grr1 might localize at kinetochores, suggesting that the SCF Grr1 complex could be a novel player in the control of chromosome bi-orientation at kinetochores. Finally, I could isolate intragenic suppressors, carrying a second mutation in MPS1 that restored the mitotic functions of the Mps1-3 protein. T he data gathered so far indicate that this class of suppressing mutations, unlike the extragenic suppressors above, reestablish Mps1 kinetochore localization. Since Mps1 regulates its own turnover at kinetochores by autophosphorylation, we postulated that the elevated kinase activity of the Mps1-3 mutant protein might be responsible for its displacement from kinetochores and the suppressors might re-establish kinetochore localization of Mps1-3 by decreasing its kinase activity. Surprisingly, although most of the suppressing mutations reduced the ability of Mps1-3 to phosphorylate an exogenous substrate, they did not significantly affect autophosphorylation, suggesting that Mps1-dependent phosphorylation of an unidentified kinetochore protein could influence Mps1 residence time.