Héliciane Par

Palenzuela

Ariane Abrieu

Catherine Jackson

Cécile Leduc

Nicolas Minc

d'assemblage de l'actine Dynein force exertion in bulk cytoplasm

Keywords: Cytoplasmic dynein, microtubules, force generation mechanism, intracellular transport, friction, cytoplasm

Titre:

Génération de forces par la dynéine dans le cytoplasme

Résumé:

Les microtubules et la dynéine, un des moteurs moléculaires qui lui sont associés, sont indispensables à des nombreux processus cellulaires fondamentaux, tels que le transport de vésicules et d'organelles, mais aussi la génération de forces nécessaires au positionnement du noyau et du fuseau mitotique, à la caryogamie, et aux mouvements des astres de microtubules. Le mécanisme le plus communément accepté expliquant la génération de forces par la dynéine sur les microtubules consiste en l'exertion de forces de traction sur les microtubules par la dynéine ancrée au cortex cellulaire. Cependant, de plus en plus d'études tendent à montrer que la dynéine peut aussi exercer des forces sur les microtubules directement depuis le cytoplasme, sans ancrage au cortex, et les mécanismes biophysiques de ce phénomène ne sont pas encore totalement élucidés.

Ainsi, dans ce travail, nous cherchons à identifier le mécanisme de génération de force par la dynéine dans le cytoplasme. En particulier, nous testons une hypothèse prédominante, selon laquelle la dynéine peut tracter les microtubules, au travers des forces de friction hydrodynamique générées par le transport de vésicules et organelles le long des microtubules au sein du cytoplasme visqueux et encombré.

Plus précisément, nous avons construit un test de motilité en 3D, où des billes recouvertes de dynéine peuvent se déplacer sur des microtubules, dans un milieu visqueux, loin des surfaces de la chambre de microcopie. Nous avons ainsi notamment montré, qu'une bille ainsi transportée par des dynéines peut exercer des forces sur le microtubule, le déplaçant dans la direction opposée au transport de la bille. Cette reconstitution d'un système minimal in vitro montre donc qu'un objet déplacé par la dynéine sur un microtubule exerce une force de traction sur ce microtubule, indiquant donc qu'il est envisageable que des vésicules ou organelles se comportent in vivo comme des ancres cytoplasmiques pour la dynéine, lui permettant d'exercer des forces sur un astre de microtubules. Nous nous sommes également intéressés au modèle des embryons d'oursins, où le mouvement de l'astre de microtubules après fertilisation est majoritairement dépendant des forces qu'y exercent la dynéine depuis le cytoplasme. Des expériences de centrifugation des oeufs, induisant la polarisation de la répartition des vésicules et organelles, ont été menées, afin d'étudier l'impact de différentes organelles sur le positionnement de l'astre de microtubules après fertilisation.

Mots clefs:

Dynéine, microtubules, génération de forces, transport intracellulaire, friction, cytoplasme Title:

Dynein force exertion in bulk cytoplasm

Abstract:

Microtubules and dynein, one of its associated molecular motor, organize many essential cellular processes, ranging from vesicle and organelle transport to the generation of pulling forces required for nuclear and spindle positioning, karyogamy, and microtubules aster movements. The most commonly accepted mechanism for microtubule-dynein based force-generation is dynein pulling on the extremities of microtubules from anchoring sites at the cell cortex. However, there is in vivo mounting evidence suggesting that dynein may also pull from sites in the bulk cytoplasm, without anchoring to the cortex. The biophysical mechanisms explaining this phenomenon are not yet fully elucidated.

Hence, in this work, we seek to identify the quantitative basis of force generation by dynein in bulk cytoplasm. In particular, we test a predominant hypothesis, that dynein may pull on microtubules from the hydrodynamic friction forces associated with the dynein-dependent transport of vesicles to the minus-end of microtubules in the viscous and crowded cytoplasm.

More precisely, we developed a 3D motility assay, where dynein coated beads can move on microtubules, in a viscous medium away from coverslip surfaces. We thus showed, that a dynein transported bead can exerce forces on a microtubule, displacing the microtubule in the opposite direction of the moving bead. This in vitro minimal system reconstitution thus showed that dynein transported objects can exert traction forces on microtubules, implying that it is conceivable that, in vivo, vesicles or organelles can act as cytoplasmic anchors for dynein, allowing this motor to exert forces on a microtubule aster. We were also interested in the sea urchin embryo model, where the microtubule aster movement after fertilization mainly depends on forces exerted by dynein from the cytoplasm. Egg centrifugation experiments, inducing polarization of the distribution of vesicles and organelles, were carried out to study the impact of different organelles on the positioning of the microtubule aster after fertilization.

Table of contents

Introduction

I -The cytoskeleton and its associated motors A -A dynamic cytoskeleton

The cell cytoskeleton, contrary to what its name implies, is a very dynamic network, allowing cells to establish and maintain their shape, divide, and in some cases, move in their environment. The cell cytoskeleton is divided into three main classes of filaments: microtubules (MTs), actin and intermediate filaments (IFs). This manuscript will briefly address IFs and actin and mainly focus on MTs. The cytoskeleton has a key role in exerting forces inside the cell, for example through polymerisation and association with molecular motors. As the in vitro studies of these force generation mechanisms is very relevant to our work, they will be discussed at length in the next chapter.

The genes for actin and tubulin appeared in the common ancestor for life on earth, and so actin and tubulin homologues can be found amongst eukaryotes and prokaryotes, whereas IFs genes appeared in early eukaryotes, and are impressively diversified, especially amongst animals [START_REF] Pollard | Overview of the Cytoskeleton from an Evolutionary Perspective[END_REF]. [START_REF] Pollard | Overview of the Cytoskeleton from an Evolutionary Perspective[END_REF]

-Intermediate filaments a -Intermediate filaments functions in the cell

Intermediate filaments (IFs) are especially concentrated in the nuclear periphery, from where they extend to the cell surface, and can attach to focal adhesions in mesenchymal cells, or to desmosomes and hemidesmosomes (respectively cell-cell junctions, or cell-extracellular matrix junctions) in epithelial cells [START_REF] Jones | Intermediate Filaments and the Plasma Membrane[END_REF][START_REF] Etienne-Manneville | Cytoplasmic Intermediate Filaments in Cell Biology[END_REF]. The IF network also connects the nuclear envelope to organelles such as the mitochondria and the Golgi apparatus.

Intermediate filaments are mainly known for their mechanical roles within the cell, contributing to cell shape and offering mechanical support in the cytoplasm and at the cell surface, strongly involved in cell adhesion [Pegoraro et al. 2016;[START_REF] Herrmann | Intermediate Filaments: Structure and Assembly[END_REF]. But the IFs also subtly modulate cell migration and motility, thanks to expression patterns and signaling and mechanical properties that are highly dependent on the cell type, tissue and context [Cheng and Eriksson 2016]. In addition, IFs are also involved in organizing the intracellular components. For example, they are implicated in nucleus positionning in astrocytes cells, and mitochondria localization and function [START_REF] Toivola | Cellular integrity plus: organelle-related and protein-targeting functions of intermediate filaments[END_REF][START_REF] Dupin | Cytoplasmic intermediate filaments mediate actin-driven positioning of the nucleus[END_REF].

Contrary to actin and MTs, IFs are no tracks to any motors.

IFs are categorized into six classes, according to their sequence. The class V corresponds to nuclear IFs, named lamins, whereas the five other classes consists of cytoplasmic IFs. Class I and II respectively consists of acidic and basic keratins, which are especially present in epithelia, but can also be found in non-epithelial cells, and are notably the first IFs proteins to be expressed during embryonic development [START_REF] Lehtonen | Expression of cytokeratin polypeptides in mouse oocytes and preimplantation embryos[END_REF]. Class III IFs are composed of proteins such as vimentins and desmin, which can either homodimerize or heterodimerize with other class III IFs proteins. Class IV IFs proteins, such as nestin, synemin and neurofilaments, are mainly present in neurons and muscles. Finally, class VI IFs comprises phakinin/CP49 and filensin, which are IFs proteins specific to the ocular lens.

b -IF structure

IFs are the less stiff of the three cytoskeleton filaments, with a persistence length varying from several hundred nanometers to a couple of micrometers (the persistence length is proportional to stiffness, and is defined as the length from which the correlation between the object tangents is lost) [START_REF] Schopferer | Desmin and vimentin intermediate filament networks: their viscoelastic properties investigated by mechanical rheometry[END_REF][START_REF] Nöding | Intermediate filaments in small configuration spaces[END_REF]. Apart from this important flexibility, IFs show other interesting properties, like a stretching capability, as well as stiffening after stretching [START_REF] Charrier | Mechanical Properties of Intermediate Filament Proteins[END_REF]. Keratin intermediate filaments could indeed be stretched up to three fold through atomic force microscopy (AFM), and a concomitant decrease in the filament diameter was observed [START_REF] Kreplak | Exploring the mechanical behavior of single intermediate filaments[END_REF]. These stretching properties are linked with the structure of IFs, which consist of laterally associated filaments, and is detailed in the next paragraph.

A common IFs proteins organization has been proposed based on the known sequence of desmin. IFs proteins are composed of an α-helical coiled coil, whose size is conserved, adjoined by two non α-helical end domains on each side of the coiled coil domain. These IFs proteins assemble as dimeric coiled coil, which further associate in an anti-parallel manner, meeting halfway of their total length, finally yielding tetramers that will serve as building blocks for IFs [START_REF] Herrmann | Structure and Assembly Properties of the Intermediate Filament Protein Vimentin: The Role of its Head, Rod and Tail Domains[END_REF]. This antiparallel unit organization results in non-polar filaments, which might explain why no motors have been found to be able to walk on IFs. [START_REF] Herrmann | Intermediate Filaments: Structure and Assembly[END_REF].

Contrary to MTs and actin, IFs have the property to self-assemble without nucleotides [START_REF] Steinert | Self-assembly of bovine epidermal keratin filaments in vitro[END_REF][START_REF] Renner | Reconstitution of intermediate-sized filaments from denatured monomeric vimentin[END_REF]. As shown in the figure 2 above, the IF formation is composed of several steps. During the first phase, tetramers associate laterally, usually grouped by eight, forming a unit length filament (ULF), about 58nm long [Hermann et al., 1996]. The second phase is the longitudinal annealing, where ULFs associate longitudinally, shaping a 17 nm-wide immature IF.

The third and last step is the radial compaction, yielding a 11 nm-wide mature IF [Hermann and Aebi, 2016].

C -IF dynamics

IFs assembly dynamics are very unique, differing from the typical polymerisation and depolymerisation dynamics of actin and MTs, which will be discussed later. It was first thought that the cellular IF network was extremely stable, but was then shown that the IF mesh can actually dynamically rearrange, translocate, shrink and extend. In astrocytes for example, IF transport and turnover depends on actin-dependent retrograde flows, and transport by motors on MTs [START_REF] Leduc | Regulation of microtubule-associated motors drives intermediate filament network polarization[END_REF]. In addition to elongation through polymerization, vimentin IFs have been shown to re extend through severing and end-to-end annealing [START_REF] Winheim | Deconstructing the late phase of vimentin assembly by total internal reflection fluorescence microscopy (TIRFM)[END_REF][START_REF] Hookway | Microtubule-dependent transport and dynamics of vimentin intermediate filaments[END_REF]. Finally, IFs can also experience subunit turnover, in a non-polar fashion, along all IF length [START_REF] Vikstrom | Steady state dynamics of intermediate filament networks[END_REF].

IF formation is also regulated in space and time, as indeed, post translational modifications have been shown to regulate IFs dynamics. Phosphorylation, for example, is involved in vimentin and keratin dynamics regulation [START_REF] Snider | Post-translational modifications of intermediate filament proteins: mechanisms and functions[END_REF].

-Actin a -Actin functions in the cells

Actin is present in different parts of the cells, displaying different architectures.

It is present as an actomyosin meshwork at the cell cortex, allowing cells to deform their surface through contractile forces, for example to migrate, divide or differentiate [START_REF] Chugh | The actin cortex at a glance[END_REF]. Actin is also present in the nucleus, as monomers and filaments, where it plays a role in nuclear integrity maintenance, DNA repair mechanisms and gene regulation [START_REF] Virtanen | Diverse functions for different forms of nuclear actin[END_REF][START_REF] Plessner | Dynamizing nuclear actin filaments[END_REF]. Actin can also be part of membrane protrusions such as lamellipodium and filopodia, respectively composed of branched actin or parallel actin filament bundles, and respectively enabling cells to deform to advance, or probe their environment [START_REF] Wang | Exchange of actin subunits at the leading edge of living fibroblasts: possible role of treadmilling[END_REF][START_REF] Jacquemet | Filopodia in cell adhesion, 3D migration and cancer cell invasion[END_REF]. Finally, actin can be organized as actomyosin stress fibers, contractile structures of antiparallel actin filament bundles, important for cell motility as well as motionless contractility, enabling tissue and extracellular matrix reshaping [START_REF] Pellegrin | Actin stress fibres[END_REF].

b -Actin structure

Actin is present in cells as two forms: globular monomers (G-actin) and polymerized filamentous actin (F-actin) [START_REF] Dominguez | Actin Structure and Function[END_REF]. G-actin is a 42kDa protein, with a length of ~5.4nm [START_REF] Collins | The primary structure of actin from rabbit skeletal muscle. Completion and analysis of the amino acid sequence[END_REF]. It is composed of 4 different subdomains, and in between the 2 nd and 4 th , is a nucleotide binding cleft, as shown in the figure 3 below [START_REF] Pollard | Actin and Actin-Binding Proteins[END_REF]. [START_REF] Pollard | Actin and Actin-Binding Proteins[END_REF].

As actin monomers are polar, their association forms a polar filament, with a so-called barbed end and pointed end, bearing different properties. Subunits are assembled linearly, as two intertwined right-handed helical strands. One strand revolves a full turn in 72nm, a distance referred to as actin helical pitch [START_REF] Holmes | Atomic model of the actin filament[END_REF]. F-actin persistence length is only around 10µm, because despite a high rigidity of the protein, the actin filament is very thin (with an average width of ~8nm), and so finally yields a significantly smaller stiffness than MTs [START_REF] Gittes | Flexural rigidity of microtubules and actin filaments measured from thermal fluctuations in shape[END_REF].

c -Actin dynamics

Actin monomers can self-assemble under physiological salt conditions, and need the presence of cations, as they promote subunits interaction through binding of specific sites [START_REF] Kang | Regulation of Actin by Ion-Linked Equilibria[END_REF]. Polymerisation of pure actin starts with a lag time, which depends strongly on actin concentration, and corresponds to a slow nucleation step [START_REF] Oosawa | Thermodynamics of the polymerization of protein[END_REF]. Indeed, the additions of a 2 nd and a 3 rd monomer are fast but yield an unstable assembly [START_REF] Cooper | Kinetic evidence for a monomer activation step in actin polymerization[END_REF][START_REF] Sept | Thermodynamics and Kinetics of Actin Filament Nucleation[END_REF]. The actin assembly becomes more stable after the addition of a fourth subunit.

Monomers preferentially bind ATP than ADP, and upon assembly, they quickly hydrolyze ATP into ADP-Pi, followed by a slower random release of the inorganic phosphate (Pi), as shown in the figure 4 below [START_REF] Carlier | Direct evidence for ADP-Pi-F-actin as the major intermediate in ATP-actin polymerization. Rate of dissociation of Pi from actin filaments[END_REF][START_REF] Jégou | Individual Actin Filaments in a Microfluidic Flow Reveal the Mechanism of ATP Hydrolysis and Give Insight Into the Properties of Profilin[END_REF]. Quickly upon assembly, ATP-actin is randomly hydrolyzed into ADP-Pi actin, after which the inorganic phosphate leaves the filament at a much slower rate. Adapted from [START_REF] Pollard | Actin and Actin-Binding Proteins[END_REF] After incorporation, monomers experience a conformation change, (a rotation of ~20 degrees of the subdomains 1 and 2) which could be the cause of the activation of the ATPase property of actin [START_REF] Oda | The nature of the globular-to fibrous-actin transition[END_REF].

As actin filaments are polar, actin assembly rates at the two different ends are different, with a higher assembly rate at the barbed end than at the pointed end.

When ATP is present and can bind actin monomers, the steady-state concentration will be such that the net monomer addition at the barbed end balances the net monomer loss at the pointed end, in a fascinating "treadmilling" process [START_REF] Wegner | Head to tail polymerization of actin[END_REF]. Actin-binding proteins, which are going to be described below, have a different affinity for actin as a function of the actin nucleotide state. Actin rigidity also depends on this nucleotide state, with a higher actin rigidity in the ADP-Pi state [START_REF] Isambert | Flexibility of actin filaments derived from thermal fluctuations. Effect of bound nucleotide, phalloidin, and muscle regulatory proteins[END_REF].

d -Actin-binding proteins

Various actin-binding proteins (ABPs) can interact with actin, regulating its nucleation, elongation, severing, crosslinking or branching.

Figure 5: Overview of the various actin binding proteins activities [START_REF] Pollard | Actin and Actin-Binding Proteins[END_REF].

Sequestering activity

In cells, the high actin concentration (~100µM) would imply that almost all actin would be polymerized, yielding a quite inert actin cytoskeleton [Funk et al., 2019]. But actin structures are highly dynamic in cells, due to the interaction of actin-binding proteins (ABPs) with actin. Indeed, profilin and thymosin β-4, notably, bind to actin monomers, and by sequestering them, control actin nucleation and assembly [START_REF] Kaiser | Profilin is predominantly associated with monomeric actin in Acanthamoeba[END_REF][START_REF] Xue | Structural basis of thymosin-β4/profilin exchange leading to actin filament polymerization[END_REF].

Nucleation and elongation factors

Actin nucleation and elongation are controlled in cells by proteins such as formins, the Arp2/3 complex, Spire or Ena/VASP, through various processes. Formin can nucleate actin filaments from actin monomers by stabilizing dimers, as well as strongly increase actin assembly rate from profilin-actin, while tracking the elongating barbed end in a processive way [START_REF] Kovar | Control of the Assembly of ATP-and ADP-Actin by Formins and Profilin[END_REF]. Ena/VASP can also processively track the barbed end, and speed up filaments elongation [START_REF] Hansen | VASP is a processive actin polymerase that requires monomeric actin for barbed end association[END_REF][START_REF] Edwards | Capping Protein Regulators Fine-Tune Actin Assembly Dynamics[END_REF]. The Arp2/3 complex nucleates F-actin either as branches from the side of pre-existing actin filaments, thus forming branched networks, or directly from monomers, when activated by WISH/DIP/SPIN90 [START_REF] Blanchoin | Direct observation of dendritic actin filament networks nucleated by Arp2/3 complex and WASP/Scar proteins[END_REF][START_REF] Wagner | Dip1 Defines a Class of Arp2/3 Complex Activators that Function without Preformed Actin Filaments[END_REF].

Capping proteins

Some ABPs can cap actin filaments ends, preventing monomers from assembling or disassembling, thus controlling filament length. For example, Capping Protein (CP) and gelsolin can cap the barbed end of actin filaments, whereas tropomodulin can cap the actin filaments pointed ends [START_REF] Isenberg | An actin-binding protein from Acanthamoeba regulates actin filament polymerization and interactions[END_REF][START_REF] Nag | Gelsolin: The tail of a molecular gymnast[END_REF][START_REF] Rao | Mechanism of actin filament pointed end capping by tropomodulin[END_REF].

Severing activity

Severing proteins allow for a quicker actin turnover. The two main severing proteins families are ADF/cofilin and gelsolin, but two proteins from the formin family, FRL-α and INF-2, have also been shown to exhibit severing properties [START_REF] Harris | The mouse formin, FRLalpha, slows actin filament barbed end elongation, competes with capping protein, accelerates polymerization from monomers, and severs filaments[END_REF]Gurel et al., 2014]. ADF/cofilin binds cooperatively to actin filaments, causing a local increase in the actin twist and a change of mechanical properties, which favor filament severing. This severing occurs preferentially at the border between cofilindecorated and undecorated sections, and more frequently at the border closer to the pointed end [START_REF] Mcgough | Cofilin Changes the Twist of F-Actin: Implications for Actin Filament Dynamics and Cellular Function[END_REF][START_REF] De La Cruz | How cofilin severs an actin filament[END_REF][START_REF] Wioland | ADF/Cofilin Accelerates Actin Dynamics by Severing Filaments and Promoting Their Depolymerization at Both Ends[END_REF]. Gelsolin, on the other hand, binds to the filaments sides, and causes severing by curling the filament [START_REF] Mcgough | Determination of the gelsolin binding site on F-actin: implications for severing and capping[END_REF][START_REF] Sun | Gelsolin, a Multifunctional Actin Regulatory Protein[END_REF].

Crosslinking and bundling proteins

In cells, actin filaments associate in various structures thanks to crosslinking proteins. Bundling proteins, such as fascin and fimbrin/plastin allow filaments to associate in parallel bundles, with a close proximity between each other, present in protrusive structures [START_REF] Bartles | Parallel Actin Bundles and Their Multiple Actin-bundling Proteins[END_REF][START_REF] Klein | Structure of the Actin Crosslinking Core of Fimbrin[END_REF][START_REF] Bretscher | Fimbrin, a new microfilament-associated protein present in microvilli and other cell surface structures[END_REF][START_REF] Glenney | F-actin binding and bundling properties of fimbrin, a major cytoskeletal protein of microvillus core filaments[END_REF][START_REF] Shin | Molecular Architecture of the Chick Vestibular Hair Bundle[END_REF]. On the other hand, some crosslinking proteins, as filamin, allow actin filaments to associate less closely, thanks to filamin distant actin binding domain, in actin structures at the leading edge of cells [START_REF] Matsudaira | Actin crosslinking proteins at the leading edge[END_REF]Svitkina, 2016]. Finally, α-actinin associates filaments with intermediate distance, and mixed polarity, in structures such as the cytokinetic ring and stress fibres [START_REF] Foley | The non-muscle functions of actinins: an update[END_REF][START_REF] Winkelman | Fascin-and α-Actinin-bundled networks contain intrinsic structural features that drive protein sorting[END_REF].

-Microtubules

a -MT functions in cells

MTs are highly dynamic polymers, which can organize in various structures, and on which depend various essential processes, such as cell motility, cell division, cell shape and intracellular transport and organisation [START_REF] Goodson | Microtubules and Microtubule-Associated Proteins[END_REF]. The MTs are the tracks of two molecular motors families: dynein and kinesin, which will be described at length in the next part on molecular motors.

To begin with, cell motility relies on specific MT arrangements found in cilia and flagella. These structures, composed of MTs and motors, enable the motility of various protists, and is also the basis for most metazoan sperm motility. A basal body, composed of nine MTs triplets, lies at the base of each cilia or flagella. Cilia also have important roles in organs, as for example in respiratory tracks, where they allow proper fluid movement and elimination [START_REF] Viswanadha | Ciliary Motility: Regulation of Axonemal Dynein Motors[END_REF].

Very importantly, MTs can also organize from MTs organizing centers (MTOCs). In most cases, MTs are organized as a radial array, named MT aster, with MTs minus ends oriented towards the MTOC, and MTs plus ends extending towards the cell periphery. MT asters can extend, or shrink, and are strongly involved in force exertion inside cells, as we will detail in the next chapters, respectively focusing on the in vitro characterization of MT-and motor-dependent forces, and MT-and dyneindependent aster centration after fertilization. Indeed, as detailed later, after fertilization in big eggs such as echinoderms, a MT aster grows around the male pronucleus, and exert forces which enable it to reach the cell center.

The interphase array of MTs is also of crucial importance in cells, as it participate to cell shape, and is the basis for intracellular transport, which consequently acts on intracellular organisation, and cell polarity [START_REF] Barlan | Microtubule-Based Transport and the Distribution, Tethering, and Organization of Organelles[END_REF].

But MTs are not always organized as a radial array, as for example, in higher plants, where they are organized parallel to each other, and perpendicular to the cell longer axis [START_REF] Ehrhardt | Microtubule Dynamics and Organization in the Plant Cortical Array[END_REF]. MTOCs are complex assemblies, and if the various MTOCs types contain many similar proteins, and possess the same nucleation activity, they can differ in structure and composition between cell types.

Perinuclear MTOCs, containing centrioles and yielding radial MTs arrays are called centrosomes. Other MTOCs, as for amoebozoa and fungi, are deprived from centrioles. For example, in yeast, these centrioles-free MTOCs are embedded in the nuclear membrane, and are called spindle pole bodies (SPBs) [Wu and Akhmanova, 2017].

MTs are essential for proper cell division, through the MT structure of the mitotic spindle, which emanates from two centrosomes. The centrosomes is composed of two centrioles (cylindrical structures composed of nine MTs triplets) as well as pericentriolar material. It needs to duplicate before cell division, yielding two centrosomes, which will correspond to two poles of the mitotic spindle. The number of centrosome is tightly regulated in cells, and defects in this number are strongly correlated with aberrant chromosome number in cancer daughter cells [START_REF] Hardy | Reappraisal of the Hansemann-Boveri hypothesis on the origin of tumors[END_REF][START_REF] Nigg | The centrosome cycle: Centriole biogenesis, duplication and inherent asymmetries[END_REF]. The MT mitotic spindle is capable of separating chromosomes copies, in order for each daughter cells to end up with the same gene pool. Each sister chromatid attaches to one side of the spindle, through the connection of kinetochore spindle MTs to each chromatid kinetochore, which consists in a complex protein assembly situated at the centromere, and regulates the chromatid association to the MT tips. Once attached to one side of the spindle, each chromosome copy can be pulled by the spindle MTs towards opposite sides of the cell [START_REF] Mcintosh | Mitosis[END_REF]. This force exertion mechanism will be detailed further in the chapter on dynein involvement in cell division.

Finally, at the end of cell division, during what is called cytokinesis, cells separate thanks to the constriction of an actomyosin ring, and a MT structure called midbody forms at the point of abscission. This structure, made of MT and several proteins, not only links the future daughter cells, but is also involved in the cytokinetic activity itself, contributing for example to selective vesicle transport [START_REF] Chen | Resurrecting remnants: the lives of post-mitotic midbodies[END_REF].

b -MT structure

MTs have a significantly higher stiffness than actin and IFs, with a persistence length of more than 1mm [START_REF] Gittes | Flexural rigidity of microtubules and actin filaments measured from thermal fluctuations in shape[END_REF]. They are hollow tubes of ~25nm of diameter, with lengths ranging from less than 1µm to more than 100µm.

MTs are made of heterodimers of α-tubulin and β-tubulin. These dimers associate longitudinally, forming a protofilament. These protofilaments further connect with each other, each α-tubulin bonding with their neighbor α-tubulin, forming a total of 13 protofilaments on average, although this number can vary as a function of the species, cell type and cell stage [START_REF] Chaaban | A microtubule bestiary: structural diversity in tubulin polymers[END_REF]. As each protofilament is slightly shifted from its neighbor, the MT filament resemble a helix, except, for 13 protofilaments MTs, in the seam region, where a little gap exists between two protofilaments, with α-tubulins of one protofilament facing β-tubulin of the neighboring protofilament. As dimers are formed by two different proteins, MTs are polar. The MT side with apparent β-tubulins is called the MT plus end, and the MT side with apparent α-tubulins is called the MT minus end.

Figure 6: MTs: from a tubulin heterodimer to a MT polymer. Dimers are assembled in filaments, which further associate laterally [START_REF] Goodson | Microtubules and Microtubule-Associated Proteins[END_REF].

c -MT nucleation

The beginning of tubulin assembly, referred as nucleation, is not kinetically favorable, and thus requires the presence of regulatory proteins. Nucleating factors and Microtubules Associated Proteins (MAPs) allow cells to control the place and time of MT polymerisation, and polymerise even at low tubulin concentration. Indeed, if as little as 1µM tubulin is required for MT elongation, MT nucleation without MAPs would necessitate a tubulin concentration considerably higher than this critical concentration [Voter and Erikson, 1984;[START_REF] Wieczorek | Microtubuleassociated proteins control the kinetics of microtubule nucleation[END_REF]. Additionally, MT nucleation efficiency increases non-linearly with tubulin concentration [START_REF] Kuchnir Fygenson | Spontaneous nucleation of microtubules[END_REF]. This nucleation regulation is not yet fully understood, but is orchestrated in vivo by two different proteins type: γ-tubulin ring complexes (γ-TuRCs) and microtubule-associated proteins (MAPs).

On the one hand, γ-TuRCs are composed of several γ-tubulin small complex (γ-TuSCs), which are a combination of two γ-tubulin subunits, associated through two γ-tubulin complex proteins (GCP), most frequently GCP2 and GCP3. These γ-TuSCs associate together, with GCP2 and GCP3 sometimes replaced by GCP4, GCP5 and GCP6 [START_REF] Guillet | Crystal structure of γ-tubulin complex protein GCP4 provides insight into microtubule nucleation[END_REF][START_REF] Tovey | Microtubule nucleation by γ-tubulin complexes and beyond[END_REF]. γ-TuRCs seem to allow MT nucleation by supplying a template for the formation of a 13 protofilament MT, as shown in the figure 7 below. Indeed, they provide exposed γ-tubulin, arranged in a helical manner, where α-tubulin from dimers can dock, initiating the 13 protofilament structure, and providing dimers enough proximity to increase the strength of their otherwise weak lateral interaction [START_REF] Aldaz | Insights into microtubule nucleation from the crystal structure of human γ-tubulin[END_REF][START_REF] Kollman | Microtubule nucleating γ-TuSC assembles structures with 13-fold microtubule-like symmetry[END_REF]. γ-TuRCs can form in cytosol, or directly on MTOCs [START_REF] Kollman | Microtubule nucleating γ-TuSC assembles structures with 13-fold microtubule-like symmetry[END_REF][START_REF] Lynch | Activation of the γ-Tubulin Complex by the Mto1/2 Complex[END_REF][START_REF] Cota | MZT1 regulates microtubule nucleation by linking γTuRC assembly to adapter-mediated targeting and activation[END_REF][START_REF] Vogt | The γTuRC components Grip75 and Grip128 have an essential microtubule-anchoring function in the Drosophila germline[END_REF]. [START_REF] Tovey | Microtubule nucleation by γ-tubulin complexes and beyond[END_REF].

On the other hand, some MAPs have been shown to be MT nucleation factors, causing an increase in the lateral and longitudinal contacts between dimers: TPX2 homologues, Tog domain protein like XMAP215 and CLIP associated proteins (CLASPs), calmodulin-regulated spectrin associated protein 2 (CAMSAP)/Patronin, and p150Glued [START_REF] Al-Bassam | Regulation of Microtubule Dynamics by TOGdomain proteins XMAP215/Dis1 and CLASP[END_REF][START_REF] Roostalu | Complementary activities of TPX2 and chTOG constitute an efficient importin-regulated microtubule nucleation module[END_REF][START_REF] Thawani | XMAP215 is a microtubule nucleation factor that functions synergistically with the γ-tubulin ring complex[END_REF][START_REF] Roostalu | Microtubule nucleation: beyond the template[END_REF].

As represented in the figure 8 below, γ-TuRCs and MAPs can act alone, or in combination, yielding a more efficient nucleation, thus based both on template supply and stabilization of the nascent MT lattice. The smaller stable portion of the lattice is called nucleus, and refers to the smaller size after which the addition of a dimer becomes kinetically favorable [START_REF] Roostalu | Microtubule nucleation: beyond the template[END_REF][START_REF] Tovey | Microtubule nucleation by γ-tubulin complexes and beyond[END_REF]. These MAPs, favorable to MT nucleation, also compete with nucleation inhibitors, such as the catastrophe factors mitotic centromere-associated kinesin (MCAK) and EB1, offering the possibility for an even finer tuning of nucleation [START_REF] Wieczorek | Microtubuleassociated proteins control the kinetics of microtubule nucleation[END_REF] [ [START_REF] Tovey | Microtubule nucleation by γ-tubulin complexes and beyond[END_REF].

Nucleation can also occur from preexisting MTs. For example, the augmin complex can recruit γ-TuRCs to a MT side, in a TPX2 dependent manner [START_REF] Goshima | Augmin: a protein complex required for centrosome-independent microtubule generation within the spindle[END_REF]Petry et al., 2012].

Whereas in vivo nucleation is controlled and favored by several proteins, MT can nucleate in vitro in a minimal solution of tubulin dimers, GTP and Mg 2+ [START_REF] Voter | The Kinetics of Microtubule Assembly. Evidence for a Two-Stage Nucleation Mechanism[END_REF], and is known to be favored by MT stabilizing drugs [START_REF] Hamel | Interactions of taxol, microtubule-associated proteins, and guanine nucleotides in tubulin polymerization[END_REF][START_REF] Arnal | How does taxol stabilize microtubules?[END_REF], glycerol [START_REF] Shelanski | Microtubule Assembly in the Absence of Added Nucleotides[END_REF] and GMPCPP, a GTP non-hydrolysable analogue [START_REF] Hyman | Role of GTP hydrolysis in microtubule dynamics: information from a slowly hydrolyzable analogue, GMPCPP[END_REF][START_REF] Wieczorek | Microtubuleassociated proteins control the kinetics of microtubule nucleation[END_REF].

d -MT dynamics

MT dynamics are quite fascinating, with a plus end more dynamic than the minus end, and alternating phases of slow polymerisation and quick depolymerisation, a phenomena named dynamic instability. The transitions between MT growth and shortening are called catastrophes, and the transitions between MT shortening and growth are called rescues [START_REF] Mitchison | Dynamic instability of microtubule growth[END_REF]. This spontaneous disassembly originates from the hydrolysis of GTP and the structural changes it implies, as described in the next paragraph.

Figure 9: MT dynamics at the minus and plus ends. GTP tubulin is incorporated into the MT lattice, and will progressively be hydrolyzed. Phases of growth and shrinkage alternate, through rescues and catastrophes. The MT plus ends is more dynamic than the MT minus end [START_REF] Akhmanova | Microtubule minus-end regulation at a glance[END_REF].

Indeed, a tubulin dimer can bind two GTP molecules through its two α-tubulin and β-tubulin nucleotides binding pockets (respectively named N-site and E-site), but only the β-tubulin site can exchange and hydrolyze GTP [START_REF] Nogales | Tubulin and FtsZ form a distinct family of GTPases[END_REF]. When tubulin concentration exceeds the critical one, dimers can assemble. Assembly takes place only when β-tubulin is bound to GTP, as the critical concentration for GDP tubulin assembly is unachievably high [START_REF] Howard | Mechanics of Motor Proteins and the Cytoskeleton[END_REF]. Soon after assembly, the βtubulin captured GTP will release its phosphate, as the newly incorporated tubulin acts as a GTPase activating protein for the previously incorporated dimer [START_REF] Nogales | High-Resolution Model of the Microtubule[END_REF][START_REF] Melki | Continuous Monitoring of Pi Release Following Nucleotide Hydrolysis in Actin or Tubulin Assembly Using 2-Amino-6mercapto-7-methylpurine Ribonucleoside and Purine-Nucleoside Phosphorylase as an Enzyme-Linked Assay[END_REF]. The MT lattice is thus composed mainly of GDP tubulin, with the exception of the very end of the MT, where GTP has not yet been hydrolyzed. Interestingly, GTP tubulin and GDP tubulin had been shown to have different conformations [START_REF] Alushin | High resolution microtubule structures reveal the structural transitions in αβtubulin upon GTP hydrolysis[END_REF][START_REF] Goodson | Microtubules and Microtubule-Associated Proteins[END_REF]. Indeed, the hydrolysis of GTP and loss of inorganic phosphate leads to a compaction of the GTP β tubulin binding pocket, the E-site. Thus, whereas GTP tubulin can be straight in a protofilament, GDP tubulin is slightly curved, accumulating strain and thus favoring MT destabilization. This dynamic instability is finely tuned in cells through the action of microtubule-binding proteins (MTBPs), as described in the next part.

The majority of the tubulin is assembled from the MT tips, but it has also been shown that dimers can be incorporated along the MTs, at lattice defects sites [Schaedel et al., 2018]. [START_REF] Alushin | High resolution microtubule structures reveal the structural transitions in αβtubulin upon GTP hydrolysis[END_REF].

e -Regulation of MT through MTBPs and the tubulin code

MTBPs favoring MT disassembly

GTP hydrolysis isn't the only actor of MT lattice destabilization. Indeed, specific proteins can destabilize MTs through different mechanisms.

To begin with, severing proteins can break MTs, thus exposing a GDP tubulin lattice, which will thus undergo a catastrophe [START_REF] Sharp | Microtubule-severing enzymes at the cutting edge[END_REF]. These proteins, such as katanin, fidgetin and spastin, are member of the AAA family (ATPases Associated with diverse cellular Activities) [START_REF] Roll-Mecak | Microtubule severing enzymes[END_REF].

Sequestering proteins also play a role in inhibiting MT polymerisation, as they bring about a lowering of the concentration of tubulin dimers which can be used for polymerisation. Stathmin, for example, favors the association of monomers into a curved dimer, which cannot be incorporated into the MT lattice [START_REF] Cassimeris | The oncoprotein 18/stathmin family of microtubule destabilizers[END_REF].

Finally, some proteins can directly destabilize the MT tip, such as some kinesins, which can remove tubulin subunits at the tip. This facilitates catastrophe, and even disassembles GMPCPP or taxol stabilized MTs [START_REF] Desai | Kin I Kinesins Are Microtubule-Destabilizing Enzymes[END_REF][START_REF] Walczak | Microtubule-Depolymerizing Kinesins[END_REF]. Stathmin, as an addition to its well-known sequestering role, can also cap MTs, binding to exposed protofilaments at MTs tips, which prevents dimers addition and promotes catastrophes [START_REF] Gupta | Mechanism for the catastrophe-promoting activity of the microtubule destabilizer Op18/stathmin[END_REF].

MTBPs favoring MT assembly

Even if the mode of action of each MT stabilizer is not yet known, they all share the presence of several MT binding domain (MTBD), indicating that at least a part of their stabilizing properties is due to the lateral or longitudinal crosslinking of protofilaments. It is also worth noting that MT stabilizers can potentially act either by promoting MT polymerisation, preventing catastrophe, facilitating rescues, or stalling MT dynamics, inhibiting both shrinkage and growth. The way stabilizers promote MT growth rate is still debated. Some can bind simultaneously to MT plus tips and to free dimers, facilitating the free dimer incorporation in a tethering mechanism [START_REF] Ayaz | Dynactin binding to tyrosinated microtubules promotes centrosome centration in C. elegans by enhancing dynein-mediated organelle transport[END_REF]. They could also, through lowering the departure rate of dimers from the lattice (koff, MT), increase the proportion of incorporated dimers [START_REF] Gardner | Rapid Microtubule Self-assembly Kinetics[END_REF]. Finally, these stabilizers can act alone or in synergy [START_REF] Zanic | Synergy between XMAP215 and EB1 increases microtubule growth rates to physiological levels[END_REF][START_REF] Gupta | Promoting microtubule assembly: A hypothesis for the functional significance of the +TIP network[END_REF].

The MTBPs favoring MT polymerisation range from Tog domain proteins such as XMAP215 and CLASP (which were already discussed as nucleation promoting factors) [START_REF] Al-Bassam | Regulation of Microtubule Dynamics by TOGdomain proteins XMAP215/Dis1 and CLASP[END_REF], to CAP-GLY containing proteins such as CLIP170 [START_REF] Arnal | CLIP-170/Tubulin-Curved Oligomers Coassemble at Microtubule Ends and Promote Rescues[END_REF][START_REF] Steinmetz | Capturing protein tails by CAP-Gly domains[END_REF]. The MT-kinetochore linker NDC80 also plays a role in stabilizing MTs [START_REF] Umbreit | The Ndc80 kinetochore complex directly modulates microtubule dynamics[END_REF][START_REF] Varma | The KMN protein networkchief conductors of the kinetochore orchestra[END_REF], as well as proteins with calponin-homology (CH) domain, such as the MT growing end tracking protein EB1 [START_REF] Komarova | Mammalian end binding proteins control persistent microtubule growth[END_REF]. Last but not least, STOP proteins, and the MAPS tau, MAP2 and MAP4 also act as MT stabilizer [START_REF] Bosc | STOP Proteins[END_REF][START_REF] Dehmelt | The MAP2/Tau family of microtubule-associated proteins[END_REF].

MTBPs with functions not linked to dynamic instability

MTBPs also have other properties than stabilizing or destabilizing the MT lattice. Some have crosslinking activities, as the MAP65/Ase1/PRC1 family, which can bundle antiparallel MTs [START_REF] Bieling | A Minimal Midzone Protein Module Controls Formation and Length of Antiparallel Microtubule Overlaps[END_REF][START_REF] Subramanian | Insights into anti-parallel microtubule crosslinking by prc1, a conserved non-motor microtubule binding protein[END_REF]. Some MTs stabilizers can also crosslink MTs, as tau for example, which can bundle MTs by coating the negatively charged MTs [START_REF] Melki | Interaction of microtubule-associated proteins with microtubules: yeast lysyl-and valyl-tRNA synthetases and .tau. 218-235 synthetic peptide as model systems[END_REF]. This lateral association of MTs can also be caused, in vitro, by the depletion effect caused by crowding agents like polyethylene glycol [START_REF] Sanchez | Spontaneous motion in hierarchically assembled active matter[END_REF].

MTBPs also play a role as connectors and regulators of cytoskeletal elements. Indeed, the three cytoskeleton elements should not be considered independent, but rather, engaged in a crosstalk, essential for a range of cellular process [START_REF] Dogterom | Actin-microtubule crosstalk in cell biology[END_REF]. Crosstalk between actin and MTs encompasses crosslinking of MTs and actin (as mediated by the plakin proteins for example [START_REF] Suozzi | Spectraplakins: Master orchestrators of cytoskeletal dynamics[END_REF][START_REF] Applewhite | The actinmicrotubule cross-linking activity of Drosophila Short stop is regulated by intramolecular inhibition[END_REF]); MTs ends anchoring to actin networks (involving protein such as myosin X [START_REF] Weber | A microtubule-binding myosin required for nuclear anchoring and spindle assembly[END_REF]); MT growth guided by actin bundles [START_REF] López | Actin-microtubule coordination at growing microtubule ends[END_REF] and actin nucleation and elongation from MT plus tips (controlled by proteins assembly of formin, CLIP170 and EB1 [START_REF] Lewkowicz | The microtubule-binding protein CLIP-170 coordinates mDia1 and actin reorganization during CR3-mediated phagocytosis[END_REF][START_REF] Henty-Ridilla | Accelerated actin filament polymerization from microtubule plus ends[END_REF]).

MTBPs can also play a role in controlling the number of protofilaments of the MT lattice, as for example doublecortin, which drives the assembly of 13 protofilaments MTs, stabilizing both longitudinal and lateral connections [START_REF] Fourniol | Template-free 13-protofilament microtubule-MAP assembly visualized at 8 Å resolution[END_REF].

Finally, MTBPs also include motors (which will be detailed in the next part), proteins regulating motor activities (such as ensconsin and tau [START_REF] Barlan | The microtubule-binding protein ensconsin is an essential cofactor of kinesin-1[END_REF][START_REF] Dixit | Differential Regulation of Dynein and Kinesin Motor Proteins by Tau[END_REF]) and even some organelle binding proteins [START_REF] Vedrenne | Phosphorylation Controls CLIMP-63-mediated Anchoring of the Endoplasmic Reticulum to Microtubules[END_REF]Gurel et al., 2014]. [START_REF] Bodakuntla | Microtubule-Associated Proteins: Structuring the Cytoskeleton[END_REF].

Figure 11: Overview of the variety of MAPs and their properties. MAPs can crosslink MTs (A), connect them to membranes (B), sever MTs or protect them from severing (C), crosslink MT with other cytoskeletal elements (D), control protofilament number (E), regulate motor activities (F) or bundle MTs (G)

The tubulin code

Another level of MT regulation is based on what is called the tubulin code. Indeed, despite the highly conserved sequence of α and β tubulin among species, MTs exhibit very different structures or properties as a function of species, cell type or cell cycle. This mainly relies on the existence of different tubulin isotypes, and a wide range of post translational modifications (PTMs). These different isotypes and PTMs can tune the MT mechanical properties, its polymerization and depolymerisation dynamics, as well as the MT interactions with MAPs [START_REF] Janke | The tubulin code and its role in controlling microtubule properties and functions[END_REF].

Tubulin isotypes number varies a lot as a function of species, and the human genome possesses 9 isotypes for each α and β tubulin. Some MT architectures rely only on specific isoforms, such as the specific MT array allowing platelets assembly for coagulation, assembled from β1tubulin isoform only [START_REF] Schwer | A lineage-restricted and divergent β-tubulin isoform is essential for the biogenesis, structure and function of blood platelets[END_REF].

Tubulin PTMs can occur specifically or not on free dimers or assembled dimers, allowing for another layer of MT regulation. Some PTMs found in tubulin were already described in other proteins. It includes acetylation, methylation, phosphorylation, ubiquitylation, palmitoylation and polyamination [START_REF] L'hernault | Chlamydomonas .alpha.-tubulin is posttranslationally modified by acetylation on the .epsilon.-amino group of a lysine[END_REF][START_REF] Park | Dual Chromatin and Cytoskeletal Remodeling by SETD2[END_REF][START_REF] Eipper | Rat Brain Microtubule Protein: Purification and Determination of Covalently Bound Phosphate and Carbohydrate[END_REF][START_REF] Ren | Parkin Binds to α/β Tubulin and Increases their Ubiquitination and Degradation[END_REF]Ozols and Caro, 2017;[START_REF] Song | Transglutaminase and Polyamination of Tubulin: Posttranslational Modification for Stabilizing Axonal Microtubules[END_REF]. But other PTMs were actually discovered in tubulin. They encompass the enzymatic addition of amino acid, such as glycine, glutamate, tyrosine, and the removal of an amino acid, such as tyrosine [START_REF] Redeker | Polyglycylation of tubulin: a posttranslational modification in axonemal microtubules[END_REF][START_REF] Edde | Posttranslational glutamylation of alpha-tubulin[END_REF][START_REF] Arce | Incorporation of l-Tyrosine, l-Phenylalanine and l-3,4-Dihydroxyphenylalanine as Single Units into Rat Brain Tubulin[END_REF]Rodríguez et al., 1973;[START_REF] Hallak | Release of tyrosine from tyrosinated tubulin. Some common factors that affect this process and the assembly of tubulin[END_REF].

Figure 12: The tubulin code is based on various tubulin isotypes and a range of PTMs. These PTMs include phosphorylation, methylation, acetylation and polyamination, but also tyrosination, detyrosination, polyglycylation and polyglutamylation [START_REF] Janke | The tubulin code and its role in controlling microtubule properties and functions[END_REF].

B -Cytoskeletal motors

Molecular motors are proteins which convert chemical energy into mechanical motion, allowing them to step on their preferred tracks: actin for myosin motors, and MTs for dyneins and kinesins. This manuscript will briefly address myosin and kinesin, and will give more details about dynein.

These motor families often have a specific directionality, as for example, dynein, which can only walk towards the minus end of MTs. Another characteristic of motors is their processivity, that is to say a property to walk for several steps on their tracks before being released from them. Motors are of vital importance in cells, involved in various aspects that will be quickly described in this part, with the exception of dynein dependent transport and dynein-dependent force exertion on the cytoskeleton, which will be detailed in the next chapters. (respectively myosin V, kinesin-1 and cytoplasmic dynein) [START_REF] Carter | Crystal clear insights into how the dynein motor moves[END_REF].

-Myosin

Myosin is primarily known to be the motor responsible for the force generation in muscle cells (myosin II), organized in structures called sarcomeres. But the myosin family also plays a role in force generation in non-muscle cells, with implication in stress fibers contraction, cytokinesis ring constriction, and cortex contraction [START_REF] Weber | Antibody to myosin: the specific visualization of myosin-containing filaments in nonmuscle cells[END_REF][START_REF] Hu | Long-range selforganization of cytoskeletal myosin II filament stacks[END_REF][START_REF] Henson | The ultrastructural organization of actin and myosin II filaments in the contractile ring: new support for an old model of cytokinesis[END_REF][START_REF] Chugh | The actin cortex at a glance[END_REF]. Finally, myosin is involved in transport, predominantly oriented towards the actin filaments barded ends (as for example myosin which can bring Ena/VASP to the filopodia tips), with the exception of myosin VI displacement, oriented towards the pointed end of actin filaments [START_REF] Wells | Myosin VI is an actin-based motor that moves backwards[END_REF][START_REF] Tokuo | Myosin X transports Mena/VASP to the tip of filopodia[END_REF].

There exist dozens of myosin families but they all share a common building plan, as shown in the figure 14 below. The myosin head is composed of the motor domain ATPase, the actin-binding domain and the lever arm (which is a helix containing one or several calmodulin binding sites, also called IQ motif based on the letter code of the two first amino acids of the sequence). Some myosins contain a coiled coil region following the level arm, which allows them to dimerize. Finally, the C-terminal region of myosins enables them to bind to their specific target [START_REF] Sweeney | Structural and functional insights into the Myosin motor mechanism[END_REF].

Figure 14: Functional domains of myosin families. Each myosin has specificities, but they share a common organisation: a head which can bind actin, and comprises the motor domain, followed by a helix with calmodulin binding sites, in some cases a coiled coil region which can allow dimerization, and finally the C-terminal region, enabling the motor to bind its specific target [START_REF] Sweeney | Structural and functional insights into the Myosin motor mechanism[END_REF].

Myosin exerts forces through the conversion of the chemical energy of ATP to mechanical energy. Indeed, the ATP cycle causes a change in conformation of the motor domain, which is transmitted and amplified by the converter region, and transmitted to the myosin lever arm, enabling it to apply forces [START_REF] Sweeney | Structural and functional insights into the Myosin motor mechanism[END_REF].

-Kinesin

Kinesin was first identified in the squid giant axons and optic nerves in 1985 [START_REF] Vale | Identification of a novel forcegenerating protein, kinesin, involved in microtubule-based motility[END_REF]. Kinesins can be classified in 14 families, which all share the same conserved motor domain [START_REF] Lawrence | A standardized kinesin nomenclature[END_REF].

Kinesin is involved in transport (mainly towards the plus end of MTs, with the exception of the kinesin-14 family) but also MT dynamics regulation, as well as chromosome dynamics in meiosis and mitosis [START_REF] Carter | Crystal clear insights into how the dynein motor moves[END_REF][START_REF] Klinman | Walking Forward with Kinesin[END_REF].

It is composed of a heavy chain (120KDa) and a light chain (70KDa), present in two copies, forming a homodimer dimerised through their coiled coil region, as represented in the figure 15 below [START_REF] Vale | The Molecular Motor Toolbox for Intracellular Transport[END_REF][START_REF] Carter | Crystal clear insights into how the dynein motor moves[END_REF]. The heavy chain includes the motor ATP hydrolysis region and the MT binding domain (MTBD), and is sufficient for kinesin walking on MTs. The light chain is involved in cargo binding (along with the tail of the heavy chain) and motor activty regulation. Even if kinesin sequence is different from other motors, kinesin catalytic core presents a structural resemblance to the one of myosin [START_REF] Kull | Crystal structure of the kinesin motor domain reveals a structural similarity to myosin[END_REF]. [START_REF] Vale | The Molecular Motor Toolbox for Intracellular Transport[END_REF] Kinesin force exertion is based on conformational changes caused by nucleotide binding and hydrolysis, which will change the kinesin affinity for the MT, and bias its movement towards one end of the MT, which is the plus end for most kinesins. Indeed, when ATP is bound to kinesin motor domain, kinesin affinity for the MT is high, and it is the ATP hydrolysis that, through a conformational change of the kinesin, allows it to detach from the MT in order to make a step. When the phosphate is released, it brings about a conformational change of the neck linker, which will make it more mobile, and its docking will biases kinesin movement toward MT plus end [START_REF] Clancy | A universal pathway for kinesin stepping[END_REF][START_REF] Rice | A structural change in the kinesin motor protein that drives motility[END_REF].

Kinesin is a very processive motor, able to walk hundreds of steps [START_REF] Howard | Movement of microtubules by single kinesin molecules[END_REF][START_REF] Block | Bead movement by single kinesin molecules studied with optical tweezers[END_REF][START_REF] Vale | The Way Things Move: Looking Under the Hood of Molecular Motor Proteins[END_REF] with an 8nm step size, which corresponds to the tubulin dimers longitudinal size [START_REF] Svoboda | Direct observation of kinesin stepping by optical trapping interferometry[END_REF]Vale and Michigan, 2000;[START_REF] Yildiz | Kinesin Walks Hand-Over-Hand[END_REF]. These steps are more probably performed with a hand over hand mechanism, where the leading kinesin head alternates [START_REF] Asbury | Kinesin Moves by an Asymmetric Hand-Over-Hand Mechanism[END_REF][START_REF] Yildiz | Kinesin Walks Hand-Over-Hand[END_REF] than in an inchworm fashion, where the leading kinesin head would always be the same [START_REF] Hua | Distinguishing Inchworm and Hand-Over-Hand Processive Kinesin Movement by Neck Rotation Measurements[END_REF], although theory would predict that when one kinesin is submitted to a very large force (>18pN), it would slide following an inchworm manner [START_REF] Shao | On the hand-over-hand mechanism of kinesin[END_REF]. Kinesin can also sidestep from one protofilament to another one on the MT lattice, a process that could allow it to bypass obstacles placed on the MTs track [START_REF] Mitra | A Brownian Ratchet Model Explains the Biased Sidestepping of Single-Headed Kinesin-3 KIF1A[END_REF] Kinesin velocity is about 1µm/s in cells, and it has been shown with optical trapping experiments that a single kinesin can exert up to 6pN when walking alone [START_REF] Svoboda | Force and velocity measured for single kinesin molecules[END_REF][START_REF] Mallik | Teamwork in microtubule motors[END_REF]. Strikingly, kinesin is actually more efficient working alone than in groups, contrary to dynein, as detailed in the next part.

Kinesin activty is also finely tuned, for example by regulatory proteins (such as the glutamate-receptor-interacting protein (GRIP1)), by the tubulin nucleotide sate, or by tubulin PTMs [START_REF] Setou | Glutamate-receptor-interacting protein GRIP1 directly steers kinesin to dendrites[END_REF][START_REF] Hammond | Posttranslational Modifications of Tubulin and the Polarized Transport of Kinesin-1 in Neurons[END_REF]Nakata et al., 2010;[START_REF] Hirokawa | Kinesin superfamily proteins (KIFs): Various functions and their relevance for important phenomena in life and diseases[END_REF]. Kinesin-1 dimers, for example, are auto inhibited, and are only activated by the binding of specific partners [START_REF] Sweeney | Motor Proteins[END_REF]. Another way to regulate kinesin activity would be the viscosity of the cytoplasm itself, as it was shown in vitro that small crowders stopped kinesin whereas large crowders did not [Sozanski et al. 2015].

-Dynein

Dynein was first identified in the cilia of Tetrahymena pyriformis in 1965, and is very different from myosins and kinesins [Gibbons and Rowe, 1965].

They are two types of dyneins: axonemal and cytoplasmic dyneins. Axonemal dyneins are specifically present in cells presenting cilia or flagella, and generate the sliding of MTs inside these motility structures, responsible for their beating.

Cytoplasmic dyneins are separated in two categories. Cytoplasmic dynein 1, or intracellular cytoplasm dynein, is the main actor for minus end directed transport, and, very importantly, can generate forces inside the cells, notably for aster and spindle positionning. As stated before, as cytoplasmic dynein 1 force generation is at the core of our work, its cellular functions will be further detailed in the next chapters.

Cytoplasmic dynein 2, also called Intraflagellar transport (IFT) dynein, transports cargoes from the axoneme tip to the basal body of the cilia [START_REF] Roberts | Emerging mechanisms of dynein transport in the cytoplasm versus the cilium[END_REF].

The description below will mainly focus on the common aspects of dyneins, and more specifically on cytoplasmic dynein 1 characteristics. Dynein is a large and complex motor, whose dimer is about 1.4MDa, and belongs to the AAA proteins family, ATPases Associated with various cellular Activities.

Even if the functions of the different dynein families are diverse, they share the same mechanochemical cycle and are composed of the same type of polypeptides chains: heavy chains, intermediate chains, and light intermediate chains, with a conserved structure of the heavy chain [Cho and Vale, 2011;[START_REF] Schmidt | Review: Structure and mechanism of the dynein motor ATPase[END_REF][START_REF] Grotjahn | Setting the dynein motor in motion: New insights from electron tomography[END_REF]. The biggest part is the heavy chain (~500KDa), and is composed of the motor domain, the tail, the stalk, the strut (also named buttress) and the C-terminal domain. The motor domain is composed of six AAA domains forming a ring, and is the minimal part required for dynein motion on MTs.

The N-terminal of the AAA ring is the linker, which is the mechanical element enabling the dynein powerstroke. The stalk, emanating from the ring near the AAA4 unit, is a ~15nm long coiled coil region allowing the dynein to attach to its track through its MT binding region at its tip. The tail is needed to dimerize two heavy chains together, as well as binding to each heavy chains the intermediate and light intermediates chains.

In cytoplasmic dynein 1, each heavy chain can bind the light intermediate chain DYNC1L1, up to three light chains (DYNLRB, DYNLL, and DYNLT, also found as Roadblock or LC7, LC8, and TCTEX), and the intermediate chain (DYNC1I) [START_REF] Cianfrocco | Mechanism and Regulation of Cytoplasmic Dynein[END_REF]Robert, 2018]. These various chains are key to binding cargo directly or through cargo adaptors proteins [Robert, 2018]. [START_REF] Roberts | Functions and mechanics of dynein motor proteins[END_REF].

In the two last decades, efforts have been made to decipher the dynein mechanochemical cycle, based on structural studies, from X-ray crystallography and cryo-electron microscopy, to biochemical and single molecule assays. The mechanochemical cycle relies on the transmission of the movement following ATP binding and hydrolysis in the motor domain, causing a powerstroke from the linker, and a change in affinity of the MTBD, allowing the motor to advance on the MT track toward its minus end [START_REF] Kon | The 2.8 Å crystal structure of the dynein motor domain[END_REF]. This movement transmission is particularly challenging, as there is an important distance between the hydrolysis site and the MTBD of the dynein.

The motor domain consists in 6 AAA+ units, but only the four first unit (AAA1 to AAA4) possess functional nucleotide binding region, and it has furthermore been shown by biochemical and mutations assays that AAA1 is the main functional unit of this motor domain [START_REF] Kon | Distinct Functions of Nucleotide-Binding/Hydrolysis Sites in the Four AAA Modules of Cytoplasmic Dynein[END_REF].

The dynein mechanochemical cycle can be summarized as follows, as shown in the figure 17 below [START_REF] Cianfrocco | Mechanism and Regulation of Cytoplasmic Dynein[END_REF].

In the beginning of the cycle, the AAA1 nucleotide binding pocket is empty, in an open conformation, and the dynein has a high affinity for the MT through its microtubule binding domain (MTBD). AAA1 and AAA2 are away from each other, and the linker overlaps the motor ring at the level of AAA5 (step 1 in Figure 17).

When ATP binds AAA1, AAA1 and AAA2 can get closer. This conformational change of the ring leads, through interaction of the buttress to the coiled coil stalk, to a conformation change of the MTBD, weakening its association with the MT [START_REF] Gibbons | The affinity of the dynein microtubule-binding domain is modulated by the conformation of its coiled-coil stalk[END_REF][START_REF] Carter | Structure and Functional Role of Dynein's Microtubule-Binding Domain[END_REF]Kon et al., 2009;[START_REF] Redwine | Structural basis for microtubule binding and release by dynein[END_REF][START_REF] Bhabha | Allosteric Communication in the Dynein Motor Domain[END_REF][START_REF] Schmidt | Structure of human cytoplasmic dynein-2 primed for its powerstroke[END_REF]. ATP binding to AAA1 also causes the linker to displace toward AAA2 in a pre-power stroke position, from a straight conformation (step1) to a bent conformation (step 4) [START_REF] Schmidt | Structure of human cytoplasmic dynein-2 primed for its powerstroke[END_REF].

ATP is hydrolyzed into ADP + Pi, and the MTBD, detached from its track, diffuse near the MT, with a bias toward its minus end, possibly increased by the linker previous position change, or the angle between the stalk and the MT [START_REF] Mizuno | Dynein and kinesin share an overlapping microtubulebinding site[END_REF][START_REF] Carter | Structure and Functional Role of Dynein's Microtubule-Binding Domain[END_REF]. The MTBD still has a weak affinity to the MT tracks at this point, so it can only softly interact with the MT (steps 5 and 6 in Figure 17). This interaction between the MTBD and the MT causes a conformational change of the MTBD, resulting in its high affinity to the MT, and thus a strong attachment of the dynein to its track (step 7). This change in conformation is transmitted to the ring through the stalk coiled coil and the buttress, causing a distancing of AAA5 and AAA6 from AAA1, the release of Pi from AAA1, and the return of the linker almost to a post-power stroke position [START_REF] Lin | Structural mechanism of the dynein power stroke[END_REF]. This switch corresponds to the dynein power stroke, allowing it to exert forces, for example to pull a cargo, or displace MTs between themselves in axonemes. The linker then finishes moving to the post-power stroke position, replacing itself near a hydrophobic site in AAA5 [START_REF] Schmidt | Insights into dynein motor domain function from a 3.3 Å crystal structure[END_REF]. A gap is created again between AAA1 and AAA2, resulting in ADP release, and the repetition of the complete cycle. Adapted from [START_REF] Cianfrocco | Mechanism and Regulation of Cytoplasmic Dynein[END_REF] Dynein step size ranges from 8nm to 32nm, [START_REF] Kinoshita | Step Sizes and Rate Constants of Single-headed Cytoplasmic Dynein Measured with Optical Tweezers[END_REF]. Dynein dimers can step 8nm forwards, 8nm backwards, or perform 5nm sidesteps, which corresponds to the tubulin lateral size. A backward step was found to have a lower probability than a forward step, and a lateral step the same as a forward step [START_REF] Ando | Small stepping motion of processive dynein revealed by load-free high-speed single-particle tracking[END_REF]. These backward and lateral steps could seem counterproductive, but actually gives dynein stepping flexibility, allowing it to bypass obstacles on the MT lattice, as for example MAPS [START_REF] Dixit | Differential Regulation of Dynein and Kinesin Motor Proteins by Tau[END_REF]. Dynein can walk processively, and it has been shown that the C sequence end of its motor domain is of major importance in dynein processivity regulation [Numata et al., 2011].

If the cytoplasmic dynein heavy chain C-terminal 380-kDa fragment has been shown to be sufficient to drive MT gliding [Nishiura et al., 2004]; in vivo, cytoplasmic dynein predominantly walks processively on MTs as a dimer, as coordination between two dynein heads is necessary for dynein processive stepping. Indeed, for a processive MT gliding in a classic in vitro gliding assay, it was shown that approximately five single headed dyneins were needed, whereas only one dynein dimer could achieve the same goal [Shima et al., 2006]. Dynein can employ various stepping fashions, like a hand-over-hand motion, an inchworm motion, or nonalternating one head steps, using one or multiple protofilaments [START_REF] Toba | Overlapping hand-over-hand mechanism of single molecular motility of cytoplasmic dynein[END_REF][START_REF] Shibata | A Single Protofilament Is Sufficient to Support Unidirectional Walking of Dynein and Kinesin[END_REF][START_REF] Kubo | How Cytoplasmic Dynein Couples ATP Hydrolysis Cycle to Diverse Stepping Motions: Kinetic Modeling[END_REF]. Dynein work more efficiently when in group than alone, contrary to kinesin. Indeed, a mammalian cytoplasmic dynein alone can exert a force of 1pN (which is substantially less than the 6pN force exertion of kinesin), but when working in groups, unlike kinesins, they have increased strength and persistence and can exert up to 20pN [START_REF] Hendricks | Force measurements on cargoes in living cells reveal collective dynamics of microtubule motors[END_REF][START_REF] Blehm | In vivo optical trapping indicates kinesin's stall force is reduced by dynein during intracellular transport[END_REF]Rai et al., 2013;[START_REF] Mallik | Teamwork in microtubule motors[END_REF].

This might be due to the fact that, contrary to kinesin, dynein has a gear like response to load, taking smaller steps when under higher load [START_REF] Mallik | Cytoplasmic dynein functions as a gear in response to load[END_REF]. This can enable groups of dynein to share better the load as, when a dynein is slowed down by the load, faster dyneins can take over. Moreover, dynein detachment rate decreases with increasing loads. Thus, under high load, dyneins will not detach right away, but stop and catch bond on the MT. The largest the dynein group is, the longest it can catch bond [START_REF] Leidel | Measuring molecular motor forces in vivo: implications for tug-of-war models of bidirectional transport[END_REF][START_REF] Mallik | Teamwork in microtubule motors[END_REF] Dynein ATPase activity increases strongly in presence of MTs, with for example a seven fold augmentation of the ATPase rate of the dynein motor domain in presence of 10µM MTs, as compared to an absence of MTs. [START_REF] Kon | The 2.8 Å crystal structure of the dynein motor domain[END_REF].

Dynein activity can also be controlled by MAPs, which can be especially useful in the polarized transport in neurons, notably in dendrites, where MTs polarity are mixed in vertebrates. For example, it has been shown that MAP9 (also known as ASAP for aster-associated protein) inhibits dynein and kinesin-1, but not kinesin-3 [START_REF] Monroy | Competition between microtubule-associated proteins directs motor transport[END_REF][START_REF] Monroy | A Combinatorial MAP Code Dictates Polarized Microtubule Transport[END_REF].

Dynein regulation is also finely tuned by proteins such as activators or cargo adaptors, and this part will be detailed in the next chapter on dynein-dependent transport on MTs.

II -In vitro characterization of MT-and motordependent force generation

A wide range of forces can be exerted in cells through the action of MT dynamics and association with molecular motors. Indeed, MTs can exert pushing forces through polymerisation against the cell cortex, pulling forces at the cortex generated either by cortically anchored dyneins or through MT capture and depolymerisation, as well as pulling forces exerted by the motion of dynein transported cargos along MTs in the viscous cytoplasm. These force generation mechanisms are essential in cells, for example for nuclear and spindle positioning, and if the first force generation mechanisms have been extensively studied in vitro, it

is not yet the case of the last one, on which we thus chose to focus our in vitro reconstitution work.

A -In vitro studies on single MTs

-Gliding assays to reconstitute the association of motors and MTs

The use of in vitro gliding assays allowed for a better knowledge of motor properties, such as stepping characteristics, and resistance to load, as described in the previous chapter on motor properties. The principle of gliding assays is either to attach motors on the coverslip surface, for MTs to be able to glide on them, either to bind MTs to the coverslip surface, for motors to be able to walk on them.

A typical example or the use of gliding assays is the assessment of motor properties, such as the identification of the structural elements necessary for motor force exertion through the completion of its mechanochemical cycle. For example, a study focused on the impact of specific mutations on dynein motility, in a gliding assay where MTs were flowed on a surface coated with various mutated dyneins [Kon et al., 2004]. Four of the six ATPase modules of the dynein motor were mutated, in order to assess which of the modules were needed for dynein motility. Mutation on the dynein first ATPase module impaired MTs movements on the dynein carpet, whereas mutations on the second, third or fourth ATPase units led to reduced MT gliding velocities. This allowed to conclude that the first ATPase unit was necessary for dynein motility, and that the other three ATPase units were more likely to act as regulators of dynein activity.

A sophistication of this type of gliding motility assay consists in the study of MT gliding on motors anchored in a lipid bilayer [START_REF] Grover | Transport efficiency of membrane-anchored kinesin-1 motors depends on motor density and diffusivity[END_REF][START_REF] Lopes | Membrane mediated motor kinetics in microtubule gliding assays[END_REF]. For example, as shown in the figure 19 below, streptavidin kinesin-1 motors could be attached in a biotinylated supported lipid bilayer. MT gliding velocities were found to be proportional to the motor density and membrane viscosity [START_REF] Grover | Transport efficiency of membrane-anchored kinesin-1 motors depends on motor density and diffusivity[END_REF]. It suggested that the MTs lower gliding velocities at lower motor densities were due to slippage of the motors in the lipid bilayer. This could have relevance in vivo, as membrane cargo transport could be regulated by the coupling of the cargo to the motor.

Figure 19: Representation of a gliding assay where MTs can glide on kinesins anchored in a lipid bilayer. Kinesin tagged with a streptavidin-binding peptide could be attached to lipids tagged with streptavidin and forming part of a lipid bilayer on a coverslip. Kinesins walking toward the MT plus end will pull the MT in the opposite direction. Adapted from [START_REF] Grover | Transport efficiency of membrane-anchored kinesin-1 motors depends on motor density and diffusivity[END_REF].

In vitro assays where motors would glide on immobilized MTs were also carried out, for example in order to study kinesin-5 (Eg5 in vertebrates) motility.

Indeed, this kinesin is involved in the formation of the bipolar mitotic spindle, and can experience two behaviours: it can either diffuse without preferred orientation on the MTs, either walk towards the MT plus end in an ATP-dependent fashion. A minimal assay, where MTs were immobilized on the coverslip surface, indeed showed that Eg5 tetramers would diffuse on these MTs without preferred directionality [START_REF] Kapitein | Microtubule cross-linking triggers the directional motility of kinesin-5[END_REF]. But, notably, when the kinesin tetramer diffusing on the MT would encounter and bind to a freely diffusing MT, it would mediate its directional transport towards the immobilized MT plus end, and would thus displace this MT cargo on the immobilized MT. This switch in Eg5 behaviour might allow for a more efficient and less energy consuming sliding of antiparallel MTs in the mitotic spindle.

-Force measurements of motor-dependent transport on MT

In vitro force measurement have been of extremely useful to characterize motor properties, notably to estimate their force generation capabilities inside the cells. Several of these important results were mentioned in the last chapter on the cytoskeletal motor properties. Several methods can be used, such as gliding assays performed in viscous medium, or coupled to the use of an optical trap.

A typical example of in vitro studies on motor force exertion, is the study of MTs motion on a motor carpet in a viscous medium [START_REF] Hunt | The force exerted by a single kinesin molecule against a viscous load[END_REF]. The movement of MTs on a kinesin carpet were studied, as a function of the MT length, kinesin coating density, and medium viscosity. At low motor densities, when viscosity was increased, the MT gliding velocity was lower for longer MTs, as longer MTs would undergo a larger drag force from the viscous fluid. The experiment was also carried out at very low kinesin densities, where a single kinesin could support the gliding of a MT, allowing to deduce that a single kinesin molecule could exert a force of approximately 4pN.

As already mentioned when motor properties were described, in vitro assays could also allow to measure the stall forces of motors, through the optical trapping of motor coated beads walking on MTs. The stall force is defined as the force necessary to stop the motion of the motor. Such a technique was used to measure the stall forces of group of motors carrying beads in vitro, and led to the conclusion that the stall force of a kinesin or dynein motor were respectively of the order of 6pN and 1pN [Rai et al., 2013].

-Force generation though MT polymerisation

MT growth was also shown to be able to generate forces by pushing through polymerisation against a barrier. This mechanism is for example at the core of nucleus positionning in yeast Schizosaccharomyces pombe [START_REF] Tran | A Mechanism for Nuclear Positioning in Fission Yeast Based on Microtubule Pushing[END_REF]. The first in vitro force measurements for this mechanism originated from experiments of MT polymerisation against a solid or soft barrier. Study of the buckling of growing MTs against the rigid wall of a microfabricated chamber showed that single MTs could exert forces up to 4pN, and that the polymerisation velocity decreased when the applied force increased [Dogterom and Yurke, 1997]. The growth of MTs inside lipid vesicles was also shown to deform these vesicles, and the analysis of the concomitant MT buckling inside the vesicles suggested a force exertion of more than 2pN [START_REF] Funk | Profilin and formin constitute a pacemaker system for robust actin filament growth[END_REF]. This force exertion mechanism was also investigated at the scale of a reconstituted aster, as detailed in the next section.

-Force generation through depolymerisation and association with molecular motors

It was also shown in vitro, that MTs can exert pulling forces through depolymerisation, and association with molecular motors. This force generation mechanism is of particular interest, for example in positionning of the mitotic spindle, as detailed in the next chapter on dynein functions in cells, and has been studied in vitro at the scale of a minimal aster, as detailed in the next section.

To begin with, in vitro experiments studied the depolymerisation of biotinylated MTs grown on a chamber surface, and on which could bind streptavidin beads, along the length of the MTs [START_REF] Grishchuk | Force production by disassembling microtubules[END_REF]. These beads could be trapped in an optical trap, and when MTs would depolymerise from their plus end, the MTs would shrink until their plus end encountered the bead which was fixed on the MT and trapped in the optical tweezer. The MT depolymerisation would thus exert a pulling force on the bead, which could be measured through the concomitant small displacement of the bead in the optical trap. The maximal MT depolymerisation force assessed with this technique was 5pN.

Experiments also focused on the study of MT attachments, and MTs dynamics under tension. For example, association of MTs to the kinetochore Dam1 complex was shown to be sufficient to generate a load bearing attachment [START_REF] Franck | Tension applied through the Dam1 complex promotes microtubule elongation: a direct mechanism for length control in mitosis[END_REF]. The experiment was based on the application of tension on the growing MT plus end tip, through the attachment of the MT plus end to a bead coated with the Dam1 complex, and trapped in an optical tweezer. As shown in the figure 20 below, the MT was attached to the coverslip surface through its biotinylated seed, and the stage was moved, leading to the application of a tension force on the MT plus tip.

This study additionally showed that, when the tensile force applied to the MT tip was increased from 0.5 to 2pN, MT dynamics were impacted, with a lower catastrophe rate, and a higher rescue rate. [START_REF] Franck | Tension applied through the Dam1 complex promotes microtubule elongation: a direct mechanism for length control in mitosis[END_REF].

Figure 20: Representation of an in vitro experiment allowing to apply tension forces on the MT plus end. A MT is grown from a MT biotinylated seed, which enable the MT attachment to the surface, as represented in the darker region of the red MT. A Dam1 coated bead is trapped in an optical tweezer, and can attach to the MT plus end through the Dam1 complex. A tension force is then applied on the MT plus end, through the motion of the motorized stage

Motors were also shown to be able to depolymerise MTs, exerting pulling forces on them. The experiment set-up consisted of MKAC (mitotic centromereassociated kinesin) coated beads, which, trapped in two laser beams, could capture the two ends of a MT, away from the chamber surface, as shown in the figure 21 below [START_REF] Oguchi | The bidirectional depolymerizer MCAK generates force by disassembling both microtubule ends[END_REF]. The first observation was that, if one bead was released from the optical trap, the MT would depolymerise under the action of the depolymerizing kinesin coated bead, which would keep its connection to the MT tip.

The MT shrinking velocity was proportional to the amount of kinesin coated on the beads. Then, very interestingly, when the two beads at both MT tips were kept in the optical traps, the depolymerizing kinesin coated beads would exert pulling forces on the MT, which could be measured by the small displacement of the beads inside the optical traps. It thus showed that MT depolymerisation induced by MCAK could exert pulling forces on MTs, on the order of magnitude of 1pN per motor, which can be of great relevance in cells, notably for force generation during chromosome segregation. (A) When one bead was released, the depolymerizing kinesin coated bead would lead to the shortening of the MT. (B) When the two beads were kept in the optical trap, the depolymerisation activity of the kinesin coated on the beads would pull on the MTs, exerting a force which could be assessed through the measure of the beads displacement in the traps. Adapted from [START_REF] Oguchi | The bidirectional depolymerizer MCAK generates force by disassembling both microtubule ends[END_REF].

It was also shown that dyneins anchored at the border of a microfabricated chamber, could capture the MT plus end, and exert forces on this shrinking MTs. This was shown using an optically trapped bead, from which a MT could grow, and contact the border of the microfabricated chamber [Laan et al., 2012]. When contacting the barrier, the MT would be captured by dyneins, and depolymerise, and this would displace the bead in the optical trap, revealing that the dynein could exert an average pulling force of 2pN on the MT.

B -In vitro studies at the scale of a MT aster

MT asters are radial structure of MTs emanating from a microtubule organizing center (MTOC). MTs are dynamic, and can thus elongate or shrink. MT asters are essential to several essential processes, such as the positionning of the nucleus, mitotic spindles, or the establishment of cell polarity through MT dependent transport.

The study of aster growth and positionning is of particular interest, as the growing MT aster have to properly position itself after fertilization. Here some in vitro studies on MT aster growth and aster force generation mechanisms for aster centration will be presented. The in vivo study of MT aster positionning will be extensively discussed in the next designated chapter.

-Reconstitution of aster growth in vitro

Aster growth in vitro can be studied thanks to MT nucleation from various structures, such as purified centrosomes, or artificial MTOCs. These artificial MTOCs can for example consist of biotinylated MT seeds attached on a streptavidin bead and from which MTs can elongate [START_REF] Holy | Assembly and positioning of microtubule asters in microfabricated chambers[END_REF]. They can also be made of beads coated with the nucleation factor XMAP215, which was already presented in the first part of the introduction [START_REF] Popov | XMAP215 is required for the microtubule-nucleating activity of centrosomes[END_REF]. In Xenopus egg extract, it is also possible to nucleate a MT aster from chromosomes, or from beads coated with Aurora Kinase A, which is involved in MT nucleation, notably during spindle assembly [START_REF] Gruss | The mechanism of spindle assembly[END_REF][START_REF] Tsai | Aurora A Kinase-Coated Beads Function as Microtubule-Organizing Centers and Enhance RanGTP-Induced Spindle Assembly[END_REF]Ishihara et al., 2014;[START_REF] Colin | Actin-Network Architecture Regulates Microtubule Dynamics[END_REF].

Contrary to the usual simple representation of MT asters, all MT minus ends do not necessarily originate from the MTOC. Indeed, it was shown in Xenopus laevis egg extracts that MTs can be nucleated along the length of preexisting MTs [Petry et al., 2012]. Interestingly, nucleated MTs had a low angle respective to the MT they were nucleated from, and their plus ends were oriented in the same direction as the MT they were nucleated from. It has then been shown, also in Xenopus laevis egg extracts, that a MT aster can grow from an Aurora Kinase A coated bead, and that, after the MT nucleation phase on this artificial MTOC, MTs were predominantly nucleated away from the MTOCs, at sites along the aster MTs [Ishihara et al., 2014].

MT asters could grow up to diameters of hundreds of micrometers, and, interestingly, when two asters would grow in close proximity, MT growth was inhibited at the border region between the two asters and the aster growth was thus inhibited on this front.

They also observed that outward MT sliding, supported by dynein motors, contributed to the aster growth rate, but dynein inhibition through addition of the dynactin dominant negative fragment p150-CC1 showed that MT sliding was dispensable for the generation of MTs asters of hundreds of micrometers.

Figure 22: Representation of the model for aster growth in large cells, based on MT nucleation from a MTOC, followed by MT nucleation along the aster MTs. MTs are represented in green, and the MTOC in yellow [Ishihara et al., 2014].

As the minus end-directed motor kinesin-14 is known to be involved in spindle pole focusing, the formation of MT asters from a minimal system composed of only tubulin, kinesin-14, ATP and GTP was investigated in vitro [START_REF] Hentrich | Microtubule organization by the antagonistic mitotic motors kinesin-5 and kinesin-14[END_REF]. It showed that the minus end directed kinesin-14 could support MT crosslinking and sliding, leading to the formation of MTs asters from growing MTs in vitro.

In vitro studies also focused on the regulation of MT aster growth, as for example the regulation of MT aster dynamics by the actin network [START_REF] Colin | Actin-Network Architecture Regulates Microtubule Dynamics[END_REF].

In this study, Xenopus eggs extract with intact actin filaments, was mixed with mineral oil, yielding droplets of tens of micrometers of diameter. Centrosomes, promoting the growth of MTs asters, and the Arp2/3 activator Scar-PWA, promoting the nucleation of branched actin networks, could be added to the extract. The formation of the actin branched network was faster than the one of the MTs aster, thus they could observe the impact of the presence a branched actin network on the growth of the MT aster.

They found that MTs asters had longest and fastest growing MTs in the presence of an unbranched actin network, suggesting that actin branched network could inhibit MT growth. Thus, they studied the polymerisation of single MTs grown from biotin seeds on a coverslip surface, in the absence or presence of a branched or unbranched actin network, and could indeed show that the presence of denser branched actin network would disturb MT elongation, decreasing MT average length and growth rate, and increasing the MT catastrophe frequencies.

-Aster centering based on pushing forces

In addition to studies focusing on the force generation of single polymerizing MTs, experiments also aimed at reconstituting MT aster positionning through MT polymerisation pushing forces. MT asters could indeed be grown, through the growth of tens of MTs from purified centrosomes as well as from artificial MTOCs (based on stabilized MT seeds attached to a streptavidin latex bead) [START_REF] Holy | Assembly and positioning of microtubule asters in microfabricated chambers[END_REF]. These growing MTs asters were able to position themselves at the center of closed microfabricated chambers, only due to MT pushing against the chamber borders.

However, when MTs became long enough to buckle against the chamber border, the MT aster was often displaced from the chamber center, suggesting that MTs catastrophe is an important regulator for aster positionning though MT pushing forces. The same experiment was thus conducted, but with the possibility to move the aster center, after the aster had reached the chamber center, through optical trapping of the aster center bead [START_REF] Faivre-Moskalenko | Dynamics of microtubule asters in microfabricated chambers: The role of catastrophes[END_REF]. And indeed, when the MT aster center was displaced at the chamber periphery after its initial centration, and then freed to move again, it was not able to travel towards the chamber center anymore. It was explained by the MT buckling and growing along the chamber borders, which immobilized the MT aster in the chamber. In the presence of a MT catastrophe promoter however, when the MT aster center was displaced at the chamber periphery, and then freed to move again, the aster was able to move again, exploring the chamber efficiently through MT pushing forces. After the first aster positionning through pushing forces inside the chamber, the aster center bead could be moved with an optical tweezer, and aster repositioning could be studied, in presence or absence of a MT catastrophe promoter [START_REF] Faivre-Moskalenko | Dynamics of microtubule asters in microfabricated chambers: The role of catastrophes[END_REF].

-Aster centering based on cortical pulling forces

In vitro reconstitution of MTs aster centration based on cortical pulling force generation mechanisms was also carried out. Centrosomes were placed in microfabricated chambers whose borders were coated with dyneins [Laan et al., 2012]. MTs could thus grow from these centrosomes, and reach the chamber borders. At high dynein coating density, MTs would not buckle against the barrier, suggesting that the MT had been captured by anchored dynein, which would control the MT dynamics. And, in these conditions, anchored dyneins could pull on the MTs, leading to the aster positioning at the chamber center in 95% of the cases. Very importantly, MTs were observed to slip along the chamber borders, and thus to reorient before being pulled on by anchored dyneins. This reorientation of the MTs also orients the pulling forces exerted by the anchored dyneins on the MT, and thus the net resulting pulling force is directed towards the chamber center. So here, it is the MT slippage along the border, previous to their capture by anchored dyneins, which allows the cortical pulling forces to center the aster in the chamber.

III -Dynein functions in the cell

Dynein is essential to cells at many levels. To begin with, dynein is responsible for minus end transport in cells, which is of crucial importance, as for example in neuronal transport, where cargo need to be transported over very long distances. But dynein is also deeply involved in force exertion, notably during aster positionning and mitosis. We will thus further detail in this part dynein-dependent transport in cells, as well as its regulation, and describe dynein involvement in cell division. Dyneindependent force generation for aster centration, which is the main motivation for this work, will be discussed at length in the next chapter.

A -Dynein broad implication in intracellular traffic

-Dynein persistent transport motion on MTs

Dynein is profoundly involved in intracellular traffic, transporting a large range of objects, such as RNA, virus, proteins and, last but not least, a wide array of membrane based vesicles and organelles. Interestingly, the minus end directed transport of all these elements relies only on cytoplasmic dynein, contrary to the plus end directed transport, which relies on more than 40 different kinesins. Dynein must then have a different way than kinesin to regulate the object-specific transport in the cell. Indeed, dynein can connect with adaptors, which will connect to specific cargo type, and are often dynein activators.

It has been shown that, in vivo, the dynactin complex was needed for dyneindependent vesicle transport on MTs [START_REF] Mckenney | Activation of cytoplasmic dynein motility by dynactin-cargo adapter complexes[END_REF][START_REF] Schlager | In vitro reconstitution of a highly processive recombinant human dynein complex[END_REF].

This can be linked to a conformation change of dynein dimers following dynactin binding. Indeed, it has been show that dynein dimer can exist as an inactivated form, with the motor domains stacked together, and known as the phi particle [START_REF] Torisawa | Autoinhibition and cooperative activation mechanisms of cytoplasmic dynein[END_REF]. However, when dynein dimers are linked to dynactin, the two dyneins take a more parallel conformation, which activates dynein for processive transport [Zhang et al., 2017]. It is not yet known if dynactin can impose the opening of the phi particle, or binds to a transiently open configuration of the dimer [START_REF] Roberts | Emerging mechanisms of dynein transport in the cytoplasm versus the cilium[END_REF]. Furthermore, bicaudal D homologue 2 (BICD2), a coiled coil protein, is able to increase the strength of the interaction between dynein and dynactin, and further activate dynein-dynactin activity [START_REF] Splinter | BICD2, dynactin, and LIS1 cooperate in regulating dynein recruitment to cellular structures[END_REF]. But BICD2 is actually not the only coiled coil protein able to bind and activate dynein-dynactin, several other have been identified. They are called activating adaptors, as they activate dynein-dynactin, and connect it to specific cargoes, thus enabling the control of a cargo-specific and efficient dynein dependent transport [START_REF] Reck-Peterson | The cytoplasmic dynein transport machinery and its many cargoes[END_REF].

Dynactin is a large complex, of 1.1MDa, composed of 23 subunits. It is built around an actin-like filament, which is capped by the capping protein CAPZ at its barbed end, and the actin related protein ARP11 at its pointed end, and connects to several other subunits, not detailed here.

Activating cargo adaptors do not all share common sequences, but they all share a long coiled coil, a protein binding site allowing them to connect with their cargo, and a binding site for dynein light intermediate chain C terminus [Schroder et al., 2014;Schroder et al., 2016;[START_REF] Gama | Molecular mechanism of dynein recruitment to kinetochores by the Rod-Zw10-Zwilch complex and Spindly[END_REF]. Some adaptors, such as the RAB-interacting lysosomal protein (RILP), can bind to dynein-dynactin, but might not be able to further activate the complex, as their coiled coil region is not as long as the one of activating adaptors [START_REF] Jordens | The Rab7 effector protein RILP controls lysosomal transport by inducing the recruitment of dynein-dynactin motors[END_REF]. In terms of velocity and force, it has been shown that the formation of the DDB complex (Dynein dimer, Dynactin, BICD2) as well as the dynactin-dependent recruitment of two dynein dimers in the complex increases the stall force of the complex. This increase in dynein force generation can allow dynein to win in a tug of war against kinesin. Velocity, on the other hand, is not directly linked to the number of dimers recruited, but rather, is predominantly regulated by cargo adaptors, and the tail-tail interactions between dynein dimers recruited by dynactin [Belyy et al., 2016;[START_REF] Urnavicius | Cryo-EM shows how dynactin recruits two dyneins for faster movement[END_REF][START_REF] Elshenawy | Cargo adaptors regulate stepping and force generation of mammalian dynein-dynactin[END_REF].

It should also be underlined that groups of dynein do not necessarily need interaction with partners such as dynactin and cargo adaptors for persistent motion, as it has been shown in vitro that dynein groups alone can transport beads persistently [Rai et al., 2013;[START_REF] Mallik | Teamwork in microtubule motors[END_REF]. Moreover, it has been shown that dynein can cluster into domains on phagosomes as they mature, and that this clustering increases the dynein group stall force, allowing dyneins to win the tug of war against kinesins, and transport quickly the phagosomes towards lysosomes for pathogen degradation [Rai et al., 2016]. [Rai et al., 2016].

Dynein activation also relies on other regulators, which are not involved in cargo binding: NudE/NudEL and Lis1, which interact directly with the dynein motor domain [START_REF] Reck-Peterson | The cytoplasmic dynein transport machinery and its many cargoes[END_REF][START_REF] Canty | Activation and Regulation of Cytoplasmic Dynein[END_REF]. Lis1 regulate dynein, as it has been shown to be involved in essential dynein-dependent cell functions, such as dynein recruitment at MTs plus tip, initiation of cargo minus end transport and proper spindle positionning [START_REF] Jha | Combinatorial regulation of the balance between dynein microtubule end accumulation and initiation of directed motility[END_REF][START_REF] Moon | LIS1 controls mitosis and mitotic spindle organization via the LIS1-NDEL1dynein complex[END_REF]. Lis1/NudEL dynein regulation remains unclear, as Lis1 had been seen to inhibit or activate dynein. This mechanism begins to be clarified, as binding of Lis1 to the AAA3 part of the motor domain brings about different MTBD affinity changes as a function of the AAA3 nucleotide state, thereby decreasing or increasing dynein velocity [START_REF] Desantis | Lis1 Has Two Opposing Modes of Regulating Cytoplasmic Dynein[END_REF]. Interestingly, Lis1 major role could be to activate dynein by favoring an open conformation of the dimers, preventing them to go back to a phi conformation, and favoring the recruitment of dynactin and cargo adaptors [START_REF] Elshenawy | Huntingtin-associated protein 1 (HAP1) interacts with the p150Glued subunit of dynactin[END_REF][START_REF] Htet | LIS1 promotes the formation of activated cytoplasmic dynein-1 complexes[END_REF]. In support of this hypothesis, Lis1 has been seen to dissociate from dynein-dynactin complex after favoring their formation, and does not colocalize with moving dyneins [START_REF] Elshenawy | Huntingtin-associated protein 1 (HAP1) interacts with the p150Glued subunit of dynactin[END_REF][START_REF] Egan | Lis1 is an initiation factor for dynein-driven organelle transport[END_REF].

A last level or regulation of dynein-dependent transport is the recruitment of dynein to the MT [START_REF] Carter | How dynein and dynactin transport cargos: a structural perspective[END_REF]. Lis1 has been demonstrated to be involved in dynein recruitment at MTs plus tips, as well as dynactin, notably through dynactin p150 glued subunit interaction with the plus end tracking EB1 protein as well as CLIP170 [START_REF] Watson | Microtubule plus-end loading of p150(Glued) is mediated by EB1 and CLIP-170 but is not required for intracellular membrane traffic in mammalian cells[END_REF][START_REF] Duellberg | Reconstitution of a hierarchical +TIP interaction network controlling microtubule end tracking of dynein[END_REF][START_REF] Jha | Combinatorial regulation of the balance between dynein microtubule end accumulation and initiation of directed motility[END_REF].

Dynein is thus able to transport towards the minus end of MTs a large range of objects, through various specific adaptors. We will briefly detail the transport of RNA, viruses and proteins, and describe more in detail membrane based vesicles and organelles transport.

-Objects carried by dynein dependent transport a -RNA transport

To begin with, dynein is involved in RNA transport, which is of high importance, as mRNA localization within the cell enables a spatial control of gene expression.

RNA are transported as ribonucleoproteins (RNPs) [START_REF] Bullock | Messengers, motors and mysteries: sorting of eukaryotic mRNAs by cytoskeletal transport[END_REF][START_REF] Reck-Peterson | The cytoplasmic dynein transport machinery and its many cargoes[END_REF]. Coupling of mRNA to dynein is achieved in flies thanks to the Egalitarian (Egl) protein, and in filamentous fungi, through mRNA hitchhiking on early endosomes [START_REF] Dienstbier | Egalitarian is a selective RNA-binding protein linking mRNA localization signals to the dynein motor[END_REF][START_REF] Haag | Membrane-Coupled mRNA Trafficking in Fungi[END_REF]

b -Virus transport

Objects transported by dynein include viruses, which can use dynein at different steps of their life cycle, for example either for DNA replication, or dispersion of the virus away from the infection area. All virus families contain members which are known to use dynein at some point of their cycle. For example, DNA and RNA virus are transported by dynein towards the nucleus, where they can replicate [START_REF] Cao | Circovirus transport proceeds via direct interaction of the cytoplasmic dynein IC1 subunit with the viral capsid protein[END_REF]; and rabies virus uses dynein-dependent transport on axons to disseminate after infection [START_REF] Klingen | Double-labeled rabies virus: live tracking of enveloped virus transport[END_REF]. Binding of viruses to dynein can be direct or indirect, with the respective examples of viruses connecting dynein directly though a viral capsid protein, or viruses transported within vesicles [START_REF] Radtke | Plus-and minus-end directed microtubule motors bind simultaneously to herpes simplex virus capsids using different inner tegument structures[END_REF][START_REF] Klingen | Double-labeled rabies virus: live tracking of enveloped virus transport[END_REF].

c -Protein transport

Dynein motors also enable minus end directed transport of several proteins type. First, they can notably transport cytoskeletal elements such as IFs and MTs themselves. It has indeed been shown that dynein is involved in the subcellular localization of the IF vimentin, as well as the MT transport in rats axons [START_REF] Ahmad | Cytoplasmic dynein and dynactin are required for the transport of microtubules into the axon[END_REF][START_REF] Hookway | Microtubule-dependent transport and dynamics of vimentin intermediate filaments[END_REF]. Second, even if small proteins can quickly diffuse in the cytoplasm, some transcription factors can be transported towards the nucleus for gene expression regulation. It is for example the case when, after translation of transcription factor at sites of nerve injury, these transcription factors are brought by dynein to the nucleus for specific genes expression [START_REF] Seksek | Translational diffusion of macromolecule-sized solutes in cytoplasm and nucleus[END_REF][START_REF] Perry | Local translation in neuronal processes--in vivo tests of a "heretical hypothesis[END_REF]. Third, dynein is implied in centrosome coupling with the nucleus, and in supplying necessary elements to the centrosome. Indeed, BICD2 and dyneindynactin have been shown to be recruited to nuclear pore complex (NPCs) and thus to enable to keep centrosomes and nucleus close before mitosis [START_REF] Splinter | Bicaudal D2, dynein, and kinesin-1 associate with nuclear pore complexes and regulate centrosome and nuclear positioning during mitotic entry[END_REF]. Additionally, dynein can bring to the centrosomes essential elements for MT nucleation and polymerisation, such as CDK5RAP2, which is involved in γ tubulin recruitment and MT assembly [START_REF] Jia | Dynamic recruitment of CDK5RAP2 to centrosomes requires its association with dynein[END_REF]. Finally, dynein enables cells to form aggresomes that can thus be eliminated by autophagy [START_REF] Johnston | Cytoplasmic dynein/dynactin mediates the assembly of aggresomes[END_REF].

Aggresomes are aggregates of misfolded proteins, which appear when pathways to refold them, or to get rid of them through proteolysis, were submerged [START_REF] Garcia-Mata | Hassles with taking out the garbage: aggravating aggresomes[END_REF]. Misfolded proteins are recognized and connected to dynein-dynactin through three possible adaptors in order to allow aggresomes formation [START_REF] Kawaguchi | The deacetylase HDAC6 regulates aggresome formation and cell viability in response to misfolded protein stress[END_REF][START_REF] Reck-Peterson | The cytoplasmic dynein transport machinery and its many cargoes[END_REF].

d -Membrane vesicles and organelles transport

There is a wide range of membrane vesicles and organelles in the cell, and their movements and subcellular localization very often rely on cytoplasmic dynein. Specific Rab proteins bind to some of these membrane cargoes. They are small GTPases governing the regulation of the formation, transport and fusion of these membrane cargoes. To do so, these Rab proteins bind to effectors, as for example Rab6, which can bind to the BICD1 effector, leading to the tethering of the Rab6 containing cargo to dynein-dynactin for minus end transport [START_REF] Matanis | Bicaudal-D regulates COPIindependent Golgi-ER transport by recruiting the dynein-dynactin motor complex[END_REF][START_REF] Hutagalung | Role of Rab GTPases in membrane traffic and cell physiology[END_REF].

Mitochondria

Mitochondria present bidirectional transport on MTs, and can travel long distances, in order to accumulate at places of high metabolic need in the cell, as for example at sites of axon growth. Dynein, which is necessary for mitochondria minus end directed movement, binds mitochondria through two adaptors: the motor adaptor Milton (TRAK in humans), which then binds to the mitochondria adaptor Miro (RHOT in humans) [Melkov and Abdu, 2018]. As TRAK exhibits a region similar to BICDL1, it could act not only as an adaptor, but as an activating adaptor of dynein [START_REF] Schlager | In vitro reconstitution of a highly processive recombinant human dynein complex[END_REF].

Endoplasmic reticulum

The endoplasmic reticulum (ER) network is composed of the nuclear envelope, rough sheets, and smooth tubules. As these membrane elements are interconnected, sharing a common lumen, the ER can be considered as the biggest cell organelle. The ER is a major site for calcium storage, lipid metabolism and protein synthesis, folding and modifications [START_REF] Eisen | Source and sinks for the calcium released during fertilization of single sea urchin eggs[END_REF][START_REF] Fagone | Membrane phospholipid synthesis and endoplasmic reticulum function[END_REF][START_REF] De La Cruz | How cofilin severs an actin filament[END_REF][START_REF] Samtleben | Direct Imaging of ER Calcium with Targeted-Esterase Induced Dye Loading (TED)[END_REF][START_REF] Reid | Diversity and selectivity in mRNA translation on the endoplasmic reticulum[END_REF]. Continuous to the nuclear envelope, the rough ER (RER) is composed of cysternal sheets, and, contrary to tubular ER, contains a large number of ribosomes [START_REF] Matsuura | Immunoelectron Microscopy of the Outer Membrane of Rat Hepatocyte Nuclear Envelopes in Relation to the Rough Endoplasmic Reticulum[END_REF].

The smooth ER (SER), is made of curved and thin tubular structures, which are typically interconnected with three way junctions, and continuously rearrange.

Proteins and lipids synthetized in the ER will be targeted to their final destination through the endoplasmic reticulum Golgi intermediate compartment (ERGIC) and the Golgi [START_REF] Appenzeller-Herzog | The ER-Golgi intermediate compartment (ERGIC): in search of its identity and function[END_REF].

The ER network is very dynamic, and its minus end transport relies on dynein [START_REF] Woźniak | Role of kinesin-1 and cytoplasmic dynein in endoplasmic reticulum movement in VERO cells[END_REF]. ER association with dynein is indirectly mediated in fungi, where the ER hitchhikes on other dynein bound vesicles, such as early endosomes [START_REF] Guimaraes | Peroxisomes, lipid droplets, and endoplasmic reticulum "hitchhike" on motile early endosomes[END_REF]. However, it is not yet known if mammalian cells use hitchhiking or more direct interactions in order to recruit dynein to the ER [START_REF] Salogiannis | Hitchhiking: A Non-Canonical Mode of Microtubule-Based Transport[END_REF].

Golgi apparatus

The Golgi apparatus is composed of stacks of membrane, mainly located near the nucleus, but also in the cytoplasm for some species and cell types. Secreted proteins move from the cis-Golgi to the trans-Golgi, and Golgi-derived vesicles are transported towards lysosomes, endosomes or to the plasma membrane. Dynein is involved in Golgi positioning and movements, implying several adaptors according to the cases, mostly from the BICD family, as well as Rab6 [START_REF] Reck-Peterson | The cytoplasmic dynein transport machinery and its many cargoes[END_REF].

Endosomes and lysosomes

Cells can internalize parts of their membrane, for example parts containing receptors and their ligands, in a process called endocytosis. These endocytosis vesicles are directed to the endolysosomal system, whose movements rely on dynein [START_REF] Burkhardt | Overexpression of the dynamitin (p50) subunit of the dynactin complex disrupts dynein-dependent maintenance of membrane organelle distribution[END_REF][START_REF] Harada | Golgi vesiculation and lysosome dispersion in cells lacking cytoplasmic dynein[END_REF]. Early endosomes are labelled with Rab5, which can be recognized either by the FHF protein complex, which contain an activating adaptor of the Hook family, or by HTT proteins (huntingtin) and HAP1 (Huntingtin-associated protein 1), and connect dynein by this mean [Engelender et al., 1997;[START_REF] Guo | Rab5 and its effector FHF contribute to neuronal polarity through dynein-dependent retrieval of somatodendritic proteins from the axon[END_REF]. Late endosomes and lysosomes, for their part, interact with the GTPase Rab7, which in turn allows an association with dynein through ORP1L and RILP [START_REF] Jordens | The Rab7 effector protein RILP controls lysosomal transport by inducing the recruitment of dynein-dynactin motors[END_REF][START_REF] Johansson | Activation of endosomal dynein motors by stepwise assembly of Rab7-RILP-p150Glued, ORP1L, and the receptor betalll spectrin[END_REF][START_REF] Pu | Mechanisms and functions of lysosome positioning[END_REF].

Recycling endosomes, which allow internalized receptors to be recycled at the plasma membrane, are labelled with Rab11, which can then interact with dynein adaptors such as RAB11FIP3 [START_REF] Horgan | Rab11-FIP3 is critical for the structural integrity of the endosomal recycling compartment[END_REF][START_REF] Welz | Orchestration of cell surface proteins by Rab11[END_REF][START_REF] Mckenney | Activation of cytoplasmic dynein motility by dynactin-cargo adapter complexes[END_REF]. Signaling endosomes, for their part, internalize receptors linked to their ligand, allowing for signaling cascade in the cell, and seem to share the same adaptors as early and late endosomes [START_REF] Delcroix | NGF signaling in sensory neurons: evidence that early endosomes carry NGF retrograde signals[END_REF][START_REF] Saxena | The small GTPase Rab7 controls the endosomal trafficking and neuritogenic signaling of the nerve growth factor receptor TrkA[END_REF].

Melanosomes

Melanosomes are lysosomes related organelles containing pigments, and their dynein-dependent movement in cells relies on dynein, and supposedly on the activator adaptor NINL (ninein like protein) [START_REF] Dona | NINL and DZANK1 Co-function in Vesicle Transport and Are Essential for Photoreceptor Development in Zebrafish[END_REF].

Autophagosomes

Autophagosomes are vesicles formed after swallowing of proteins and organelles predestined to be treated by autophagy. They are several candidate activating adaptors for dynein-dependent autophagosomes movement [START_REF] Reck-Peterson | The cytoplasmic dynein transport machinery and its many cargoes[END_REF].

Peroxisomes

Peroxisomes are involved in numerous metabolic activities, such as fatty acid catabolism and reduction of the reactive oxygen species (ROS) hydrogen peroxide.

Peroxisomes motile fraction is low, and this motile fraction is transported either by hitchhiking, or through binding of the cargo activating adaptor TRAK2 and splice variants of the GTPase RHOT1 [START_REF] Neuhaus | Why do peroxisomes associate with the cytoskeleton?[END_REF][START_REF] Okumoto | New splicing variants of mitochondrial Rho GTPase-1 (Miro1) transport peroxisomes[END_REF].

Lipid droplets

Lipids droplets allow cells to store energy as well as precursors of the membrane lipids. They mostly diffuse freely, but can also be actively transported on MTs by kinesin and dynein [START_REF] Targett-Adams | Live cell analysis and targeting of the lipid droplet-binding adipocyte differentiation-related protein[END_REF]. They can be coupled to dynein either through BicD adaptors, or through hitchhiking [START_REF] Larsen | BicaudalD actively regulates microtubule motor activity in lipid droplet transport[END_REF][START_REF] Guimaraes | Peroxisomes, lipid droplets, and endoplasmic reticulum "hitchhike" on motile early endosomes[END_REF].

Nuclei

Finally, dynein is also implicated in nuclear positionning, notably for mitosis and after embryo fertilization, but that role will be treated in details in the next parts.

Dynein is also involved in nuclear movement in interphase cells, such as nuclei positionning in developping polynucleated myofibers, or nucleus movements during cell migration [START_REF] Tsai | Dual subcellular roles for LIS1 and dynein in radial neuronal migration in live brain tissue[END_REF][START_REF] Folker | Translocating myonuclei have distinct leading and lagging edges that require kinesin and dynein[END_REF][START_REF] Cadot | Moving and positioning the nucleus in skeletal muscle -one step at a time[END_REF].

B -Dynein involvement in cell division

In this part, we will detail dynein involvement in cell division itself, whereas the next chapter will tackle dynein implication in aster centration, notably after fertilization.

To begin with, dynein is involved in centrosomes separation on the nuclear envelope (NE) at the end of G2 and beginning of prophase. Indeed, even if this separation is mainly supported by the kinesin Eg5, through MT antiparallel sliding, dynein also participates in this separation, and can rescue it in the absence of Eg5 [START_REF] Tanenbaum | Mechanisms of centrosome separation and bipolar spindle assembly[END_REF][START_REF] Raaijmakers | Nuclear envelopeassociated dynein drives prophase centrosome separation and enables Eg5independent bipolar spindle formation[END_REF]. Moreover, tethering of the centrosomes to the NE is insured by dynein. Indeed, dynein recruitment to the NE relies on the dynein activating adaptor BICD2 and its interaction with RANB2, which connects the complex to the nuclear pore [START_REF] Splinter | Bicaudal D2, dynein, and kinesin-1 associate with nuclear pore complexes and regulate centrosome and nuclear positioning during mitotic entry[END_REF]. This complex can in turn recruits Nup133, CENP-F (centromere protein F) and Nde1/L1, which can interact with the dynein regulator Lis1 [START_REF] Bolhy | A Nup133-dependent NPC-anchored network tethers centrosomes to the nuclear envelope in prophase[END_REF][START_REF] Hu | Dynein recruitment to nuclear pores activates apical nuclear migration and mitotic entry in brain progenitor cells[END_REF]. Thus, this pathway not only enables dynein recruitment to the NE, but also activates dynein, which must be of interest in this process requiring the generation of high forces.

Second, dynein also plays a role in nuclear envelope breakdown (NEBD), which consists in the disassembly of the nuclear envelope after centrosomes separation, and allows MTs to contact chromosomes. It consists in a phosphorylation-dependent dismantling of the lamina, as well as in the breaking of the lipid bilayer, caused by MT-and dynein-dependent invaginations of the membrane near the centrosomes. It has been shown that, even if NEBD can happen without MT or active dynein at the NE, MTs and dynein allow to speed up the operation, thus enabling the mitosis robust continuation [START_REF] Georgatos | Nuclear envelope breakdown in mammalian cells involves stepwise lamina disassembly and microtubule-drive deformation of the nuclear membrane[END_REF][START_REF] Salina | Cytoplasmic dynein as a facilitator of nuclear envelope breakdown[END_REF][START_REF] Hebbar | Lis1 and Ndel1 influence the timing of nuclear envelope breakdown in neural stem cells[END_REF]].

Then, dynein is implied at the kinetochores, which are the main attachment sites of the MTs to the chromosomes.

Dynein is indeed recruited to the kinetochores, and had been shown to be involved in MT-kinetochore attachment stabilization, proper chromosome alignment and chromosomes transport towards spindle poles, but it is not necessarily the kinetochore associated pool of dynein who itself is needed [START_REF] Schmidt | Functional analysis of cytoplasmic dynein heavy chain in Caenorhabditis elegans with fast-acting temperature-sensitive mutations[END_REF][START_REF] Yang | Kinetochore dynein is required for chromosome motion and congression independent of the spindle checkpoint[END_REF][START_REF] Varma | Direct role of dynein motor in stable kinetochore-microtubule attachment, orientation, and alignment[END_REF][START_REF] Raaijmakers | Function and regulation of dynein in mitotic chromosome segregation[END_REF].

Dynein has an implication in silencing the spindle assembly checkpoint (SAC), enabling chromosome separation and continuation of the process. An important step for checkpoint silencing is the removal of SAC components such as MAD1 from the kinetochores, in dynein-dependent and dynein-independent fashions that are not yet fully described [START_REF] Hoffman | Microtubule-dependent changes in assembly of microtubule motor proteins and mitotic spindle checkpoint proteins at PtK1 kinetochores[END_REF][START_REF] Kuijt | Conditional targeting of MAD1 to kinetochores is sufficient to reactivate the spindle assembly checkpoint in metaphase[END_REF].

Besides, dynein is involved at the spindle in various ways. The mitotic spindle is composed of three different types of MTs: MTs ranging from the centrosomes to the cell cortex are named astral MTs, MTs reaching the kinetochores from the centrosomes are called kinetochore MTs or k-fibers, and finally MTs going from the centrosomes toward the midzone, and overlapping with the MTs coming from the other centrosome are called polar MTs. This overlap is enabled by motors which allow antiparallel MT sliding, and is essential for spindle formation. Even if the kinesin Eg5 is the main driver of this MT sliding, some evidence show that dynein can act as an antagonist to kinesin and thus contribute to this MT overlap [START_REF] Blangy | Phosphorylation by p34cdc2 regulates spindle association of human Eg5, a kinesinrelated motor essential for bipolar spindle formation in vivo[END_REF]Kashina et al., 1995;[START_REF] Tanenbaum | Dynein, Lis1 and CLIP-170 counteract Eg5-dependent centrosome separation during bipolar spindle assembly[END_REF][START_REF] Ferenz | Dynein antagonizes eg5 by crosslinking and sliding antiparallel microtubules[END_REF].

Dynein has a role in spindle pole focusing, which is the process of MTs minus ends accumulation at the two spindle poles of the mitotic spindle. Dynein-dependent MTs focusing can follow different paths. Dynein can transport MTs towards spindle poles, through various possible MT-dynein linker [START_REF] Heald | Spindle assembly in Xenopus egg extracts: respective roles of centrosomes and microtubule self-organization[END_REF][START_REF] Khodjakov | Minus-end capture of preformed kinetochore fibers contributes to spindle morphogenesis[END_REF]. Dynactin is a possible candidate, which can bind dynein and MTs through its p150 glued subdomain, but whose depletion may or may not lead to focusing defects [START_REF] Waterman-Storer | The p150Glued component of the dynactin complex binds to both microtubules and the actin-related protein centractin (Arp-1)[END_REF][START_REF] Gaglio | Opposing motor activities are required for the organization of the mammalian mitotic spindle pole[END_REF][START_REF] Raaijmakers | Systematic dissection of dynein regulators in mitosis[END_REF]. NuMA is a serious candidate, as it can bind MTs, accumulates at the spindle pole in a dynein dependent fashion, and as its depletion can lead to focusing defects [START_REF] Merdes | Formation of spindle poles by dynein/dynactin-dependent transport of NuMA[END_REF][START_REF] Haren | Direct binding of NuMA to tubulin is mediated by a novel sequence motif in the tail domain that bundles and stabilizes microtubules[END_REF][START_REF] Silk | Requirements for NuMA in maintenance and establishment of mammalian spindle poles[END_REF].

Spindle positionning strongly involves dynein, and is of crucial importance, as it will define the cell division plane. Indeed, a decentered mitotic spindle will yield an asymetric division, which can be essential, for example in order to generate asymetric daughter cells, with different concentrations of cell fate factors, in the context of embryonic development or cell differentiation. On the other hand, a centered mitotic spindle yields a symetric division, for example crucial for tissue morphogenesis and maintenance [START_REF] Morin | Mitotic spindle orientation in asymmetric and symmetric cell divisions during animal development[END_REF].

Division plane positionning control can rely on various MTs-dependent forces, such as MTs pushing against the cortex, or dynein-dependent pulling forces from the cortex or from the cytoplasm. This part will detail dynein force exertion from the cortex, and the other force exertion mechanisms will be discussed in the next chapter.

Dynein can indeed exert forces on spindle astral MTs, and thus displace the spindle, as it has been shown that astral MTs can dock on dynein-dynactin enriched sites at the cortex [START_REF] Busson | Dynein and dynactin are localized to astral microtubules and at cortical sites in mitotic epithelial cells[END_REF]].

Recruitment of dynein to the cell cortex is tightly regulated. Dynein recruitment factors at the cortex can take the shape of a belt, opposite crescents, or many sites such as in tricellular junctions [START_REF] Bosveld | Epithelial tricellular junctions act as interphase cell shape sensors to orient mitosis[END_REF]. The distribution of these factors is very important, as dynein repartition at the cortex will contribute in defining the spindle positionning, and thus the cell division orientation and positionning, yielding symetric or asymetric division. Both intrinsic cues, linked with cell polarity, and extrinsic cues, linked with the cell environment, regulate the repartition of cortical force generators. Notably, experiments based on HeLa cells adhesion on fabricated adhesive micro-patterns showed that actin retraction fibers, which are present at adhesion sites, can determine cell division orientation [START_REF] Théry | The extracellular matrix guides the orientation of the cell division axis[END_REF].

In C. elegans, the first embryonic division is asymetric, and it has been shown that spindle positionning relies on cortical pulling forces, and that the pulling asymmetry was due to a larger number of force generators at the posterior pole [Grill et al., 2001;Grill et al., 2003].

In metazoan cells, the major pathway recruiting dynein and dynactin to the cortex is the one of a conserved ternary complex, which is Gαi / LGN / NuMA in humans, Gαi / Pins / Mud in Drosophila, and Gα / GPR1/2 / LIN-5 in C elegans [START_REF] Kotak | Cortical dynein is critical for proper spindle positioning in human cells[END_REF][START_REF] Di Pietro | Regulation of mitotic spindle orientation: an integrated view[END_REF]. The heterotrimeric G protein Gαi binds the plasma membrane through myristoylation, acts as an anchor for the other partners, and its repartition is not polarized, contrary to LGN and NuMA. LGN, the leucine-glycine-asparagine repeat protein complex, can bind various proteins, including Gαi and NuMA [START_REF] Mochizuki | Identification and cDNA cloning of a novel human mosaic protein, LGN, based on interaction with G alpha i2[END_REF][START_REF] Di Pietro | Regulation of mitotic spindle orientation: an integrated view[END_REF]. Finally, NuMA stands for nuclear and mitotic apparatus, and is a coiled coil protein which can interact with LGN, dynein and MTs [START_REF] Du | A mammalian Partner of inscuteable binds NuMA and regulates mitotic spindle organization[END_REF][START_REF] Haren | Direct binding of NuMA to tubulin is mediated by a novel sequence motif in the tail domain that bundles and stabilizes microtubules[END_REF][START_REF] Kotak | Cortical dynein is critical for proper spindle positioning in human cells[END_REF]. As LGN can only connect to Gαi when bound to GDP, and possesses a guanine dissociation inhibitory activity, the stability of the complex can be regulated by proteins like the GTPase activating protein RGS14, or the guanine exchange factor Ric8a [START_REF] Willard | Return of the GDI: the GoLoco motif in cell division[END_REF][START_REF] Woodard | Ric-8A and Gi alpha recruit LGN, NuMA, and dynein to the cell cortex to help orient the mitotic spindle[END_REF]. The polarized localization of LGN and NuMA relies on polarity factors, as well as proximity of the metaphase plate. The high level RAN-GTP around chromatin has indeed been shown to prevent NuMA/LGN localization at the cortex regions neighboring the chromosome plate [START_REF] Kiyomitsu | Chromosome-and spindle-pole-derived signals generate an intrinsic code for spindle position and orientation[END_REF][START_REF] Bird | RanGTP and CLASP1 cooperate to position the mitotic spindle[END_REF]. Finally, it has been shown in HeLa cells that dynein/dynactin localization at the cortex was actually dynamic, with an alternating presence to one crescent to the other, yielding spindle oscillations, and its concomitant centration. This is due to the Polo-like kinase (Plk1), which localizes at the spindle poles, and, when one of the spindle pole is close enough from the cortex, can disrupt dynein/dynactin recruitment to NuMA/LGN [START_REF] Kiyomitsu | Chromosome-and spindle-pole-derived signals generate an intrinsic code for spindle position and orientation[END_REF]. As dynein/dynactin is still present on the opposite NuMA/LGN crescent, the spindle is pulled by the anchored dyneins towards this opposite crescent, and the process can repeat from this side. When the spindle assembly checkpoint is satisfied, the two phases of anaphase can take place: first, sister chromatids separate, and move away from each other, in a process mainly insured by MT depolymerizing activity at the kinetochore and the spindle poles, where distance between spindle poles is conserved. Second, the spindle poles move away from each other, further dragging the chromatids away from each other.

In the first phase of anaphase, chromatid tracking of depolymerizing MTs is likely to be carried out by proteins like Ndc80 and the Ska complex, which are able to attach to depolymerizing MTs, and that have been shown to colocalize to kinetochores throughout anaphase [START_REF] Mcintosh | Fibrils connect microtubule tips with kinetochores: a mechanism to couple tubulin dynamics to chromosome motion[END_REF][START_REF] Welburn | The human kinetochore Ska1 complex facilitates microtubule depolymerization-coupled motility[END_REF].

Dynein might play an indirect role in this phase, by bringing factors such as MTs depolymerizing proteins to the spindle poles [START_REF] Gaetz | Dynein/dynactin regulate metaphase spindle length by targeting depolymerizing activities to spindle poles[END_REF].

Finally, in the second phase of anaphase, dynein might play a role in spindle centering and elongation, but the scope of its contribution is not yet clear. In anaphase, dynein is recruited to the cortex through NuMA and its interaction with Factin binding protein 4.1 family. Contrary to metaphase recruitment, this one is independent of LGN and Gαi, and is not dynamic [START_REF] Kiyomitsu | Cortical dynein and asymmetric membrane elongation coordinately position the spindle in anaphase[END_REF].

This dynein-dependent MT attachment to the cortex is important for anaphase spindle centration, and maybe also for spindle elongation, even if the major elongation driver are the pushing forces from the midzone of the spindle [START_REF] Kotak | NuMA phosphorylation by CDK1 couples mitotic progression with cortical dynein function[END_REF].

IV -Aster centration after fertilization

A -The MT aster centration after fertilization in large embryos

The MT aster motion after fertilization is a very interesting model for the study of the MT-dependent force generation mechanism, notably in large embryos such as the sea urchin zygote, where the MT aster will grow, and find the embryo center with high precision.

The sea urchins embryos present many advantages as model organisms.

Indeed, the embryo is translucent, and thus easy to image, in live photon microscopy for example. The gametes are easy to collect, and female gamete can typically be kept for one day, and male gamete for a week. Eggs are also big and solid enough to tolerate mechanical treatments, such as injection, cutting, or centrifugation. Sperm is easily activated through the active mixing and oxygenation of sperm in sea water, and a couple of drops of this mix can fertilize a very large number of eggs. The embryos thus formed can develop in simple artificial sea water, without the need to add any special growth medium. Additionally, the division pattern and cell cycle timing of these echinoderm embryos are very reproducible. Finally, they can easily absorb fluorescent dyes, and be injected with mRNA. The sea urchin embryos is thus a system allowing for quantitative studies of the mechanisms involved in embryonic cleavages and cell division. This system also has some disadvantages, such as the fact that sea urchins do not resist to changes in aquarium water temperature changes, and that male and female sea urchins cannot be differentiated. The major drawback of this system however, is that it is not well suited for forward genetic studies. Indeed, the generation time of sea urchins is several years long, which would render it very difficult to establish genetically modified lines.

We will detail the MT aster centration after fertilization in the sea urchin Paracentrotus lividus, and begin by briefly describe fertilization itself. When a motile male gamete encounter an egg which has not yet been fertilized, the sperm head will enter the egg, and a fertilization envelope will emanate from the sperm entry point, surrounding all the egg, and thus preventing polyspermy.

MTs will then nucleate from the male pronucleus centrosome, leading to the growth of a MT aster. MT nucleation however, is unlikely to take place only at the sperm MT aster center, as it was observed that MT asters in large cells such as the embryos of Zebrafish and Xenopus laevis seem to be bushy, and that MT density at the aster outskirts was equal or higher to the one closer to the MT aster center [START_REF] Mitchison | Growth, interaction, and positioning of microtubule asters in extremely large vertebrate embryo cells[END_REF]. Mechanisms explaining this phenomena include the nucleation of MT along the aster MTs themselves, the release of MTs from the aster center and their sliding towards the aster periphery, possibly mediated by dynein, as well as the capture of MT which would have been nucleated away from the aster center, for example at the cell cortex. The first mechanism is likely to be the one explaining this MT aster bushy structure. Indeed, MT nucleation on MTs themselves, away from the centrosome, has already been observed in other organisms such as higher plants, whereas MTs were not typically observed between the growing asters and the cell cortex in Zebrafish nor Xenopus embryos [START_REF] Kirik | TONNEAU2/FASS Regulates the Geometry of Microtubule Nucleation and Cortical Array Organization in Interphase Arabidopsis Cells[END_REF]. Plus, as already discussed previously, it was later shown in an in vitro study using Xenopus eggs extract, that more than half of the aster MTs were nucleated away from the aster center [Ishihara et al., 2014;[START_REF] Field | Assembly of spindles and asters in Xenopus egg extracts[END_REF].

After sperm entry in the sea urchin Paracentrotus lividus, the MT aster will grow and move towards the aster center, and its trajectory can be separated in three steps.

The first step is the penetration phase, where the small MT aster moves slowly, without favored directionality. After this short phase, the second step consists in the directed motion of the MT aster towards the cell center, at a constant velocity, averaging 5µm/min. During this phase, the MT aster travels almost all of the distance separating it from the aster center. This velocity is slightly higher than the aster MTs growth rate, which is around 4µm/min [Tanimoto et al., 2016]. The deductions that can be made from this constant velocity, about the force generation mechanism at play, will be discussed in the continuation of this section. When the aster MTs reach the female pronucleus, the female pronucleus is transported towards the aster center, presumably in a dynein-dependent manner. As the female pronucleus position and the sperm entry point are both random, the aster MTs reaches the female pronucleus more or less early through the centration process, and thus pronuclei fusion can take place during or at the end of the MT aster centration [START_REF] Chambers | The Movement of the Egg Nucleus in Relation to the Sperm Aster in the Echinoderm Egg[END_REF][START_REF] Peng | Differential Regulation of Disheveled in a Novel Vegetal Cortical Domain in Sea Urchin Eggs and Embryos: Implications for the Localized Activation of Canonical Wnt Signaling[END_REF]. Finally, during the last step of the sperm trajectory, the MT aster slows down as it approaches and reaches the cell center.

Figure 30: MT aster centration after fertilization in sea urchin embryos. The MT aster nucleates from the male pronucleus centrosome. The aster grows, and move with a persistent directionality towards the cell center. When the female pronucleus is reached by the aster MTs, it is transported along the MTs, towards the MT aster center, and the two pronuclei can fuse.

We will discuss here on which elements rely this MT aster centration in large cells, and will detail in the next section, focusing on centration models, the specific mechanism proposed to sustain this force exertion.

The aster centration in the sea urchin had been found to be dependent of dynein and MTs, and independent of actin. Indeed, treatment with nocodazole, leading to MT disassembly, and ciliobrevin D, a dynein inhibitor, both stopped the MT aster motion, whereas latrunculin B treatment, leading to actin depolymerisation, did not impaired the aster centration [Tanimoto et al., 2016]. Treatment with taxol, a MT stabilizing drug, also inhibited the sperm aster motion [START_REF] Schatten | Taxol inhibits the nuclear movements during fertilization and induces asters in unfertilized sea urchin eggs[END_REF]. In the Caenorhabditis elegans embryos, pronuclear centration was also found to be dependent on dynein, as shown by depletion of the dynein heavy chain or the p150 dynactin subunit with RNAi [START_REF] Gönczy | Cytoplasmic dynein is required for distinct aspects of MTOC positioning, including centrosome separation, in the one cell stage Caenorhabditis elegans embryo[END_REF].

One can thus wonder what is the more specific nature of these MT-and dynein-dependent forces, which enable MT aster centration in large embryos.

First, these forces do not seem to rely on MT pushing mechanisms, as shown by laser ablation experiments on growing MT aster in sea urchin embryo. Indeed, when MTs were cut on one side of the embryo, parallel to the initial aster centration trajectory, the aster deviated towards the side were MTs were longer (because left untouched by the laser ablation). Such results are not be consistent with MT pushing from the cortex, as if the main force exertion at play here was MTs pushing, after laser ablation, the aster would have been pushed toward the laser ablation site. Plus, when MTs were cut at the front of the migrating aster, the aster backed up or stopped, and continued on its initial centration path when MTs had regrown at the aster front [Tanimoto et al., 2016]. Second, these forces did not seem to rely on pulling forces from the cortex either. Indeed, immunostaining of sea urchin eggs performed during the constant velocity motion phase of the sperm aster centration, showed that the aster MTs were far from the opposite cortex on the opposite side of the sperm entry point [Tanimoto et al., 2016]. Thus, the dynein dependent force on which relies the MT aster centration in large embryos cannot be mainly based on dynein pulling on MTs from the cortex.

Third, a number of experiments showed that these dynein-and MT-dependent forces enabling the MT aster centration were exerted from the cytoplasm, and, very importantly, scaled with MT length.

The very first evidence of a mechanism based on dynein pulling from the cytoplasm and scaling to MT length is the colcemid based experiment of Hamaguchi and Hiramoto in the sand dollar echinoderm embryo [Hamaguchi and Hiramoto, 1986]. As shown in the figure 32 below, after fertilization, MT were depolymerized thanks to photo deactivable colcemid, and the MT sperm aster regrowth could then be controlled by shining UV in specific zones of the egg. Results showed that the sperm aster could center in the zone where MTs were allowed to regrow, in the direction of the longest MTs, mimicking aster centration after fertilization. Very interestingly, this aster centration behaviour was unchanged whether asters were contacting the cortex or not, showing that MTs aster can be centered relying solely on dynein pulling on MTs directly from the cytoplasm. C) When this zone is moved, the asymetric aster is able to center in this zone, whether the zone touches the cortex or not. Adapted from [Hamaguchi and Hiramoto, 1986].

Another experiment exploited another light-induced chemical local treatment to create asymmetric asters and study this dynein force exertion from cytoplasm. This study was based on photo activable Combretastatin 4A, which was used to depolymerize MTs from one side of the aster in the large two cell stage blastomeres of zebrafish. This asymmetric aster thus moved toward the longer MTs, without MTs touching the cortex [START_REF] Wühr | A Model for Cleavage Plane Determination in Early Amphibian and Fish Embryos[END_REF]. Another experiment argued for MT aster centration through MT lengthdependent forces, and was based on the study of MT asters positioning in embryos of controlled shape [Minc et al., 2011;Tanimoto et al., 2016]. Sea urchins embryos were placed in microfabricated wells of various shapes just after fertilization, and the trajectory of the MT asters was tracked. It was shown that asters would still find the center of the embryo, but following trajectories that depended on the aster shape, as shown in the figure 34 below. For example, when the sperm entry was situated along a symmetry axis, such as the corner of a square, the MT aster trajectory was straight until it reached the square center, but when the sperm entered away from the corner of a rectangle, the MT aster first moved perpendicularly to the rectangular side, and then turned sharply, parallel to the rectangle side, until it reached the rectangle center. These results could be reproduced by 3D simulations based on MT lengthdependent forces. These experiments and simulations thus suggest that MT asters orient their trajectory thanks to their capability to sense the cell shape, through MT length-dependent pulling forces. [Tanimoto et al., 2016].

Finally, we can underline another argument supporting the MT lengthdependent pulling forces for aster centration in large embryos. Indeed, the observed constant velocity of centering asters itself can be explained by length-dependent pulling forces. A 1D model was used, where an aster consisted of a MT at the rear and a MT at the front of the aster, and MTs polymerised at a constant speed, and stopped growing when reaching the cell boundary. The velocity of the aster thus corresponds to the ratio between the MTs length-dependent forces and the aster drag. Two conditions were found to yield a constant aster velocity. In the first possible case, the aster velocity becomes constant when it reaches the growth rate of the aster, and the difference between the MTs length at the front or the rear thus becomes constant. In the second possible case, the aster velocity becomes constant when the ratio between the lengths of the front MT and the rear MT gets constant. In both cases, forces at play have to be relatively high in order to generate the typical sea urchin embryo MT aster motion of a couple of tens of micrometers in only ten minutes.

All of these experiments show that in large cells, dynein can exert pulling forces from the cytoplasm, which scale to MT length, and can lead to aster centering. These MTs length-dependent forces can enable aster centration, and also orient division axis with respect to cell shape [Minc et al., 2011].

The most accepted model explaining forces scaling with MT length is that longer MTs can recruit more dynein all along their length, thus resulting in MT aster displacement in the direction of the longest MTs of the aster. The mechanism suggested to explain how dyneins can exert forces directly from the cytoplasm will be explained in the centration model part below.

B -Centration models

After detailing the aster centration mechanism in large embryo, which is very likely to be based on MT length-dependent pulling forces, we will present here the basis of the different centration mechanisms relying on MTs and dynein, and more specifically on what is likely to rely on the MT length-dependent pulling forces.

It is also interesting to mention that nucleus positioning can be driven by forces that are independent of centrosomes and associated astral MTs, such as in the case of nucleus centration in oocytes. Indeed, at the end of the growth of the mouse oocyte in the ovary, the nucleus is observed to move to the cell center, in the absence of centrosome and MTs [START_REF] Szollosi | Absence of centrioles in the first and second meiotic spindles of mouse oocytes[END_REF]. This process has been found to be dependent on the myosin Vb dependent diffusion of actin coated vesicles, from which actin filaments are nucleated from formin 2 [START_REF] Almonacid | Active diffusion positions the nucleus in mouse oocytes[END_REF]. This leads to a gradient of propulsion forces, which are higher at the cortex than at the cell center, and can thus displace the nucleus to the cell center. This process was shown to be non-specific, leading to the centration of the 30µm mouse nucleus, but also of oil droplets from 5 to 35µm [Colin et al., 2020]. Additionally, in the mouse oocyte, the meiotic spindle forms at the cell center, where the nucleus was positioned, but then have to be displaced toward the cell periphery, in order to yield two subsequent meiotic asymmetric divisions, leading to the formation of a large oocyte and small polar bodies. The asymmetric positioning of the spindle during meiosis is here F-actin dependent, notably through the softening of the cell cortex [START_REF] Chaigne | A soft cortex is essential for asymmetric spindle positioning in mouse oocytes[END_REF].

This softening relies on two mechanisms: an increase in the cell cortex plasticity, based on the thickening of F-actin at the cortex in an Arp2/3 dependent manner, and a reduction in cortical tension, through myosin II exclusion from the cortex.

Centration and decentration pattern can thus rely on mechanisms independent of MT asters growing from centrosomes.

-MT pushing at the cortex

Aster centration movements mainly rely on MTs and MTs associated motors such as dynein. Pushing forces from the cortex can lead to aster centering, as astral MTs can polymerise against the cortex, concomitantly pushing against it [START_REF] Tran | A Mechanism for Nuclear Positioning in Fission Yeast Based on Microtubule Pushing[END_REF][START_REF] Tolić-Nørrelykke | Positioning and elongation of the fission yeast spindle by microtubule-based pushing[END_REF]. This mechanism has been reproduced in vitro, as presented previously, but is likely to be relevant to small cells, such as Schizosaccharomyces pombe cells, where small astral MTs can sustain pushing at the cortex without buckling. [START_REF] Tran | A Mechanism for Nuclear Positioning in Fission Yeast Based on Microtubule Pushing[END_REF].

This MT pushing mechanism has also been proposed to maintain the position of the spindle of C. elegans in metaphase and anaphase [START_REF] Garzon-Coral | A force-generating machinery maintains the spindle at the cell center during mitosis[END_REF].

In this study, they used magnetic beads to pull on centrosomes, to measure for the first time the forces associated with spindle positionning maintenance. Using RNAi, and comparing embryo at their one cell or two cell stage, they found that the force maintaining the spindle was proportional to MTs number, and inversely proportional to C. elegans cell size. Approximating the polymerisation force of one MT pushing against the cortex to 1pN [Dogterom and Yurke, 1997], they computed that around 200 MTs pushing against the cortex would be needed to maintain the C. elegans metaphase spindle with the aster centering stiffness they measured.

-Dynein pulling from the cortex

The most documented mechanism for aster and spindle positioning is cortical pulling, whose mechanism was detailed in the previous chapter [START_REF] Gönczy | Mechanisms of asymmetric cell division: flies and worms pave the way[END_REF]McNally, 2013]. It is based on the fact that dynein, anchored to the cortex, can pull on MTs reaching the cortex from the MT aster, and thus control the aster positioning.

As already presented in a previous chapter, this process has been reproduced in a minimal in vitro system, where a minimal aster could center in a microchamber whose walls were coated with dynein [Laan et al., 2012].

Figure 36: Sketch of dynein/dynactin recruitment to the cell cortex, enabling dynein to contact the astral MTs plus ends, and exert pulling forces on the spindle. Dynein/dynactin is anchored to the cell cortex through

LGN/NuMA/Gαi. Its recruitment is inhibited by proximity of the metaphase plate, as well as the proximity of the Plk1 decorated spindle poles (symbolized by red inhibition arrows) [START_REF] Raaijmakers | Function and regulation of dynein in mitotic chromosome segregation[END_REF].

In large cells, such as embryos and early blastomeres, however, MTs have been shown to exert dynein-mediated pulling forces, but do not extend from the centrosome to the cortex, suggesting that forces are exerted directly in bulk cytoplasm [START_REF] Wühr | How does a millimeter-sized cell find its center?[END_REF]Kimura and Kimura, 2011;[START_REF] Mitchison | Growth, interaction, and positioning of microtubule asters in extremely large vertebrate embryo cells[END_REF]Tanimoto et al., 2016]. The next subpart will detail this mechanisms.

-Dynein pulling from the cytoplasm

As seen previously, centration of MT aster in large embryos mainly rely on dynein and MT-dependent pulling forces, which have been found to scale with MT length.

Studies thus focused on searching what could be the cytoplasmic anchors of this dynein force generation on MTs. One option is that dynein could pull, from the MT track, on other cytoskeletal elements such as F-actin or Intermediate Filaments web [START_REF] Reinsch | Mechanisms of nuclear positioning[END_REF]]. Experiments still need to be carried out on IF implication, but, as mentioned earlier, studies have shown that F-actin is not indispensable for dynein force exertion on MTs from the cytoplasm [Tanimoto et al., 2016;Cheng and Ferrell, 2019].

An alternative predominant hypothesis is that the cytoplasmic anchors of dynein pulling on MTs are the dynein cargoes themselves. Indeed, the transported cargoes can interact hydrodynamically with the viscous cytoplasm, producing friction forces that pull on MTs. As shown on the figure 37 below, the longer the MT, the more dynein transported cargoes can be recruited to the MT, which can lead to the MT aster centration. This hypothesis is supported by simulations and experiments, as detailed below. For example, simulations showed that the friction forces exerted by the transport of 100nm vesicles at typical speed of 2µm/s were sufficient to center the MT aster in the 1mm Xenopus embryo [Longoria and Shubeita, 2013]. As MTs can grow longer on the side opposite to the fertilization entry, more dynein transported cargo will be recruited to these MTs, exerting a total asymetric force (Fnet), enabling the aster to center [Longoria and Shubeita, 2013].

This hypothesis is notably supported by experiments in C. elegans. It was shown that the dynein/dynactin dependent transport of organelles was necessary for the generation of pulling forces exerted from the cytoplasm for correct centrosome positioning [START_REF] Barbosa | Dynactin binding to tyrosinated microtubules promotes centrosome centration in C. elegans by enhancing dynein-mediated organelle transport[END_REF]. Indeed, mutation on the p150 glued CAP-Gly subdomain of dynactin did not seem to inhibit the capability of dynein to exert cortical pulling forces from the cortex, but, rather, inhibited the recruitment of dynein transported cargoes on MTs, which is normally mediated through dynactin p150 glued CAP-Gly subdomain binding to MT plus end tracking proteins, or tyrosinated tubulin. This inhibition of the dynein cargo recruitment to MTs resulted in a reduction of the endosomes minus end transport, as well as aster centration defects. Furthermore, it was shown, also in C. elegans that the minus end transport of lysosomes and endosomes was involved in aster centration. Indeed, RNAi was used in order to perturb the recruitment of cargoes to dynein, and concomitant changes in traffic and aster centration were observed. First, RNAi against the dynein light chain dyrb-1 were used, as dynein light chains are known to be involved in cargo binding.

It showed that Dyrb-1 was needed for centrosome centration, as well as the minus end transport of early endosomes, yolk granules and lysosomes. Second, they used RNAi against Rab7 and RILP proteins, which connect lysosomes to dynein, and observed a consequent decrease in lysosomes minus end transport, as well as a reduction of the aster centration velocity. Moreover, they found that endosomes minus end motion was correlated in time with centrosome displacement.

All of these results suggest that the dynein dependent transport of organelles can indeed generate forces on the aster MTs, leading to aster centration.

Research needs to be carried in this direction to continue unravelling this mechanism, notably to identify the main cytoplasmic anchors for dynein force exertion in cytoplasm. An interesting candidate, as discussed in the next part, is endoplasmic reticulum (ER), as it has been seen to accumulate near the aster center throughout sea urchin embryo sperm aster centration [START_REF] Terasaki | Organization of the sea urchin egg endoplasmic reticulum and its reorganization at fertilization[END_REF].

B -Discussion on dynein pulling in the viscous cytoplasm

-Properties of the cell cytoplasm

The generation of MT length scale-dependent pulling forces is very likely to rely on the friction forces associated with dynein dependent organelles transport. Thus, it is important to discuss some characteristics of the cell cytoplasm, as the friction forces generated on moving organelles will depend on them.

When an object is moved in a viscous medium, such as the cell cytoplasm, a friction force is applied to this object, in the opposite direction of the object motion, as shown in the figure 38 below. When a vesicle is transported by dynein towards the MT minus end, a friction force is applied on this vesicle, directed towards the MT plus end, and can lead to MT pulling towards its plus end. [Longoria and Shubeita, 2013].

If the vesicle is considered as a sphere, then the friction force associated with its movement can be described thanks to the Stokes law, with 𝜐 the viscosity of the medium experienced by the vesicle, and 𝑅 𝑣𝑒𝑠𝑖𝑐𝑙𝑒 and 𝑣 𝑣𝑒𝑠𝑖𝑐𝑙𝑒 the respective vesicle radius and velocity :

𝐹 𝐹𝑟𝑖𝑐𝑡𝑖𝑜𝑛 𝑣𝑒𝑠𝑖𝑐𝑙𝑒 = 6𝜋 𝜐 𝑅 𝑣𝑒𝑠𝑖𝑐𝑙𝑒 𝑣 𝑣𝑒𝑠𝑖𝑐𝑙𝑒
The friction force experienced by the vesicle, and thus potentially allowing the dynein transported vesicle to pull on the MTs, is proportional to the viscosity experienced by the vesicle, and the vesicle radius and velocity.

The cytoplasm is a complex fluid, whose viscosity is scale-dependent, so that big objects such as MT asters are unlikely to feel the same viscosity as vesicles, which themselves surely do not experience the same viscosity as the molecular motors. Indeed, viscosity measurements made with different probe sizes yielded different results [START_REF] Kalwarczyk | Comparative Analysis of Viscosity of Complex Liquids and Cytoplasm of Mammalian Cells at the Nanoscale[END_REF]. For example, measures done in sea urchins eggs using molecular size probes such as fluorescent tubulin, or fluorescein, yielded viscosities of a couple of cP [START_REF] Salmon | Diffusion coefficient of fluorescein-labeled tubulin in the cytoplasm of embryonic cells of a sea urchin: video image analysis of fluorescence redistribution after photobleaching[END_REF][START_REF] Periasamy | Picosecond rotation of small polar fluorophores in the cytosol of sea urchin eggs[END_REF].

Experiments done with a medium size probe (100nm in diameter) in Caenorhabditis elegans embryos lead to a viscosity measure of 1000cP [START_REF] Daniels | Probing Single-Cell Micromechanics In Vivo: The Microrheology of C. elegans Developing Embryos[END_REF]. Finally, measures carried out with big probes of 5 to 9µm of diameter, in sea urchin and Drosophila melanogaster embryos, yielded viscosities around 1000-3000cP [Hiramoto, 1969;[START_REF] Doubrovinski | Measurement of cortical elasticity in Drosophila melanogaster embryos using ferrofluids[END_REF]. Additionally, in the context of an embryo, the cytoplasm viscosity can also depend on the time after fertilization [Hiramoto, 1969]. Finally, it is also possible that the cytoplasm could behave as a non-Newtonian fluid. In Newtonian fluids, the viscosity only varies as a function of temperature and pressure. In non-Newtonian fluids however, viscosity additionally varies as a function of shear rate, meaning that the viscosity experienced by an object can change as a function of the stress applied to this object. For example, in shear thinning fluids, such as tomato sauce, viscosity decreases as a function of shear rate, whereas in shear thickening fluids, such as mixtures of cornstarch and water, viscosity increases as a function of shear rate.

-Pulling on MTs through the friction forces associated with dyneintransported cargoes

MT length-dependent forces generated by dyneins can, by converting aster shape into an oriented force, enable aster centration, notably in large cells [START_REF] Wuhr | A model for cleavage plane determination in early amphibian and fish embryos[END_REF]Minc et al., 2011;Tanimoto et al., 2016]. This model was investigated by various theoretical modeling, initiated with the work of Kimura and Onami [Kimura and Onami, 2005;[START_REF] Hendricks | Force measurements on cargoes in living cells reveal collective dynamics of microtubule motors[END_REF]Longoria and Shubeita, 2013;[START_REF] Letort | Centrosome centering and decentering by microtubule network rearrangement[END_REF]. They indeed showed that, in C. elegans, aster centration simulations based on dynein pulling from the cytoplasm matched better the observed aster centration than simulations based on MT pushing mechanism [Kimura and Onami, 2005].

However, this simulation only took into account the drag of the pronucleus, and not the one of the whole aster, as at the time, there was no available measurements of the force needed to displace a MT aster. This simulation also did not include yet the cytoplasmic flows created by the motion of the nuclear centrosomes complex (NCC) and the transported vesicles, which were taken into account by the following work of Shinar et al. They incorporated these cytoplasmic flows as well as cellular confinement to the simulations, which showed that cytoplasmic pulling could allow aster centration, and that the generated minus end directed flows resembled the observed minus end motion of yolk granules [START_REF] Shinar | A model of cytoplasmically driven microtubule-based motion in the single-celled Caenorhabditis elegans embryo[END_REF].

Nonetheless, even if dynein cytoplasmic pulling is strongly supported by experiments and modelling, work is still needed in order to demonstrate that cargo do accumulate on longer MTs, and that the friction force from cargo minus end transport is sufficient to displace a MT aster.

In order to appreciate if friction forces from cargo minus end transport could be sufficient to displace a MT aster, one need to assess the amount of force necessary to move this MT aster. The best way to assess this MT aster drag force is to measure it in vivo, as it would be particularly hard to approximate. Indeed, a MT aster is not a simple object, it can behave as a porous medium, whose fluid permeability decreases when increasing the MT number composing the aster [Nazockdast et al., 2017]. Furthermore, viscosity in the cytoplasm is scaledependent, so any viscosity measurement done for small objects in the cytoplasm would very unlikely represent the one experienced by a MT aster [START_REF] Kalwarczyk | Comparative Analysis of Viscosity of Complex Liquids and Cytoplasm of Mammalian Cells at the Nanoscale[END_REF]. In addition, this viscosity can also depend on the time after fertilization [Hiramoto, 1969].

Recent studies thus focused on directly assessing the force associated with aster centration [De Simone et al., 2018;[START_REF] Tanimoto | Physical forces determining the persistency and centring precision of microtubule asters[END_REF]. The first study, in C. elegans embryos, quantified female pronucleus and male aster movements, and, using the female pronucleus as a probe, estimated the aster drag coefficient and centering net force [De Simone et al., 2018]. The second study, in sea urchins embryos, used magnetic beads manifesting minus end motion and sticking to centrosomes, to apply forces to centering asters. It allowed to directly measure the force necessary to stall the aster movement, which was found to be approximately 600pN [START_REF] Tanimoto | Physical forces determining the persistency and centring precision of microtubule asters[END_REF].

Figure 39: Determining sperm aster centration force in sea urchin embryo. Magnetic beads are injected in a sea urchin egg, which is then fertilized. The beads are transported to the aster center, where they stick. A force is applied on the beads with a magnet, and the force necessary to block the aster motion (the aster stall force) can be deduced from the intercept of the aster speed as a function of the force applied [START_REF] Xie | Cytoskeleton Force Exertion in Bulk Cytoplasm[END_REF].

This amount of force could possibly be attained through vesicle friction force, as dynein groups can collectively apply forces of the order of 10-20 pN [Rai et al., 2013[START_REF] Hendricks | Force measurements on cargoes in living cells reveal collective dynamics of microtubule motors[END_REF]. Thus, an overabundance of dozens of dynein cargoes at the aster front as a comparison to the number of dynein cargoes at the rear of the aster, could theoretically be sufficient to drive the aster centration. However, it remains impossible to confirm it for the moment, as it is not yet known how efficiently dynein pulling is transmitted to the MTs, and even more so to the MTs aster.

To begin with, asters are not simple structures where MTs all emanate from the MTOCs, but rather, as discussed before, bushy structures, where MTs are predominantly nucleated away from the MTOCs, and sometimes are not even connected to the MT aster network [Ishihara et al., 2014]. Thus, it is likely that a dynein transported cargo does not always convey its pulling force to the MT aster, but can instead rearrange the aster network, for example by promoting MT transport towards the aster periphery.

Furthermore, the transport of a cargo on a MT in the complex and crowded cytoplasm and surrounding dense MT network might not allow vesicles to efficiently exert pulling forces on the MTs. Indeed, cargo motion on a MT in very close proximity to other parallel MTs could be comparable to the motion of a sphere in a closed tube, which would restrain the cargo pulling efficiency on the MT.

Inversely, the pulling efficiency might be improved as cytoplasm elements could align along the long and thin MTs, and thus facilitate their movement, in a shear thinning mechanism experienced in non-Newtonian fluids, where viscosity decreases as a function of shear rate. Nevertheless, improvement of the pulling efficiency through shear thinning does not seem very likely, as MTs are not alone, but part of an aster, that they can be branched, and finally that they are not bare, but connected with motors transporting cargoes and various proteins.

Pulling efficiency might also be increased, as transported cargo could almost be trapped in the very crowded cytoplasm, thus pulling better on MTs [Luby-Phelps, 1999]. This could be particularly the case for semi continuous endomembranes like the endoplasmic reticulum (ER).

Another argument for an ER based dynein pulling mechanism is that ER has been seen to accumulate at the aster center throughout aster centration, which is not clearly the case for other cargo [START_REF] Terasaki | Organization of the sea urchin egg endoplasmic reticulum and its reorganization at fertilization[END_REF][START_REF] Reinsch | Mechanisms of nuclear positioning[END_REF][START_REF] Audhya | A role for Rab5 in structuring the endoplasmic reticulum[END_REF]. This accumulation of the dynein cytoplasmic anchor at the aster center during aster centration would imply a progressive exhaustion of the cytoplasmic anchor, and thus a decrease of the pulling force throughout aster centration. This is consistent with a study in sea urchin, which showed that centering forces indeed decrease after the aster center has reached close to the cell center [START_REF] Sallé | Asymmetric division through a reduction of microtubule centering forces[END_REF]. This study was based on the injection of magnetic beads in the sea urchin eggs, which displayed a persistent dynein dependent minus end transport.

In this experiment, these beads were maintained at the cell cortex after injection. It did not perturb the MT aster centration, until the aster had almost reached the cell center, but would then provoke the MT aster decentration motion towards this dynein beads cortical domain. These results are consistent with the fact that the main cytoplasmic anchor on which relies aster centration, would accumulate at the aster center throughout centration, and thus would not be available anymore to exert pulling forces when the aster had reached the cell center.

Results and discussions I -In vitro reconstitution of dynein force exertion in bulk cytoplasm

A -Results

MT can exert forces by pushing at the cortex through their polymerisation force, or by pulling from the cortex through their association with cortex anchored dynein, or, finally, by pulling directly from the cytoplasm, in a dynein-dependent fashion, where forces scale to MT length. The best rationale explaining this last type of force exertion is that friction forces are generated by the dynein-dependent cargo transport on MTs in the viscous and crowded cytoplasm, thus pulling on the MT aster.

If dynein pulling on MTs from anchoring sites has been shown to be able to displace a minimal aster in a 2D in vitro reconstitution [Laan et al., 2012], there was no yet, to our knowledge, in vitro reconstitution showing that dynein cargo transport would be able to pull on MTs without being anchored to the cortex . This is thus what we intended to achieve, working with dynein-coated beads and stabilized MTs, in a viscous medium, away from coverslip surfaces. We therefore showed that the bead motion on the MT would indeed cause the MT to be pulled in the other direction. The bigger the bead, and the smaller the MT, the more important the MT motion was. We then wanted to get closer to an in vivo conformation, for example in the case where more dynein transported cargoes would be recruited on the longer MTs of a sperm aster, allowing it to center in the embryo through the dynein mediated cargo friction forces. We thus could, thanks to the use of an optical trap, build minimal artificial asters, for example yielding asters consisting of two MTs emanating from one bead. When placing two dynein coated beads on one MT, and only one dynein bead on the other MT, we could observe, concomitant to the dynein coated beads motion, a subsequent displacement of the minimal aster towards the side where more beads were pulling on it.

RESULTS

A 3D motility assay to study dynein force exertion in bulk.

Multiple in vivo studies have suggested that cytoplasmic dynein may exert pulling forces on microtubules (MTs) directly from bulk cytoplasm, without contacting the cortex [START_REF] Xie | Cytoskeleton Force Exertion in Bulk Cytoplasm[END_REF]. This mode of force exertion may have a prevalent function in many cells, as it is thought to naturally arise from motor-driven transport of vesicles, organelles and larger endomembrane networks in the viscous cytoplasm. Such forces have for instance been predicted to contribute to the outward transport of MTs that have been released from centrosomes, potentially promoting aster expansion and organization [START_REF] Ishihara | Microtubule nucleation remote from centrosomes may explain how asters span large cells[END_REF][START_REF] Cheng | Spontaneous emergence of cell-like organization in <em>Xenopus</em> egg extracts[END_REF][START_REF] Keating | Microtubule release from the centrosome[END_REF]. They may also apply a net force to asters and cause them to move, when MTs are connected to the centrosome, with no requirement for MTs to contact the cell surface [START_REF] Xie | Cytoskeleton Force Exertion in Bulk Cytoplasm[END_REF][START_REF] Hamaguchi | Analysis of the Role of Astral Rays in Pronuclear Migration in Sand Dollar Eggs by the Colcemid-UV Method[END_REF][START_REF] Kimura | Computer simulations and image processing reveal length-dependent pulling force as the primary mechanism for C. elegans male pronuclear migration[END_REF]. Because longer MTs could accumulate more cargos, such pulling forces have been proposed to increase with MT length, providing a shape-sensing design for aster centration and orientation in large eggs and early blastomeres [START_REF] Minc | Influence of cell geometry on division-plane positioning[END_REF][START_REF] Tanimoto | Shape-motion relationships of centering microtubule asters[END_REF][START_REF] Wuhr | A model for cleavage plane determination in early amphibian and fish embryos[END_REF][START_REF] Pierre | Generic Theoretical Models to Predict Division Patterns of Cleaving Embryos[END_REF]. Simple theoretical considerations suggest that a cargo moving in bulk is akin to a micro-swimmer, generating a hydrodynamic drag force scaled to its speed, its size and cytoplasm viscosity [START_REF] Kimura | Intracellular organelles mediate cytoplasmic pulling force for centrosome centration in the Caenorhabditis elegans early embryo[END_REF][START_REF] Longoria | Cargo Transport by Cytoplasmic Dynein Can Center Embryonic Centrosomes[END_REF]. To date, however, the lack of minimal reconstitution of dynein bulk pulling has hampered deciphering the basic designs of this essential mode of MT force exertion, its general relevance and its limitations.

In vitro surface motility assays based on motors attached to a coverslip, have populated the cytoskeleton literature in recent years, delineating essential principles of force generation in real cells [START_REF]Mechanics of Motor Proteins and the Cytoskeleton[END_REF]. We designed a 3D bulk motility assay to study how an object transported along a MT in a viscous medium, away from any fixed anchoring point, may create forces on the MTs and cause MT movement. Taxolstabilized and fluorescently labelled MTs were mixed with dynein-coated fluorescent beads in a viscous medium, supplemented with ATP, and flowed into a microscopy chamber (Figure 1A and STAR Methods) [START_REF] Delatour | Arp2/3 Controls the Motile Behavior of N-WASP-Functionalized GUVs and Modulates N-WASP Surface Distribution by Mediating Transient Links with Actin Filaments[END_REF]. The motor domain of Dictyostelium discoideum cytoplasmic dynein was biotinylated in vivo, and then purified [START_REF] Numata | C-sequence of the Dictyostelium cytoplasmic dynein participates in processivity modulation[END_REF][START_REF] Imai | Direct observation shows superposition and large scale flexibility within cytoplasmic dynein motors moving along microtubules[END_REF].

The dynein molecules were bound to streptavidin beads through their biotin moiety.

Prior to each assay, dynein activity was quantified by performing MT gliding assays on dynein-coated coverslips and on carpets of surface-anchored dynein-coated beads (Figures S1A-C and STAR Methods). During assay optimization, we considered using different viscous agents, and selected methylcellulose (MC), because other commonly used crowders tended to over-bundle MTs, and/or strongly affect dynein activity (Figure S1D) [START_REF] Shimizu | Steady-State Kinetic Study on the Inhibition of the Adenosinetriphosphatase Activity of Dynein from Tetrahymena Cilia by Glycerol[END_REF][START_REF] Terashita | Reaction mechanism of 21S dynein ATPase from sea urchin sperm. I. Kinetic properties in the steady state[END_REF]. The range of MC concentrations was determined to ensure reproducible pipetting and chamber loading, while limiting MT and bead sedimentation, and also to test the influence of medium viscosity on bulk forces (see hereafter). Given these considerations, each assay allowed to capture in general ~5 events of bead gliding during a period of ~1 hour, after which most beads had reached the minus-end of MTs, where they remained bound.

At chamber mid-height, several tens of micrometers away from either chamber coverslip surfaces, MTs that appeared horizontal and to which a dynein-coated bead had bound, were immediately imaged by time-lapse fluorescent microscopy.

Remarkably, the sole motion of a dynein-coated bead 0.5µm in diameter along a MT of ~30µm in length, in a viscous medium, was sufficient to cause a concomitant marked steady MT movement in the opposite direction in the microscope frame of reference (Figure 1B and Movie S1). Tracking both bead and MT ends positions revealed that, in the majority of cases, cargo moved at near-constant speeds, causing a resultant constant MT speed; and that both motions immediately stopped once the cargo reached the MT minus-end (Figure S1E). We also noticed events in which the cargo moved, and then stopped before reaching the end of the MT. These arrests presumably reflected the detachment or stalling of motors carrying the beads. Yet, in such events, the MT also moved concomitantly and in opposite direction with the cargo, and stopped when the cargo stopped. We conclude that dynein-driven cargo motion in bulk is sufficient to pull and displace MTs.

Taxol-stabilized MTs typically ranged from 10 to 50 µm in length. We noticed that beads with the same size moving at similar speeds on a short versus a long MT, tended to displace the short MT significantly more than the long one (Figure 1C-1D and S2A). Accordingly, by computing the net velocity of the beads, Vbead, and of the MT, VMT, with respect to the surrounding fluid (e.g. in the microscope frame of reference), we found that the speed ratio |Vbead / VMT|, increased with MT length in a near-linear manner (Correlation coefficient, R=0,79, R 2 = 0.63, Figure 1D). Importantly, even though the net velocity of dynein motors along MTs exhibited some variability, likely reflecting the number of motors engaged between the bead and the MT, it did not significantly affect the dependence of the bead-to-MT speed ratio on MT length (Figure S2B-S2C). Accordingly, this behavior was also unaffected when using kinesin-coated beads, with reversed polarity and net velocity on MTs (Figure 1D and S2D). Finally, this trend was also mostly similar in single versus small MT bundles of 2-3 MTs, suggesting that MT length, and not radius was the most relevant parameter dictating MT motion in response to cargo bulk force (Figure S2E). These data are consistent with the notion that bulk MT displacement, in response to a cargo viscous force, is determined by the MT's hydrodynamic drag coefficient that linearly scales with its length, with little influence from its diameter [START_REF]Mechanics of Motor Proteins and the Cytoskeleton[END_REF][START_REF] Happel | Low Reynolds number hydrodynamics: with special applications to particulate media[END_REF].

The efficiency of MT propulsion depends on cargo size.

An important aspect of bulk hydrodynamic forces is that they are predicted to increase with the cargo drag coefficient, which increases with cargo size [START_REF] Happel | Low Reynolds number hydrodynamics: with special applications to particulate media[END_REF]. We thus repeated the 3D motility assay in the same conditions, but with beads of different diameters. Accordingly, for a similar range of MT lengths, we found that larger beads caused a larger concomitant MT displacement than smaller beads (Figure 2A-2B and Movie S1). These results directly demonstrate that the motion of a cargo in bulk, will drive a consequent MT displacement that respects a balance of hydrodynamic forces between the two objects, which behave as an isolated system, so that, |γMT*VMT|= |γbead*Vbead│, with γ the viscous drag coefficients. Importantly, because both drag coefficients are proportional to the medium viscosity, this parameter should not influence the bead-to-MT speed ratio. Accordingly, varying viscosity by changing the concentration of MC did not influence the bead-to-MT speed ratio (Figure 2C and S2F-S2G). Thus, although viscosity will linearly influence the drag forces that motors will have to overcome, it has no influence on the speed ratio between the cargo and the MT. With the ranges of cargo size, motor velocity and medium viscosity that we explored in our assays, we estimated based on Stokes formula, that net bulk forces ranging from a few hundredths to a few tenths of pN were applied to MTs. These modest forces are sufficient to move MTs of tens of micrometers at typical speeds of few tenths of µm/s. In the cell context, where several motors collectively move cargos of similar sizes at velocities of ~1 µm/s in a cytoplasm typically 100-1000x more viscous than water [START_REF] Hiramoto | Mechanical properties of the protoplasm of the sea urchin egg. II. Fertilized egg[END_REF], the resulting forces are expected to be significantly higher, on the order of several pN, comparable to forces exerted by cortex-anchored motors [START_REF] Rai | Molecular adaptations allow dynein to generate large collective forces inside cells[END_REF][START_REF] Grill | The distribution of active force generators controls mitotic spindle position[END_REF].

Since the extent of MT propulsion appeared to depend solely on MT length and bead diameter, we next sought to determine if all our data followed a master curve, as a function of the MT-length-to-bead-radius ratio (LMT/Rbead), which is a first-order approximation for the ratio of their viscous drag coefficients (Figure S3A and STAR Methods). For this, we computed an MT-to-motor speed ratio (|VMT/v*motor|), that we termed "efficiency ratio", as it represents how much of the motor activity is converted into MT movement and is comprised between 0 and 1. A value close to 0 corresponds to a situation where the motor activity is mostly converted into bead movement, with little MT motion, much like a vesicle trafficking on a static MT network. Conversely, a value close to 1 corresponds to a large MT motion, and little bead motion, mimicking MT outward transport [START_REF] Ishihara | Microtubule nucleation remote from centrosomes may explain how asters span large cells[END_REF][START_REF] Cheng | Spontaneous emergence of cell-like organization in <em>Xenopus</em> egg extracts[END_REF][START_REF] Keating | Microtubule release from the centrosome[END_REF] . By plotting this efficiency ratio, as a function of LMT/Rbead, for all bead and MT sizes, we found that our experimental conditions covered all values of efficiency (Figure 2D). To compare our data with a simple theoretical situation, we computed this curve assuming the medium was a Newtonian fluid, and neglected potential effects such as the hydrodynamic coupling between the bead and the MT, or the rotation of the bead as it moves along the MT (STAR Methods). We find that our data are systematically above this theoretical curve, but follow the same trend (Figure 2D). In fact, our data can be well fitted by the same theoretical formula, with a 2.3-fold reduction of the MT drag coefficient relative to the bead drag coefficient (Figure 2D, STAR Methods). This reduction of the effective MT drag coefficient favors its movement, and could be caused by the non-Newtonian nature of the medium, and an effect known as "shear thinning", where the MC polymers in the medium would be locally stretched along the highly anisotropic MT shape, thereby reducing its viscous friction as it moves [START_REF] Tomioka | Effects of concentration and degree of polymerization on the rheological properties of methylcellulose aqueous solution[END_REF][START_REF] Chhabra | Drag on cylinders in shear thinning viscoelastic liquids[END_REF]. Alternatively, this effect could also be caused by scale-dependent viscosity, when the size of the object (here the radius of the MT) approaches the mesh size of the polymer solution [START_REF] Brochard Wyart | Viscosity at small scales in polymer melts[END_REF].

Collective effects of multiple cargos pulling on MTs and minimal "asters".

To tackle another physiological situation where more than one cargo is applying bulk forces to a MT, we investigated the collective effect of several beads moving on a MT. Such experiment is relevant to aster centration, where it has been proposed that longer MTs could accumulate more cargos, thereby applying larger forces to centrosomes [START_REF] Tanimoto | Shape-motion relationships of centering microtubule asters[END_REF][START_REF] Wuhr | A model for cleavage plane determination in early amphibian and fish embryos[END_REF][START_REF] Kimura | Computer simulations and image processing reveal length-dependent pulling force as the primary mechanism for C. elegans male pronuclear migration[END_REF]. Given that, in our 3D motility assay, the number of events where two beads encounter a MT naturally was very low, we used an optical trap to place two beads on the MT (Figure 3A and Movie S2). We observed that MTs with two beads moving along them were displaced more than MTs of comparable length with only one bead (Figure 3B and Movie S3). Data points obtained with 2 beads also fell on the master curve of Figure 2D, considering the average motor speed and the sum of the bead radii (Figure 3C and STAR Methods). Importantly, we did not notice any obvious slowdown of MT speed, even when beads were moving close to each other, suggesting that putative hydrodynamic screening between cargos do not significantly affect the additivity of forces (Figure S3B). These data show that the forces exerted by multiple cargos are additive, providing an in vitro validation that longer MTs with more cargos may be pulled more.

We next investigated another important situation where these forces could pull in different directions, generating a tug-of-war. To do so, we built artificial, minimal asters, consisting of two or three MTs. These minimal asters were built using dyneincoated beads and the optical trap. Briefly, we first placed a bead on a MT, let it reach and stop at the minus end, and repeated this operation with the same bead on another MT (Figure S3C and STAR Methods). This resulted in a construction made of two or three MTs, with their minus ends bound to a single bead that we refer to as the "aster center". Using the optical trap, we then placed two or three other beads on MTs, near their plus ends, and tracked their movements as well as the concomitant movement of the aster center (Figure 4). This assay thus allows to reconstitute the basic elements of more complex in vivo situations, where several objects pull on several MTs, for example when organelles pull on front vs back MTs of a centering a aster [START_REF] Kimura | Intracellular organelles mediate cytoplasmic pulling force for centrosome centration in the Caenorhabditis elegans early embryo[END_REF].

Minimal asters made of two aligned, antiparallel MTs provided a "tug-of-war" situation (Figure 4A and Movie S4) illustrating that a minimal aster will move toward the region where more cargos are being transported. Following hydrodynamics force balance, the velocities of different minimal asters were here also dictated by the vectorial sum of cargo velocities (Figure 4B). To further illustrate the generalization of this principle to more complex bi-dimensional asters, we also tracked the 2D movement of a minimal aster made of three MTs and three motile beads (Figure 4C-4D and Movie S4). As above, we could detect that the movement of the aster center globally followed the 2D direction and amplitude, set by the vectorial sum of motile cargos velocities, even when the motion of beads became relatively small. We thus showed that cargos gliding on MTs of a minimal aster will displace this aster in the direction set by the vectorial sum of the hydrodynamic forces exerted on these objects, with a speed scaled to the net sum of cargos speeds.

DISCUSSION

We here report on the development of a minimal 3D bulk motility assay to study and evidence the role of bulk motor forces on MTs. This assay shows that a mobile cargo moving along a MT exerts a force on that MT from within the viscous medium, which is sufficient to displace the MT over long distances of tens of µm. By spanning bead sizes and MT lengths, we reproduce a range of physiological behavior, from bare centripetal trafficking, to MT outward propulsion reported in many cells. Reconstituted tugs-of-war show that minimal asters will move towards the region where more cargos are being transported, essentially recapitulating proposed models in vivo, for aster self-propelling motion that follows the direction of asymmetries in MT lengths and/or bulk cargo densities [START_REF] Kimura | Intracellular organelles mediate cytoplasmic pulling force for centrosome centration in the Caenorhabditis elegans early embryo[END_REF][START_REF] Tanimoto | Shape-motion relationships of centering microtubule asters[END_REF][START_REF] Wuhr | A model for cleavage plane determination in early amphibian and fish embryos[END_REF][START_REF] Kimura | Computer simulations and image processing reveal length-dependent pulling force as the primary mechanism for C. elegans male pronuclear migration[END_REF].

In our assay, cargos with all types of directionalities and sizes may effectively apply forces to move MTs. However, the important question of which specific cargos effectively promote aster centration in cells remains mostly open, although specific types of trafficking vesicles have been proposed in some systems [START_REF] Kimura | Intracellular organelles mediate cytoplasmic pulling force for centrosome centration in the Caenorhabditis elegans early embryo[END_REF][START_REF] Xie | Cytoskeleton Force Exertion in Bulk Cytoplasm[END_REF][START_REF] Barbosa | Dynactin binding to tyrosinated microtubules promotes centrosome centration in C. elegans by enhancing dynein-mediated organelle transport[END_REF]. Large cargos, like the endomembrane networks of the Endoplasmic Reticulum, may have higher effective drag coefficients, but also require more motors to be pulled at the same speed than small cargos. The efficiency of cargo-based bulk pulling for a dense MT aster network is also predicted to be affected by the surrounding MTs in the aster, because of the putative complex hydrodynamic interactions between cargos and neighboring MTs [START_REF] Nazockdast | Cytoplasmic flows as signatures for the mechanics of mitotic positioning[END_REF]. As an evidence for this, a simple assay using our optical trap, shows that the motion of a non-coated bead close to a free MT can move the MT with a speed up to ~20% of the bead speed (Figure S4A-S4C). In a dense aster, we speculate that this effect may become more important, plausibly limiting force transmission from cargos to MT asters. Our assay also indicates that the nonnewtonian nature and shear-thinning properties of a dilute polymeric solution, or mesh-size effects, could significantly reduce the longitudinal drag coefficient of MTs, thereby facilitating their displacement over that of cargos. These results may have direct relevance to the cytoplasm which is a complex non-newtonian fluid filled with polymers and membranes, and which also exhibits shear-thinning properties, and a scale-dependent viscosity [START_REF] Tsai | Passive mechanical behavior of human neutrophils: power-law fluid[END_REF][START_REF] Luby-Phelps | The physical chemistry of cytoplasm and its influence on cell function: an update[END_REF]. Minimal in vitro MT force generation assays, have paved the way to our understanding of the role of MT forces in cells [START_REF] Laan | Cortical Dynein Controls Microtubule Dynamics to Generate Pulling Forces that Position Microtubule Asters[END_REF][START_REF] Dogterom | Measurement of the force-velocity relation for growing microtubules[END_REF]. We foresee that further complexification of our initial minimal 3D bulk motility assay, potentially incorporating complex fluids closer to the cytoplasm, cytoskeletal networks or more physiological groups of active motors, will serve to decipher the key physical and biological elements promoting and limiting bulk dynein hydrodynamic force exertion in cells.

SUPPLEMENTAL INFORMATION

The Supplemental Information includes four figures and four videos. (C) Time-lapse of a 1µm diameter dynein-coated bead gliding on a short (17µm) or a long (44µm) MT. For a similar motor velocity, the shorter MT is displaced faster.

(D) Bead-to-MT speed ratio as a function of the MT length, for 1µm diameter dynein or kinesin coated beads in a 1.18% methylcellulose (MC) viscous medium (measured viscosity: 39cP).

See also Figure S1. (A) Time-lapses of a 0.5 or 3µm diameter dynein-coated bead gliding on a ~25µm MT. For a similar MT length, the larger bead will displace the MT more than the smaller bead.

(B) Bead-to-MT speed ratio plotted as a function of MT length, for dynein-coated beads of 1 µm and 3 µm diameter in a 39cP MC viscous medium. The dotted lines are linear fits to guide the eyes.

(C) Bead-to-MT speed ratio plotted as a function of MT length, for 1µm diameter dynein coated beads in a 1.18% or 1.60% MC medium (respectively yielding measured viscosities of 39cP and 124cP).

(D) MT-to motor speed efficiency ratio plotted as a function of the ratio of MT length to bead radius, for beads of diameters of 0.5, 1, 2.2, 2.8 and 3µm, in medium of viscosities of 39cP or 124cP. A fit of the data is shown as a black line, and the theoretical behavior in a Newtonian fluid as a grey dashed line.

See also Figure S2 andS3. (B) Time-lapse of one or two beads walking on ~22µm long MTs. For similar motor velocities, the MT is displaced more when two beads are walking on it.

(C) MT-to motor speed ratio plotted as a function of the ratio of the MT length to the bead number multiplied by the bead radius. Data shown are 1µm diameter beads, walking on MTs alone or by two, away from each other (the orange dots contain data with beads coated with dynein or kinesin, in a viscous medium of 39cP or 124cP).

The fit on the data of all bead sizes is shown as a black line. See also Figure S3.

STAR METHODS

CONTACT FOR REAGENT AND RESOURCE SHARING

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Nicolas Minc (nicolas.minc@ijm.fr).

METHOD DETAILS

Proteins expression and purification -Dynein

Recombinant cytoplasmic dynein was expressed in Dictyostelium discoideum, and purified following the protocol described by T. Kon et al. [36] with some modifications. This HFB380 380 kDa recombinant dynein was engineered from the Dictyostelium discoideum cytoplasmic dynein heavy chain gene as described previously [START_REF] Numata | C-sequence of the Dictyostelium cytoplasmic dynein participates in processivity modulation[END_REF].

Affinity tags were added (His6 and FLAG) at the N-terminus as well as a N-terminal BioEase tag for in vivo biotinylation. Glutathione S-transferase (GST) could also be added for dynein dimerization. Finally, a MB35 plasmid (ID 44 -Dicty Stock Center) encoding a tetracycline-controlled transcriptional activator was introduced in order to control the recombinant protein overexpression.

Cells were cultivated at 21°C in culture dishes in HL-5 medium supplemented with 10µg/mL G418 and 10µg/mL tetracycline until they reached confluence, and were then transformed through electroporation with an MB38-based plasmid containing the engineered dynein gene. 10µg/mL blasticidin was added for plasmid selection one day after electroporation. 200mL of HL5 supplemented with G418, tetracycline and blasticidine were inoculated in a confluent 10cm culture dish, and cultivated at 21°C, 200rpm. After cells had reached 10 7 cells/mL, dynein expression was induced through removal of tetracycline (centrifugation: 1,000g, 5min, 21°C), and resuspension in 400mL HL-5 supplemented only with G418 and 20µM of d-biotin for dynein biotinylation.

Cells were harvested by centrifugation (5,000g, 5min, 2°C), washed in 20mL lysis buffer (100 mM PIPES-KOH, 4 mM MgCl2, 0.1 mM EGTA, 0.9 M glycerol, 10 mM imidazole, pH 7.0), and resuspended in an equal volume of lysis buffer supplemented with 1 mM TCEP, 10 mM ABESF, 0.2 mM leupeptin, 87 µM pepstatin, 10 mM TAME and 0.1 mM ATP. Cells were sonicated (six pulses of 3s with 30s pause between each pulses) and the supernatant was collected after two successive centrifugations (18,000g, 20min, 2°C and 100,000g, 15min, 2°C) for clarification of the lysate. This lysate was gently mixed with 300 µL of pre equilibrated Ni-NTA agarose for 1h at 4°C on a rotating wheel. The mixture was loaded on a column, column flow though was discarded, and 12 column volumes (CV) of wash buffer (100mM PIPES, 4mM MgCl2, 0.1mM EGTA, 0.9M glycerol, 20mM imidazole, 1 mM TCEP, 10 mM ABESF, 0.2 mM leupeptin, 87 µM pepstatin, 10 mM TAME and 0.1mM ATP, pH 7) were flowed in and discarded. Bound proteins were then eluted with 2CV of elution buffer (100mM PIPES, 4mM MgCl2, 0.1mM EGTA, 0.9M glycerol, 250mM imidazole, 1 mM TCEP, 10 mM ABESF, 0.2 mM leupeptin, 87 µM pepstatin, 10 mM TAME and 0.1mM ATP, pH 7). The eluate was supplemented with 150mM NaCl, 5mM EGTA and 0.1mM EDTA, and then gently mixed with 100 µL of pre equilibrated AntiFLAG gel for two hours at 4°C on a rotating wheel. The gel was loaded on a column, column flow through was discarded, and the gel was washed with 10CV of PMEGS buffer (100mM PIPES, 4mM MgCl2, 5mM EGTA, 0.1mM EDTA, 0.9M glycerol, 200mM NaCl, 1 mM TCEP, 10 mM ABESF, 0.2 mM leupeptin, 87 µM pepstatin, 10 mM TAME and 0.1mM ATP, pH 7) and then 10CV of PMEG30 buffer (30mM PIPES, 4mM MgCl2, 5mM EGTA, 0.1mM EDTA, 0.9M glycerol, 1 mM TCEP, 10 mM ABESF, 0.2 mM leupeptin, 87 µM pepstatin, 10 mM TAME, 20% (w/v) trehalose and 0.1mM ATP, pH 7). Bound recombinant dynein was eluted slowly with 3CV of PMEG30 buffer supplemented with 0.25mg/mL FLAG peptide. The final eluate was flash frozen using liquid nitrogen, and stored at -80°C. Protein concentration was determined using A280 or Bradford reagent with bovine serum albumin as a standard.

-Kinesin His-tagged recombinant kinesin (pET28-mKif5B_N1665, mouse kinesin N-terminal, with motor domain and coiled coil) was expressed in Rosetta (DE3)pLysS E.coli. One liter of 2YT media supplemented with 34µg/mL chloramphenicol and 50µg/mL kanamycin was grown at 37°C, 200 rpm until the OD600nm was between 0.8 and 1 and transferred to 20°C. Protein expression was then induced for 4-5h at 20°C by addition of 0.5 mM IPTG.. Cells were harvested by centrifugation (8,000g, 20min, 20°C), and the pellet was resuspended in 40mL of lysis buffer (20 mM potassium phosphate, 150 mM NaCl, 10% glycerol (w/v), 5mM ß-mercaptoethanol, 1mM MgCl2, 0.1mM ATP.). Cells were then sonicated (5 pulses of 15s, 50% intensity, 6mm probe), and the lysate was clarified by centrifugation (80,000g, 50min, 4°C). The supernatant was then mixed with 0.250mL Ni-NTA beads (pre equilibrated in equilibration buffer: 50 mM potassium phosphate, 100mM NaCl, pH 7) and incubated for 1h at 4°C under gentle agitation. The resin was washed with minimum 5CV of wash buffer (50mM potassium phosphate, 10% glycerol, 2mM ß-mercaptoethanol, 25mM imidazole and 1M NaCl, pH 7) and the kinesin was eluted with 1CV of elution buffer (150mM imidazole, 50mM KCl, 10% glycerol and 10mM ß-mercaptoethanol, pH 7). The eluate was dialyzed against 50 mM MOPS, 250mM NaCl, 5mM ß-mercaptoethanol and 20% glycerol using a NAP-5 column (GE Healthcare), flash frozen using liquid nitrogen, and stored at -80°C. Protein concentration was determined using Bradford reagent.

-Tubulin purification and labelling Tubulin was purified from pig brains following the protocol described by Castoldi and Popov, based on cycles of polymerisation and depolymerisation and high-molarity buffer removal of associated proteins [START_REF] Castoldi | Purification of brain tubulin through two cycles of polymerization-depolymerization in a high-molarity buffer[END_REF]. Fresh pig brain tissues were homogenized in 1 volume (1ml/g) of DB buffer (50mM MES pH 6.6, 1mM CaCl2) and centrifuged at 29,000g for 1h at 4°C. The supernatant was supplemented with 1 volume of highmolarity PIPES buffer (HMPB: 1M PIPES pH 6.8, 10mM MgCl2, 20 mM EGTA) and 1 volume of glycerol and raised to a final concentration of 1.5mM ATP and 0.5mM GTP. The mixture was then incubated for 1h at 37°C to induce tubulin polymerisation.

After centrifugation at 150,000g for 30 min at 37°C, the pellet was resuspended in 0.3 volumes of cold DB buffer and incubated for 30 min at 4°C to depolymerize microtubules. After centrifugation at 4°C for 30 min at 120,000g, the supernatant containing free soluble tubulin was polymerized for a second cycle of 1h at 37°C after being supplemented as above with an equal volume of HMPB buffer, a volume of glycerol and with a final concentration of 0.5mM GTP and 1.5mM ATP. Polymerised tubulin was pelleted by centrifugation at 37°C for 30 min at 150,000g. The pellet was resuspended in 0.01 volumes of cold BRB80 buffer (80mM PIPES, 1mM EGTA, 1mM MgCl2, pH 6,8) and depolymerized for 1h at 4°C. The suspension was centrifuged for 30min at 4°C and 150,000g. The supernatant containing pure soluble tubulin was aliquoted, frozen in liquid nitrogen and stored at -80°C.

Labelling of tubulin with NHS-ester-ATTO 565 (ATTO-TEC) was performed following the protocol described by Hyman [START_REF] Hyman | Preparation of marked microtubules for the assay of the polarity of microtubule-based motors by fluorescence[END_REF]. Tubulin was first polymerized in presence of 1mM GTP and 3.5mM MgCl2 and 25% glycerol (v/v) at 37°C for 1h. Microtubules were collected by centrifugation at 35°C for 40 min at 100,000g through a cushion of 0.1M HEPES pH 8.6, 1mM MgCl2, 1mM EGTA, 60% glycerol (v/v). The pellet containing microtubules was then resuspended in labelling buffer (0.1M HEPES pH 8.6, 1mM MgCl2, 1mM EGTA, 40% (v/v) glycerol). The succinimidyl ester-coupled fluorophore (dissolved at 50mM in DMSO) was then added to a final concentration of 5mM and incubated 20 min at 37°C. Labelled microtubules were centrifuged through a cushion of 60% (v/v) glycerol in BRB80 for 40min at 35°C at 150,000g. Microtubules were depolymerised for 30min at 4°C in 50mM K-Glutamate, 0.5mM MgCl2, pH 7.0 (KOH). Tubulin was recovered by centrifugation at 4°C for 20 min at 150,000g. The solution was brought to 80mM PIPES, 4mM MgCl2, 1mM GTP and another cycle of polymerisation and depolymerisation was performed. The final pellet was resuspended in cold BRB80, aliquoted, frozen in liquid nitrogen and stored at -80°C.

-Microtubule polymerisation Tubulin and labelled tubulin (80µM total, with 7 to 20% labelling) were mixed in BRB80 with glycerol (80mM PIPES, 1mM EGTA, 1mM MgCl2, 25% glycerol, pH 6,8), and centrifuged to remove possible proteins aggregates or impurities (100,000g, 10min, 4°C). The supernatant was recovered in a microtube, 1mM GTP was added, and the mix was incubated 5min on ice, before incubation for polymerisation for 45min at 37°C. A volume of BRB80 supplemented with 40µM docetaxel (taxol analogue) was added to reach a concentration of 20µM docetaxel, and the mix was incubated for stabilization for 15 to 30 min (yielding more or less long MTs). The mix was centrifuged (100,000g, 10min, 30°C), the supernatant removed, and the pellet washed smoothly with 10µL BRB80D20 (BRB80 supplemented with 20µM docetaxel). BRB80D20 was added to the pellet, to resuspend the MTs at 47,8µM by pipetting smoothly after letting the pellet hydrate for 10min. MTs were stored at RT in the dark.

Buffers

We performed experiments in a dynein assay buffer (10mM K-PIPES, 50mM potassium acetate, 4mM MgSO4, 1mM EGTA, 10mM glucose, 25µM glucose oxydase, 6.4µM catalase, 1mM DTT, 0,4mg/mL casein, 40µM docetaxel, 1mM ATP, pH 7.0) supplemented with methylcellulose (MC) (Sigma). MC was previously prepared as a 2% solution as advised by the seller. Briefly, MC was added to half of the buffer volume heated to 80°C, agitated until particles are evenly dispersed, after which the remaining half of the buffer was added at 4°C. The mixture was then agitated at 4°C and MC dissolved when it attained the temperature at which it became water soluble. Glucose, DTT, casein, docetaxel and ATP were added after the solution cooled down to 4°C, and aliquots were stored at -20°C. Glucose oxydase, catalase and MTs were added just before experiments, to yield 1,18% or 1,60% MC.

Microscopy chambers

Chambers were made of two coverslips separated by one to three layers of parafilm.

These coverslips were previously cleaned in ultrasound baths of Hellmanex 5%, KOH 2M, and absolute EtOH, or KOH 2M, demineralized H20, and absolute EtOH (30min sonication in each solution, with H20 rinsing in between each bath). Protein solutions were injected in the microscopy chambers with a pipet into chambers of 1.5 to 2mm in width, 50-440µm in height, and 0.5-2cm in length depending on experiments, resulting in a chamber volume of 3-8µL. The chambers were finally sealed with VALAP (an even mixture of vaseline, lanolin and paraffin).

Microscopy experiments -Control experiments: gliding assay

A classical MT gliding assay was carried out to verify the functionality of the purified dynein, following the protocol described by Kon et al. [36]. Microscope chambers were constructed as described above, with one layer of parafilm between coverslips, a width of 2mm, an internal height of ~50µm, and a length of 2cm, resulting in a volume of ~6µL. The microscope chamber was covered with biotinamidocaproyl BSA, streptavidin, passivated with casein, (with buffer washes after each of these steps) and covered with 7nM biotinylated dynein. Finally, dynein assay buffer, supplemented with 400nM labelled and docetaxel-stabilized MTs, was flowed in the chamber. MTs were then free to meet dyneins at the surface and glide on them.

-Bead coating with dynein Streptavidin beads were washed in the dynein assay buffer three times in Lobind microtubes (Sigma) (a wash included the mixing of beads in buffer, followed by a 2 minutes sonication, a centrifugation at 10,000g, 4min, 4°C, and the resuspension of the pellet in buffer). Beads were then incubated with biotinylated dynein for ~1-3h in the cold room with gentle agitation (incubation with more than 50 fold the beads binding capacity). Beads used were 0.5µm (Bangs laboratory), 1µm (Bangs laboratory or Dynabeads from ThermoFisher), 2.2µm (Polysciences), 2.8µm (ThermoFisher) and 3µm (Bangs laboratory). When beads were not already fluorescent, beads could be labelled with 0.001 fold the bead binding capacity with ATTO 565-biotin after the dynein coating step.

-Bead coating with kinesin Streptavidin beads were washed as above, and then, either kinesin was incubated with 1µm beads (Bangs laboratory) for a direct adsorption on them, either beads were first incubated with biotin anti-GST, washed, and then incubated with kinesin. Each incubation consisted of ~1-3 hours of gentle agitation in a cold room, with more than 1000 fold the bead binding capacity, and each wash consisted of a centrifugation (10,000g, 4min, 4°C).

-Control experiments: bead gliding assay Streptavidin beads were coated with dynein (as previously described). Microscope chambers were constructed as described above, with two layers of parafilm between the two coverslips, a width of 2mm, an internal height of ~200µm, and a length of 0,5cm, resulting in a volume of ~3µL. The microscope chamber was first covered with biotinamidocaproyl BSA and secondly with dynein-coated beads. Finally, dynein assay buffer supplemented with 400nM labelled and docetaxel-stabilized MTs, was flowed in the chamber.

-3D motility assay experiment

The equivalent of approximately 0.005µL dynein-coated beads were centrifuged (10,000g, 6min, 20°C), re-suspended in ~3µL of 1.18% or 1.60% MC dynein assay buffer at 20°C supplemented with 12.5-25nM stabilized labelled MTs, and flowed in a hand-made chamber (width of 2mm, internal height of ~200µm, length of 0,5cm, resulting in a volume of ~3µL) which was then sealed with VALAP. We looked for events of a bead encountering a MT, focusing on MTs away from the surface (>10µm from the surface) and almost parallel to the surface. Data were acquired for ~1h.

-

3D motility assay experiments with optical trapping

The protocol was similar to the 3D motility assay described above, with the only difference that we did not looked for bead encountering MTs naturally, but instead captured beads with an home-made optical trap (IPG Ytterbium Fiber Laser (Model YLD-10-LP)) and placed them directly onto the MTs. This was particularly necessary to study the movement of two beads on a MT, or to construct a minimal aster and place several beads on it.

-Construction of an artificial minimal aster Based on the protocol of the 3D motility assay with optical trapping, we increased the complexity of the in vitro reconstitution, and constructed a minimal artificial aster with 3µm dynein coated beads. A bead was placed on a MT, and as it walked on the MT, it revealed its polarity. The bead was detached from the MT by quickly pulling on it. This first step was repeated with 1 or 2 other MTs. The last MT handled was displaced thanks to the bead still present at its minus end, and with this bead, the other one or two MT precedently handled were captured by their minus ends. It resulted in a minimal aster, where two or three MT emanated from a 3µm bead. Two or three dynein coated beads could then be subsequently placed near the plus ends of the MTs of this minimal aster, to cause aster motion.

-

Beads MSD measurement for viscosity assessment

To compute the viscosity of different media, we computed the Mean Square Displacement of beads of different sizes (1µm (Dynabeads,ThermoFisher), and 2.8µm (ThermoFisher)) in different medium. For this, beads were mixed in 1,18% or 1,60% MC dynein assay buffer, and frames were acquired every 1 or 2s for ~1min (Figure S2F). For each movies, we let the fluid equilibrate long enough, to ensure that flows were negligible, and did not affect the analysis.

-Hydrodynamic interaction experiment Beads (1.9µm diameter, streptavidin coated) were labelled with ATTO 595-biotin, and mixed with stabilized labelled MTs in a viscous medium (1.18% MC dynein assay buffer). A single bead was captured with the optical trap, placed near a MT, and kept immobile there. The motorized stage was moved parallel to the MT, at velocities ranging from 1 to 45µm/s, resulting in a situation equivalent to the trapped bead being displaced near an immobile MT. The movement of the MT that may result from the bead displacement was measured to infer the hydrodynamic coupling between the two objects.

The movements of beads were tracked on ImageJ, following the bead with a circle and measuring its centroid, using a sub-sampling of 1/100 th of the pixel size to increase the measurement precision. Velocities of walking beads and aster center bead could then be deduced from these positions.

MSD measurement for viscosity assessment

We selected beads in focus to be tracked, and checked that they were single beads , with 𝑘 the Boltzmann constant, 𝑇 the temperature and 𝑟 the beads radius.

Relationship between speeds and dimensions of MT and beads

For one bead on one MT

Let us first consider a single motor-coated bead moving along a single MT, in solution, away from all surfaces. Neglecting drift and diffusion, all movements occur along the axis materialized by the MT, which we arbitrarily choose to orient towards the MT minus end. In the laboratory frame of reference, the bead and the MT move with velocities Vbead and VMT, respectively. For the isolated system (bead + MT), the force balance yields: γMT vMT + γbead vbead=0, where γMT and γbead are the MT longitudinal drag coefficient, and the bead drag coefficient, respectively.

Based on Stoke's law, the drag coefficient of the bead is expected to scale with the bead radius, Rbead. Within our range of MT lengths, we can reasonably approximate the longitudinal drag coefficient of the MT as a linear function of the MT length, LMT [START_REF]Mechanics of Motor Proteins and the Cytoskeleton[END_REF][START_REF] Happel | Low Reynolds number hydrodynamics: with special applications to particulate media[END_REF]. We can thus write γbead = µ αbead Rbead and γMT = µ αMT LMT, where αbead and αMT are geometrical constants, and µ is the viscosity of the medium. As a consequence, the speed ratio -Vbead/VMT = αMTLMT / αbeadRbead is expected to scale with the MT-length-to-bead-radius ratio, independently of the viscosity of the medium.

In order to assess how efficiently the activity of the motor is converted into MT movement, it is convenient to compute the MT-to-motor speed ratio -VMT/v*motor, where v*motor is the speed at which the motor transports the bead along the MT, and which can be written v*motor=Vbead-VMT. Based on the previous equations, this "efficiency" ratio can be written as:

-VMT/v*motor = 1/(1+ αMTLMT / αbeadRbead), and is comprised between 0 and 1.

Fitting the experimental plot of -vMT/v*motor versus LMT/Rbead (Figure 2E) with this equation provides an estimation of αMT/αbead, which is the only free parameter of the fit. We thus find this number to be 0.020, which is approximately 2.3-fold smaller than what can be computed theoretically for a Newtonian medium, based on Stoke's equation and on our linear approximation of the MT longitudinal drag coefficient (Figure S3A). This difference may be explained by the fact that the buffer supplemented with MC may behave as a non-Newtonian fluid, and thus exhibit significant shear-thinning effects [START_REF] Tomioka | Effects of concentration and degree of polymerization on the rheological properties of methylcellulose aqueous solution[END_REF]. In addition, our theoretical computation also neglects potential complications such as the hydrodynamic coupling between the bead and the MT, and the rotation of the bead as it moves along the MT.

For two beads moving along a single MT

Considering that the drag forces of two motile beads are independent and additive, the force balance yields γMT VMT + γbead1 Vbead1 + γbead2 Vbead2 =0, and the computation of the ratio -VMT/v*motor then leads to:

-VMT/<v*motor> = 1/(1+ αMTLMT / 2αbeadRbead), where <v*motor>=(v*motor1+v*motor2)/2 is the average motor speed (Figure 3C).

For multiple beads moving on a minimal aster

We now consider the movement of N motor-coated beads on a set of MTs connected by their minus ends to a central bead. Movements are observed in the focal plane of the microscope, in two dimensions. The minimal aster is thus composed of the central bead and the MTs, and has a non-isotropic drag coefficient. Balancing the drag forces yields, projected on a x-axis parallel to the two MTs in the minimal aster: γx,aster vx,aster + γbead1 vx,bead1 +… + γbead N vx,bead N = 0, where γx,aster is the drag coefficient of the minimal aster for movements along the xaxis.

Thus vx,aster should scale with -(vx,bead1 +… + vx,bead N). (Figure 4B).

Hydrodynamic interaction experiment (Figure S4)

An optically trapped bead was displaced near a freely floating MT, by keeping the trapped bead in a fixed position, in the frame of reference of the microscope, while moving the entire chamber thanks to a motorized stage. A free bead, away from the MT and the trapped bead, was used to monitor the movement of the stage, in the frame of reference of the microscope. All movements were then determined in the frame of reference of the stage, by using the free bead as a reference.

The velocity of the trapped bead was thus measured, in the frame of reference of the stage, and so was the velocity of the MT, resulting from the hydrodynamic coupling with the trapped bead displaced parallel to it. The MT-to-bead speed ratio can be plotted as a function of the MT distance from the surface of trapped bead, and data can be fitted as a power series (Figure S4C). (E) Sketch showing the measurements done to analyse the 3D motility assay and an example of the choice for ti and tf, encompassing a constant bead and MT movement. (C) Sketch explaining the steps of construction of a minimal artificial aster. A bead is first placed on a MT, and as it walks on the MT it reveals its polarity [START_REF] Dogterom | Force generation by dynamic microtubules[END_REF][START_REF]Mechanics of Motor Proteins and the Cytoskeleton[END_REF]. The bead is then detached from the MT by sharply pulling on it. This first step is repeated with another MT [START_REF] Laan | Cortical Dynein Controls Microtubule Dynamics to Generate Pulling Forces that Position Microtubule Asters[END_REF][START_REF] Grill | Polarity controls forces governing asymmetric spindle positioning in the Caenorhabditis elegans embryo[END_REF]. This last handled MT is displaced thanks to the bead still present at its minus end, and with this bead, the other one MT precedently handled is captured by its minus ends ( 5).

KEY RESOURCES

(D) Example showing how tug-of-war events implicating multiple beads are analyzed.

B -Discussion and perspectives on the in vitro reconstitution

The result section presented the in vitro minimal system which was developed, and which consisted in the reconstitution of the force generation of dynein transported cargoes, pulling on MTs away from any surfaces or anchoring points.

This assay allowed to show that dynein-transported objects can indeed exert forces on MTs, pulling the MTs in the direction opposite to dynein transport. It also

showed that, at the scale of a very minimal aster, consisting of two MTs linked by a micrometer sized bead, dynein coated objects could also exert pulling forces on the MTs, displacing the aster towards the side where more dynein coated object were pulling. As we are going to discuss below, open questions arise from these experiments, notably the force exertion of dynein coated object at the scale of a more complex minimal aster, or the force exertion of non-spherical dynein-transported objects, for example resembling more to the ER network, which is, as detailed in the introduction and the next result section, likely to be the main cytoplasmic anchor for dynein force exertion on MTs.

As specific sets of results were already discussed in the paper, the goal of this discussion is to hypothesize some future work directions, as well as discussing some connected work that was not included in the paper, and strategies which were put aside.

The first part will focus on the discussion about the effective viscous drag of MTs, and the three other parts will focus on perspectives adding complexity to the in vitro reconstitution of dynein force generation in bulk cytoplasm, and getting closer to the physiological conditions, through the construction of more complex minimal asters, the use of non-spherical objects as cargoes, and the confinement of dynamic asters.

-The effective viscous drag of MTs relative to bead drag

An interesting question which arose from the results of the 3D motility assay, where the dynein-coated bead motion on a MT can move this MT in the other direction, was the one of the hydrodynamic friction forces exerted on each object.

Indeed, the movement of the MT was larger than expected, based on its drag calculated in a Newtonian fluid, and neglecting potential hydrodynamic interactions between the objects.

This larger MT movement would correspond to an approximately two-fold reduction of its drag coefficient. This might be due to scale-dependent viscosity, experienced when the object size is close to the viscous agent mesh size. The apparent reduction of the MT drag could also be due to a property of non-Newtonian fluids, where the viscosity coefficient depends on the shear rate. Indeed, the methylcellulose-containing viscous medium I used is likely to behave as a non-Newtonian fluid [Tomioka and Matsumura, 1983]. This viscous medium could thus exhibit shear-thinning, where the viscosity coefficient is inversely proportional to shear rate, as the polymeric methylcellulose can align along anisotropic objects such as MTs, facilitating the MT displacement.

I thus attempted to measure the longitudinal drag coefficient of the MT, compared to the drag coefficient of a bead, in the viscous medium used for the 3D motility assay, using the method that is described below, but that did not allow us to conclude, also as explained below.

The protocol was based on the capture in an optical trap of a bead linked to a stabilized MT, in the viscous medium used for the 3D motility assay (Figure 40 A).

The MT was attached to the streptavidin bead thanks to the GMPCPP stabilized biotinylated seeds from where it was polymerized. The stage was then moved, thereby putting the medium in movement and applying a drag force to the trapped bead and the MT attached to it. The small concomitant displacement of the bead in the optical trap was measured and is a readout of the friction force applied to the MT and the bead. The experiment was also done with a bead trapped without any MT attached on it, and it was assumed that the drag of the MT and the bead attached together was equal to the sum of the bead drag and the MT drag. Thus, the ratio of the drags of the bead and of the MT could be calculated, and compared to the theoretical one. This experiment suggested that the MT longitudinal drag coefficient was indeed lower than the one expected from the theory based on Newtonian fluids, but only by 30% (Figure 40 C), hence not as much as the two-fold decrease that is expected, based on the 3D motility assay. However, this set of data did not allow me to conclude clearly, as unexpected disparities in the measurements were present, maybe due to the unusual time the bead in the optical trap would take to reach its equilibrium position when submitted to the flow, as seen above in Figure 40 B.

Indeed, this type of experiment, when done on actin filaments bound to beads through formin molecules in a microfluidic setup, yielded results which were not as dispersed as ours [START_REF] Jégou | Formin mDia1 senses and generates mechanical forces on actin filaments[END_REF].

A number of arguments could explain these differences. First, in this MT setup, the streptavidin trapped bead is attached to the MT through the MT biotinylated seed.

Thus the MT does not emanate exactly perpendicularly from the bead, but is rather attached along a small MT portion parallel to the bead, and thus has to bend to align with the flow. In the actin set up, on the other hand, the formin anchoring of the actin filament on the trapped bead allowed the actin filament to directly orient with the flow, and be positioned at the bead equator, exactly perpendicular to the bead. Plus, the MT is far less flexible than actin [START_REF] Gittes | Flexural rigidity of microtubules and actin filaments measured from thermal fluctuations in shape[END_REF], thus it cannot bend as easily as actin, and will necessitate a larger distance to bend in order to align with the flow.

Thus, the MT attachment to the bead might not allow for a quick and constant alignment of the MT-bearing bead in the optical trap, which could lead to a delay for the bead to reach its equilibrium position in the optical trap when submitted to the flow.

Finally, in the MT setup, the flow applied to the MT-bearing trapped bead was created though the motion of a motorized stage, displacing the microscopic chamber.

This movement might not be as smooth as the flow generated with the microfluidic setup used in the actin study, and could have thus added noise to the measurements.

An interesting perspective would be to adapt the protocol so as to get rid of these issues. It would be possible to generate the flow using a microfluidic setup, and attach the MT to the bead through another means, in order for the MT tip to emanate from the bead equator. The protocol could also be conducted in different viscous solutions, known to behave as Newtonian or non-Newtonian fluids, and study the impact on the longitudinal drag of the MT.

-The construction of more complex minimal MT asters

Concerning the minimal aster construction, I could manage to build asters with two to three MTs emanating from a bead, and follow the motion of these minimal asters when two to three dynein coated beads moved on the aster MTs. Interesting follow-up experiments include building asters with more MTs, getting closer to real asters, and following their movement after the addition of motile dynein-coated beads on their MTs. Experiments could also tackle the MT length-dependent forces exertion at the scale of an aster, focusing on the consequences of the recruitment of more dynein coated objects on one side of the aster. An interesting experiment, as represented in the figure 41 below, would include building a symetric aster, but placing more motile beads on one side of the aster to follow, as we would expect, its consequent motion towards the side where more dynein coated beads were placed.

Another possibility is to build an asymetric aster, for example thanks to laser ablation of MTs on one side of the aster, and place this aster in a medium with a homogeneous distribution of motile dynein-coated beads. Longer MTs would thus recruit more dynein coated beads than shorter MTs, and we would expect the aster to be displaced towards the longest MTs side, mimicking MT aster centration after fertilization. These strategies could not be developed, as building an artificial aster with more than a couple of MTs could not be achieved in the available time. Indeed, several aster formation protocols were considered, and, in our hands, the more efficient and convenient protocol for the conduction of the assay allowed us to build asters consisting of only two to three MTs. The other aster construction techniques that I have tried are recapitulated below.

First, I tried mixing neutravidin with stabilized MTs polymerised from biotinylated seeds, in order to form agregates of seeds from which several MTs would emerge. However, I could not find concentrations and incubation conditions enabling the construction of asters made of several MTs. I also tried mixing first neutravidin and biotinylated seeds, and then elongate MTs from these structures. However, while I could manage to form agregates of MT seeds and neutravidin, MTs could not be elongated from these structures. Then, I tried to form MTs asters from beads, either by first coating streptavidin beads with biotinylated seeds, and then elongating the MT seeds, either by directly mixing MTs polymerised from biotinylated seeds to the streptavidin beads. This technique was not successful either, as only a very small proportion of beads were coated with a couple of very short MTs, and, even playing on concentration and incubation time, I could not obtain beads from where emanated several long MTs.

I thus turned to the construction of asters using dynein coated beads and the optical trap, directly in the microscopy chamber of the experiment. As explained in the result section, I positioned a dynein coated bead on a MT, let it reach the MT minus end in order to know the MT polarity and pulled on the bead to detach it from the MT. I then repeated this protocol with a second MT, this time not detaching the bead from the MT tip, but rather, displacing the MT thanks to the trapped bead still attached to the MT, and connecting it to the first MT minus end. Advantages of this techniques were that I could work with stabilized MTs, control the aster shape, and build the asters directly in the observation chamber, which allowed to get rid of the process of flowing already built asters in the microscopy chamber, associated with a probable risk of breaking the constructed asters. The major drawbacks of this technique however, were that the number of MTs constituting the minimal aster is limited by the difficulty of the cautious assembly of the MTs through this method, and that the aster center thus consists of a quite large object, whose drag will limit the aster motion after the placement of motile beads on its MTs. Other aster formation techniques could be tried in the future, such as the formation of denser MT asters from purified centrosomes, and would imply to master centrosome purification protocol, as well as finding ways, either to introduce stabilized MTs asters build from centrosomes into the viscous medium and into the microscope chamber without breaking them, or to polymerize these MTs asters directly in the chamber in the viscous solution, being careful to still be able to see the MTs even with unpolymerised labelled tubulin in solution.

-Reconstitution of dynein pulling through transport of non-spherical cargoes

Another fascinating follow-up of this work could be to reconstitute in vitro a system mimicking dynein pulling on MTs through the transport of Endoplasmic Reticulum. Indeed, our work showed that dynein coated beads could exerce forces on MTs through their viscous friction force, mimicking the force generation of dynein transported organelles in the viscous cytoplasm. But, as underlined in the manuscript introduction, and as suggested by in vivo experiments described in the next section, the ER is likely to be one of the main cytoplasmic anchor of this force generation mechanism, and its shape does not resemble the one of a bead. The interconnected membrane tubules of ER could be represented better than by beads, for example through the tethering of dynein to reconstituted ER network on oil droplets. It has indeed been shown that ER networks could be reconstituted from purified microsomes fractions, for example from HeLa cells, and the network would resemble more the physiological ones when the reconstitution was made on oil droplets than on rigid surfaces [START_REF] Ferencz | Shaping the endoplasmic reticulum in vitro[END_REF][START_REF] Powers | Reconstitution of the tubular endoplasmic reticulum network with purified components[END_REF]. One could thus imagine reconstructing ER networks on oil droplets, which would also contain a small concentration of tagged lipid allowing dynein to connect to this artificial cargo, and image the motion of these cargoes on MTs or reconstituted MT asters, and study the concomitant MT or MT aster motion. The 3D motility assay developed here could thus be reproduced with such elements, resembling more the ER architecture, in order to assess whether it would pull more efficiently on MT, maybe transmitting more of their motion to the MTs than dynein coated beads. Interconnected networks such as actin networks could also be used to mimick the ER network, and study the transport by dynein of such an interconnected network on a MT aster. The case of an asymetric reconstituted aster would allow to mimick, with a minimal and controlled system, the MT aster centration after fertilization.

II -Dynein force exertion during aster centration after fertilization in sea urchin embryo A -Results

In this work, we characterized the force exertion of dynein transported beads on MTs in a minimal system in vitro, but also worked on the Paracentrotus lividus sea urchin embryo model in order to try to better understand how dynein can pull in the cytoplasm. Indeed, as described in the introduction, several experiments support that sperm aster centration in sea urchin mainly relies on dynein dependent forces, scaling to MTs length, and pulling on the MTs directly from the cytoplasm.

Experiments were thus conducted, in order to appreciate the forces associated with vesicles movement, and try to affect the cargo force exertion.

-Viscosity measurement in sea urchin eggs and embryos

Viscosity of sea urchin embryos was measured, in order to assess the force exertion of vesicles and organelles. Indeed, sea urchin viscosity was already assessed in the literature, but not at the scale of objects whose size resembled the one of vesicles and organelles. The centipoise unit (cP) will be used to define viscosities, and 1cP is equivalent to 1 mPa⋅s, and corresponds to water viscosity. As described in the introduction, measurements made with very small probes, such as fluorescein molecules, yielded viscosity of a couple of cP, and measurement made with probes of 5 to 9µm in diameter yielded viscosity around 1000-3000cP, highlighting that viscosity is scale-dependent [START_REF] Kalwarczyk | Comparative Analysis of Viscosity of Complex Liquids and Cytoplasm of Mammalian Cells at the Nanoscale[END_REF][START_REF] Periasamy | Picosecond rotation of small polar fluorophores in the cytosol of sea urchin eggs[END_REF]Hiramoto, 1969;[START_REF] Doubrovinski | Measurement of cortical elasticity in Drosophila melanogaster embryos using ferrofluids[END_REF]. Thus, we used beads of 1µm in diameter, in order to measure the viscosity felt by objects resembling the size of vesicles and organelles. The principle of this assay was to first, use a magnet to pull on 1µm beads in viscous media of known viscosity, and track the beads to calculate their velocities, in order to calibrate the magnet force (Figure 43-A,B). Second, the same beads were injected in sea urchins embryos. The same magnet was used to pull on these beads, and the beads were tracked to calculate their velocity (Figure 43-C,D). Thus, the only unknown parameter was the sea urchin viscosity, which could then be deduced. Results were very dispersed, but suggested that the magnitude order of the viscosity felt by 1µm objects in the sea urchin cytoplasm, was approximately 1200cP, which is 1200 times higher than the viscosity of water, and resembles to the one of motor oil (Figure 43-E). Interestingly, we noted that this viscosity was slightly increased upon fertilization (by~25%), suggesting that reorganization of the cytoplasm at fertilization (e.g. and de novo assembly of cytoskeletal elements, like asters or endomembranes), may alter bulk fluidity (Figure 43-E).

-Affecting organelles force exertion through partitioning of the egg components with centrifugation a -Centrifugation and characterization of the centrifugation profile

Centrifugation of sea urchin eggs was carried out in order to polarize the distribution of vesicles and organelles, so as to study the impact of different organelles on the positioning of the MT aster after fertilization. This centrifugation protocol was based on previous centrifugation studies in sea urchins eggs of several species: Arbacia punctulata, Strongylocentrotus purpuratus, Paracentrotus lividus,

Sphaerechinus granularis, Parechinus microtuberculatus, Arbacia pustulosa and

Tripneustes esculenta [START_REF] Harvey | The development of half and quarter eggs of arbacia punctulata and of strongly centrifuged whole eggs[END_REF][START_REF] Harvey | Development of the parts of sea urchin eggs separated by centrifugal force[END_REF][START_REF] Anderson | A cytological study of the centrifuged whole, half, and quarter eggs of the sea urchin, Arbacia punctulata[END_REF][START_REF] Longo | Derivation of the membrane comprising the male pronuclear envelope in inseminated sea urchin eggs[END_REF][START_REF] Henson | A calsequestrin-like protein in the endoplasmic reticulum of the sea urchin: localization and dynamics in the egg and first cell cycle embryo[END_REF].

According to the species, some small differences in the organelles repartition were notable, thus we characterized the centrifugation profile in P. lividus itself.

Centrifugation of the egg on a sucrose gradient induced the partition of cytoplasm components as a function of their densities. The extent of the egg content partitioning and the egg deformation depended on the centrifugation speed and its duration (Figure 44).

The partitioning followed the repartition described in the figure below, with notable variability according to egg batches. Lighter elements were accumulated near the centripetal pole, whereas denser elements were accumulated at the centrifugal pole. A thinner granulometry zone was visible at the centrifugal pole, and the centrifugal pole was more elongated than the centripetal pole. The lighter elements notably consisted of the female pronucleus and the ER, and the denser elements of mitochondria, lysosomes and endosomes. Yolk granules, which constitute the egg energy storage, are very numerous, and thus still occupy a big part of the egg after centrifugation. The distribution of vesicles and organelles after centrifugation could be observed thanks to live imaging of labelling probes, or antibody staining after immunofixation (Figure 45). It could allow to visualize the positionning of light elements such as ER and the female pronucleus at the centripetal pole, the enrichment of denser elements such as mitochondria, lysosomes and endosomes on the centrifugal side, and the broader repartition of yolk granules.

b -Positionning of the MT aster after fertilization

The impact of organelles partitioning on the MT aster centration was then studied, as a function of the centrifugation duration, as well as the position of the sperm entry in the centrifugal pole, middle, or centripetal pole of the egg (Figure 46-B). As detailed in the introduction, the MT aster indeed grows from the sperm head after its entry in the egg and, in normal eggs, this MT aster will move and stop when the aster center reaches the center of the cell.

Here in centrifuged eggs, as shown by the MT aster trajectories, the final position of the MT aster center was not situated at the cell geometrical center, but was shifted towards the region enriched in light elements, whether the position of the sperm entry was at the centrifugal pole, middle, or centripetal pole of the egg (Figure 46-A

c -Impact of centrifugation on the first embryonic cleavage

After fertilization and MT aster centration, the embryos will experience their first cleavage. In normal sea urchins embryos, this first division is symetric, yielding two daughter cells of the same size. In the fertilized centrifuged sea urchin eggs however, the final position of the MT aster was shifted towards the centripetal pole, and the division plane was then seen to also be displaced toward this centripetal pole, where lighter elements were accumulated by centrifugation (Figure 47-A). The daughter cells relative surface areas were quantified, and the daughter cell positioned at the centripetal pole was found to be smaller than the daughter cell positioned at the centrifugal pole. This difference in daughter cells sizes was subtle for eggs centrifuged for ~7 minutes, whereas it was stronger in eggs centrifuged for ~30 minutes, whose egg components had been more powerfully partitioned (Figure 47-B).

Thus, in eggs where organelles were partitioned through centrifugation, the shift of the final MT aster position towards the centripetal pole lead to a shift of the division plane towards this centripetal pole, yielding two asymetric daughter cells.

B -Discussion and perspectives on the in vivo experiments

As described in the introduction, in sea urchins embryos, the MT aster centration after fertilization mainly relies on dynein-dependent forces, scaling with MT length, and pulling on the MTs directly from the cytoplasm [Hamaguchi and Hiramoto, 1986;Tanimoto et al., 2016]. This force exertion mechanism is thought to rely on friction forces associated with the dynein-dependent displacement of vesicles or organelles on MTs in the viscous cytoplasm [Kimura and Kimura, 2011].

Thanks to our minimal in vitro reconstitution, we could show that dyneintransported objects can indeed exert forces on MTs, pulling the MTs in the direction opposite to dynein transport; in bulk. We also wanted to better understand how dynein could pull on MTs from the cytoplasm in vivo, trying to assess which vesicles or organelles could be the main cytoplasmic anchors for dynein force exertion.

In order to affect the organelle force exertion, we carried out centrifugation studies in the sea urchin egg, which partitioned the egg vesicles and organelles, in order to study the impact of these organelles on the MT aster positionning after fertilization. These studies showed a bias of the MT aster positionning towards the centripetal pole of the sea urchins, which is enriched in light elements, and notably in endoplasmic reticulum (ER). This result rules out any important role of denser elements, like lysosomes or mitochondria, and suggests that lighter egg components, such as the ER, might be the main cytoplasmic anchors for dynein force exertion on MTs during aster centration after fertilization.

It would now be very interesting to further test the role of ER as a major cytoplasmic anchor for dynein force exertion, performing experiments that are going to be detailed below, for example focusing on disrupting the ER network, interfering with ER transport, or studying through electron microcopy the ER topology on centering asters.

First, it is important to note that egg partitioning after centrifugation, was not reached exactly to the same extent in all eggs. This might be due to inherent differences along eggs themselves, as well as possible differences in the handmade centrifugation sucrose gradient, carried out by tilting the tubes containing the sucrose solution and the eggs in artificial sea water. While the reproducibility of the sucrose density gradient could be improved, it is impossible to get rid of the variability due to differences between eggs themselves.

An interesting perspective of this centrifugation experiment would be to cut in two halves the centrifuged eggs, so as to obtain a half egg enriched in denser elements, and another half egg enriched in lighter elements (Figure 40), let their content homogenize, fertilize them, and study the MT aster positionning after fertilization. This could provide answers to further determine whether the denser or lighter organelles and vesicles are necessary for the correct sperm aster centration.

However, centrifuging and cutting is a harsh treatment for the sea urchins eggs, which would not always fertilize, and often die, and thus, the attempts I carried out did not allow to yield enough data. To conclude, this centrifugation and cutting protocol necessitates very good quality eggs. Another stimulating perspective in order to affect organelle force exertion, and further test the involvement of the ER, would be to disrupt ER structure, using atlastin mutants or reticulons, and study whether it would impact the MT aster centration after fertilization. Indeed, as detailed in the introduction, the ER is likely to be one of the main cytoplasmic anchor of the pulling force generation mechanism. Reticulons are involved in the control of the high curvature of the ER tubules, and, if overexpressed, were shown to form long and unbranched ER tubules [START_REF] Voeltz | A class of membrane proteins shaping the tubular endoplasmic reticulum[END_REF]. Atlastins are also involved in shaping the ER, and are notably needed for tubules fusion and thus their connection as en ER network [START_REF] Orso | Homotypic fusion of ER membranes requires the dynamin-like GTPase atlastin[END_REF]Hu et al., 2009].

Finally, the role of the ER could be tested by affecting dynein, and test whether it fails to accumulate to the aster center. This could be done by using drugs like Ciliobrevin D, or inject a dynactin dominant negative (the p150cc1 mutant), which would not affect dynein activity directly, but inhibit dynein/dynactin cargo transport on MTs [START_REF] Barbosa | Dynactin binding to tyrosinated microtubules promotes centrosome centration in C. elegans by enhancing dynein-mediated organelle transport[END_REF][START_REF] Mukherjee | The Perinuclear ER Scales Nuclear Size Independently of Cell Size in Early Embryos[END_REF]. A last potential assay, would be to inject dominant negatives of Rab proteins that interfere with ER/dynein connections.

For this, ongoing work in the laboratory also focuses on characterizing the ER accumulation at the aster center along the aster centration after fertilization in sea urchin. Indeed, in previous studies, as well as in the lab, the ER has already been showed to accumulate near the aster center during aster centration, using live microscopy after injection of the ER labeling Dil dye [START_REF] Terasaki | Organization of the sea urchin egg endoplasmic reticulum and its reorganization at fertilization[END_REF].

Immunofixation after aster centration also showed ER accumulation at the aster center, as well as ER alignment on MTs. A more precise observation is then ongoing using electron microscopy, to better understand the topology of ER membranes on asters, their potential connection to MTs and dynein, and potentially estimate drag forces.

Finally, another perspective developed in the laboratory is to characterize the cytoplasmic flows during aster centration, and while the aster is maintained thanks to magnetic pulling, through a technique based on the injection of magnetic beads, which are transported and maintained at the aster center, and allow to displace the MT aster with a magnet. This study on cytoplasmic flows will be very informative, as indeed, as we already mentioned in the introduction, flows are an interesting readout allowing to differentiate between the various force generation mechanisms of MTs pushing, or dynein pulling on MTs from the cortex or from cytoplasmic anchors [Nazockdast et al., 2017].

Concluding remarks

MTs

Purification of cytoplasmic dynein

Recombinant cytoplasmic dynein is expressed in Dictyostelium discoideum, and purified following the protocol described by Kon et al [Kon et al., 2009 o Cells are transferred in a 100mm petri dish with 10 mL HL5 (with tetracycline (12µg/mL) and G418 (10µg/mL)).

o Cells are incubated at 22C° for 24h.

Selection

-The medium is replaced by HL5 medium with tetracycline (12µg/mL), G418

(10µg/mL), and blasticidin S (10µg/ml) for plasmid selection.

-Cells are incubated for approximately a week at 22C°, and the medium is replaced every two days. Transformants colonies become noticeable, and approximately two days later, confluence is reached.

-After confluence is reached, cells have to be diluted approximately every three days at a 1/50 ratio.

Amplification (for a 1L culture)

-Transformed cells are transferred from a sub-confluent 100mm petri dish in a 500ml flask containing 200ml HL5 (with tetracycline (12µg/mL), G418

(10µg/mL), and blasticidin S (10µg/ml)).

-Cells are incubated two to three days at 22C°, 150rpm, until cell density reaches 10 7 cells/ml in a volume of 400 mL (generation time : ~8h)

Induction

Dynein expression is induced through the removal of tetracycline:

-Cells are washed by centrifugation (1,000g, 5min, 21°C), rinsing of the pellet with 100mL HL5, and centrifugation (1,000g, 5min, 21°C)).

-Cells are resuspended in 1L of HL5 (with G418 (10µg/mL) and d-biotin (20µM)

for dynein biotinylation).

-Cells are incubated at 22C° under agitation (150rpm) for one day, until density reaches ≈ 5-10.10 6 cells/ml. This lysate is poured into two plastic falcon tube with ~25mL each inside (glass container would break during the sonication).

Purification

-Sonication: six pulses of 10s, with a 30s pause between each pulses to avoid heating up of the sample.

Clarification

-Centrifugation (24000g, 20 min, 4°C) and collection of the supernatant -Centrifugation of the supernatant (187000g, 60 min, 4°C) and collection of the clarified supernatant

First purification column

-The lysate is gently mixed with 300µL to 2.5mL of pre equilibrated Ni-NTA agarose resin for 1h at 4°C on a rotating wheel.

-The resin is loaded on the column, and the flow through is discarded.

-The resin is washed with 12 column volumes (CV) of wash buffer (100mM PIPES, 0.1mM EGTA, 4mM MgCl2, 20mM imidazole, 0.9M glycerol, 50 mM leupeptin, 1mM mercaptoethanol, 10 mM chymostatin and pepstatin, 0.5mM PMSF, 0.1 mM ATP, pH7), and the flow through is discarded.

-Bound proteins are eluted with 2CV of elution buffer (100mM PIPES, 0.1mM EGTA, 4mM MgCl2, 250mM imidazole, 0.9M glycerol, 50 mM leupeptin, 1mM mercaptoethanol, 10 mM chymostatin and pepstatin, 0.5mM PMSF, 0.1 mM ATP, pH 7)

-The eluate is supplemented with 150mM NaCl, 0.1mM EDTA and 5mM EGTA.

Second purification column

-The eluate is gently mixed with 100-500µL of pre equilibrated AntiFLAG gel

for two hours at 4°C on a rotating wheel.

-The gel is loaded on the column, and the flow through is discarded.

-The gel is then washed with 10CV of PMEGS buffer (100mM PIPES, 5mM -The bound dynein is eluted slowly with PMEG30 buffer supplemented with 0.25mg/mL FLAG peptide.

Cryoconservation

-The eluate is centrifuged (100000g, 10 min, 4°C) and the supernatant is collected.

-The supernatant is mixed at a 10/4 ratio with the cryostorage buffer (2.5M sucrose, 5mM MgSO4, 35mM Tris-HCl, pH 7.2)

-The aliquotes are snap frozen in liquid nitrogen and stored at -80°C.

-The protein concentration can be determined using A280 or Bradford reagent with bovine serum albumin as a standard

3D motility assay

Bead coating with dynein -Streptavidin beads are washed in the dynein assay buffer (10mM K-PIPES, 50mM potassium acetate, 4mM MgSO4, 1mM EGTA, 10mM glucose, 25µM glucose oxydase, 6.4µM catalase, 1mM DTT, 0,4mg/mL casein, 40µM docetaxel, 1mM ATP, pH 7.0) three times in Lobind microtubes (Sigma). A wash includes the mixing of beads in buffer, followed by a 2 minutes sonication, a centrifugation (10,000g, 4min, 4°C) and the resuspension of the pellet in buffer.

o The Lobind microtube is changed for each wash, as it was noticed that beads would have a tendency to stick to the microtube if the microtube was used for more than one centrifugation.

-Beads are then incubated with biotinylated dynein for ~1-3h in the cold room with gentle agitation (incubation with more than 50 fold the beads binding capacity).

o Beads used were 0.5µm (Bangs laboratory), 1µm (Bangs laboratory or Dynabeads from ThermoFisher), 2.2µm (Polysciences), 2.8µm (ThermoFisher) and 3µm (Bangs laboratory).

-When beads are not already fluorescent, they can be labelled with 0.001 fold the bead binding capacity with ATTO 565-biotin after the dynein coating step.

3D motility assay experiment

-The equivalent of approximately 0.005µL dynein-coated beads are centrifuged (10,000g, 6min, 20°C) and re-suspended in ~3µL of 1.18% or 1.60% MC dynein assay buffer at 20°C supplemented with 12.5-25nM stabilized labelled

MTs.

-This mix is flowed in a hand-made chamber. The chamber consists of a coverslip, where parafilm strips are positioned, and topped with coverslips strips cut with a diamond pen. The parafilm were stuck to the coverslips through heating on a plate. Chambers thus have a width of 2mm, internal height of ~200µm, length of 0,5cm, resulting in a volume of ~3µL.

-The chamber is then sealed with VALAP, in order to avoid evaporation of the chamber content.

-Image MT aster centration (DIC and fluorescence, 30s or 60s frame interval)

PDMS chamber

Double or one sided strips of adhesive were put onto a glass coverslip in a Petri dish, and PDMS was poured inside the Petri dish. Rectangular chambers were cut, around the places were double sided adhesive was put. The chambers were then punched at their two ends, and stuck on a clean coverslip. The two holes would allow to flow the sea urchin eggs inside the chamber. The height of the chamber corresponds to the height of the adhesive used. Thickness of double sided and one sided tape are respectively 95µm and 55µm. 55µm high chambers will better immobilize centrifuged eggs, as their shape is elongated and not round as normal sea urchins eggs.

Figure 1 :

 1 Figure 1: Scale representation of an actin filament, IF and MT. Contrary to IFs, actin and MTs are polar filaments, and their respective pointed and barbed end and plus and minus ends are represented[START_REF] Pollard | Overview of the Cytoskeleton from an Evolutionary Perspective[END_REF]

Figure 2 :

 2 Figure 2: Representation of the three steps construction of an IF. Filaments first assemble laterally, then longitudinally, and finally compact laterally[START_REF] Herrmann | Intermediate Filaments: Structure and Assembly[END_REF].

Figure 3 :

 3 Figure 3: Representation of an actin monomer as a Ribbon diagram (left side) and as a space-filled model (right side). The four actin subdomains are numbered on the Ribbon diagram; and nucleotide binding cleft and barbed end side of the monomer are shown on the space filled model. Adapted from[START_REF] Pollard | Actin and Actin-Binding Proteins[END_REF].

Figure 4 :

 4 Figure 4: Scheme of the nucleotide state dynamics in an actin filament polymerised from an ADP-actin seed.Quickly upon assembly, ATP-actin is randomly hydrolyzed into ADP-Pi actin, after which the inorganic phosphate leaves the filament at a much slower rate. Adapted from[START_REF] Pollard | Actin and Actin-Binding Proteins[END_REF]

Figure 7 :

 7 Figure 7 : MT nucleation from γ-TuRCs. γ-TuSCs are composed of two γ-tubulins (shown in beige) and two GCP proteins (shown in blue), and are associated as a γ-TuRC. (A) γ-TuSCs are associated via their GCP proteins. (B) Tubulin dimers assemble on the γ-tubulin of the γ-TuRC. (C) Tubulin assembly goes on, reaching in (D) the minimum size at which assembly becomes more favorable than disassembly. (E) When the MT exceeds this minimum size, quick MT polymerisation can take place[START_REF] Tovey | Microtubule nucleation by γ-tubulin complexes and beyond[END_REF].

Figure 8 :

 8 Figure 8: Scheme representing the promotion of MT nucleation thanks to γ-TURCs (A), MAPs (B) or both (C).[[START_REF] Tovey | Microtubule nucleation by γ-tubulin complexes and beyond[END_REF].

Figure 10 :

 10 Figure 10: Conformation change of tubulin upon hydrolysis and phosphate release. The E-site compaction upon phosphate release brings about filament bending, creating lattice instability. Taxol binding reverses this conformation change, stabilizing the lattice[START_REF] Alushin | High resolution microtubule structures reveal the structural transitions in αβtubulin upon GTP hydrolysis[END_REF].

Figure 13 :

 13 Figure 13: Scale representation of the three cytoskeletal motors: (A) myosin, (B) kinesin and (C) dynein (respectively myosin V, kinesin-1 and cytoplasmic dynein)[START_REF] Carter | Crystal clear insights into how the dynein motor moves[END_REF].

Figure 15 :

 15 Figure 15: Representation of kinesin-1. Motor domains are depicted in blue, light chains and tail domains, involved in cargo attachment, are shown respectively in green and purple. Adapted from[START_REF] Vale | The Molecular Motor Toolbox for Intracellular Transport[END_REF]

Figure 16 :

 16 Figure 16: The structure of cytoplasmic dynein 1. (A) Linear representation of dynein heavy chain functional and structural domains. The six AAA units are shown in different colors. Color code of different domains is coherent within all the figure, as well as in Figure 17. (B) Two dynein heavy chains are shown dimerised, with their AAA unit forming a ring, and the intermediate, light and light intermediate chains which can bind to the tails. (C) 3D representation of the dynein motor binding to a MT tubulin dimer[START_REF] Roberts | Functions and mechanics of dynein motor proteins[END_REF].

Figure 17 :

 17 Figure 17: Representation of the cytoplasmic dynein 1 mechanochemical cycle. The color code used to depict different parts of the dynein heavy chain is the same as in the previous figure. This mechanochemical cycle allows dynein to advance on the MT, and apply force to the object attached to its tail, pulling it toward the MT minus end.Adapted from[START_REF] Cianfrocco | Mechanism and Regulation of Cytoplasmic Dynein[END_REF]

Figure 18 :

 18 Figure 18: Representation of possible dynein stepping motions on MTs: (A) hand over hand mechanism and (B) inchworm fashion. Adapted from [Reck-Peterson et al., 2012].

Figure 21 :

 21 Figure 21: Representation of the in vitro set up where two MKAC coated beads could capture the ends of a MTs.(A) When one bead was released, the depolymerizing kinesin coated bead would lead to the shortening of the MT. (B) When the two beads were kept in the optical trap, the depolymerisation activity of the kinesin coated on the beads would pull on the MTs, exerting a force which could be assessed through the measure of the beads displacement in the traps. Adapted from[START_REF] Oguchi | The bidirectional depolymerizer MCAK generates force by disassembling both microtubule ends[END_REF].

Figure 23 :

 23 Figure 23: Representation of the set up allowing to study aster centering based on MT pushing forces.After the first aster positionning through pushing forces inside the chamber, the aster center bead could be moved with an optical tweezer, and aster repositioning could be studied, in presence or absence of a MT catastrophe promoter[START_REF] Faivre-Moskalenko | Dynamics of microtubule asters in microfabricated chambers: The role of catastrophes[END_REF].

Figure 24 :

 24 Figure24: Representation of dynein dimers as auto inhibited phi particles, or dynactin activated processive dynein dimers[Zhang et al., 2017]

Figure 25 :

 25 Figure25: Representation, on the left side, of a dynein dimer linked to dynactin and BICD2. On the right side are represented two dynein dimers, linked through dynactin and BICDL1 (BICD family-like cargo adapter 1). Both BICD2 and BICDL1 activate dynein, and they respectively preferentially recruit one or two dynein dimers[START_REF] Reck-Peterson | The cytoplasmic dynein transport machinery and its many cargoes[END_REF].

Figure 26 :

 26 Figure26: Representation of the phagosome transport by kinesins and dyneins. Before dynein clustering, kinesin and dynein exert similar amount of forces (respectively represented by the red and green arrows). After clustering of dyneins, they can collectively exert more force than kinesin, and thus win the tug of war and can persistently transport the cargo toward MT minus end[Rai et al., 2016].

Figure 27 :

 27 Figure27: Representation of dynein involvement in transport. The legend details the objects transported by dynein, and specifies, in parenthesis, the adaptors enabling dynein to connect each cargo[START_REF] Reck-Peterson | The cytoplasmic dynein transport machinery and its many cargoes[END_REF]

Figure 28 :

 28 Figure 28: scheme of the dynein/dynactin recruitment to the cortex during metaphase. NuMA can bind to dynein and LGN, which is recruited to the cortex by Gαi, and can interact with the cortex proteins Dlg, Afadin [di Pietroet al., 2016]

Figure 29 :

 29 Figure 29: Scheme representing dynein pulling from the cortex. Dynein is recruited to the cortex by NuMA/LGN/Gαi, but its recruitment is inhibited by proximity of the metaphase plate, as well as the proximity of the Plk1 decorated spindle poles, causing spindle pole oscillations [McNally, 2013].

  part detailed the force generation from dynein at the cortex, other MT-dependent force generation mechanisms exist, such as pushing forces from MT polymerisation, and dynein force generation on MTs from the cytoplasm, as already mentioned in the chapter on in vitro characterization of MT-and motordependent force generation mechanisms.This part will thus detail centration models, especially focusing on dyneindependent force generation in bulk cytoplasm, and the model of MT aster centration in large embryos such as in the sea urchin zygote.

Figure 31 :

 31 Figure31: Representation of the MT laser ablation experiment in sea urchin embryos. When MTs are cut parallel to the centration axis, the MT is displaced toward the aster side where MTs are longer, then cut MTs regrow, and the aster will thus center normally again. When MTs are cut at the aster front, the MT aster goes back and then MTs re grow and the aster can center normally again. Adapted from[START_REF] Xie | Cytoskeleton Force Exertion in Bulk Cytoplasm[END_REF].

Figure 32 :

 32 Figure 32: Representation of the first experiment showing dynein force exertion on MTs from cytoplasm. (A) A sand dollar egg is fertilized, and the presence of colcemid prevents MT polymerisation. (B) Colcemid is then deactivated through UV light in a specific zone, where MTs can thus regrow. (C) When this zone is moved, the asymetric aster is able to center in this zone, whether the zone touches the cortex or not. Adapted from[Hamaguchi and Hiramoto, 1986].

Figure 33 :

 33 Figure 33: Local MT depolymerisation in two cell stage zebrafish blastomeres. First, cells are incubated in Combrestatin 4A. Then, UV light is applied locally in one side of the cell (depicted in orange), activating the Combrestatin 4A depolymerisation activity, and thus causing a local MTs depolymerisation. Asters are consequentially displaced toward the long MT side, prior to any MT contact with the cortex. Adapted from[START_REF] Xie | Cytoskeleton Force Exertion in Bulk Cytoplasm[END_REF].

Figure 34 :

 34 Figure 34 : Experimental and simulation based trajectories of MT asters in deformed eggs. Eggs were placed in microfabricated wells of various shapes after fertilization, and the MT aster trajectory was tracked[Tanimoto et al., 2016].

Figure 35 :

 35 Figure 35: scheme of spindle positionning in S. pombe through MTs pushing forces. iMTOCs are interphase MTOCs[START_REF] Tran | A Mechanism for Nuclear Positioning in Fission Yeast Based on Microtubule Pushing[END_REF].

Figure 37 :

 37 Figure 37: Representation of the cytoplasmic pulling model, where friction force of the dynein transported cargoes (Fdrag) exerce length-dependent forces on MTs.As MTs can grow longer on the side opposite to the fertilization entry, more dynein transported cargo will be recruited to these MTs, exerting a total asymetric force (Fnet), enabling the aster to center[Longoria and Shubeita, 2013].

Figure 38 :

 38 Figure 38: Representation of the friction force exerted on a vesicle transported by dynein on a MT. The friction force applied to the vesicle is directed on the opposite direction of the vesicle velocity Adapted from[Longoria and Shubeita, 2013].

Figure 1 :

 1 Figure 1: A 3D motility assay reconstituting dynein pulling on MTs in bulk.

Figure 2 :

 2 Figure 2: Impact of cargo size on MT bulk pulling efficiency.

Figure 3 :

 3 Figure 3: Collective effect of two beads pulling on a single MT

Figure 4 :

 4 Figure 4: Beads pulling on MTs in bulk can move a minimal "aster" in 2D

(

  and not aggregates) with a z stack acquisition at the end of the movie. Beads centers were tracked with the ImageJ manual tracking tool. Mean Square Displacements (MSD) were then plotted as a function of the time, and the diffusion coefficient D was deduced from the slope of the fit. This allowed to compute a diffusion coefficient, from the relation 2nD = 𝑀𝑆𝐷 ∆𝑡 , with n the number of dimensions (here n = 2 as we are measuring the MSD in 2D). The viscosity felt by the beads could then be calculated, using the Stokes-Einstein equation, for diffusion of spherical particles in a liquid at low Reynolds number: µ = 𝑘𝑇 6𝜋𝑟𝐷

Figure S2 :

 S2 Figure S2: Experimental determination of medium viscosity, comparison of kinesin and dynein coated beads velocities on MTs, and independence of the

Figure S3 :

 S3 Figure S3: Theoretical longitudinal drag coefficient of a cylinder, and method to build a minimal artificial aster. Related to Figure 3 and 4.

Figure S4 :Part 2 :

 S42 Figure S4: Hydrodynamic coupling between a MT and bead moving in its vicinity. Related to Figure 1 to 4.

Figure 40 :

 40 Figure 40: Experimental measurement of the ratio of the longitudinal MT drag to the bead drag, in the viscous medium used for the 3D motility assay. (A) Sketches representing the experiment. (B) Example of the displacement of the MT-bearing bead in the optical trap when a force was applied to it through a flow generated by moving the stage. The first equilibrium position corresponds to no force application. Then a force is applied to the trapped bead, and a second equilibrium position is attained after a couple of seconds. Then, the force application stops, and the bead thus come back to its first equilibrium position. (C) The theoretical drag ratio, based on the Stokes formula and longitudinal drag of a cylinder in a Newtonian medium, is shown in dark green, along with the measured drag ratio, shown in pale green. Dashed lines are linear fits. (D) MT-to motor speed efficiency ratio plotted as a function of the ratio of MT length to bead radius. A fit of the data is shown as a black line, the behaviour based on the measured drags is shown as a medium grey dashed line, and the theoretical behavior in a Newtonian fluid is shown as a pale grey dashed line. The fit is done based on the equation x=1/(1+αy), which come from the system force balance equation, with x=-VMT/v*motor, and y= LMT / Rbead, where VMT is the MT velocity, v*motor is the velocity of the motor on the MT, and LMT / Rbead are the MT length and the bead radius.

Figure 41 :

 41 Figure 41: Representation of different way to complicate the minimal aster conformation, with symetric or asymetric asters, in homogeneous or inhomogeneous dynein coated beads environments.

Figure 42 :

 42 Figure 42: representation of the strategies to build minimal artificial asters. Techniques tried relied on linking stabilized MTs grown from biotinylated seeds with neutravidin or streptavidin beads, or on building asters with dynein coated beads linking stabilized MTs minus ends.

Figure 43 :

 43 Figure 43: Viscosity assessment in the sea urchin embryo. (A) Magnet force calibration: overlay of fluorescent nonmagnetic (red) and magnetic (green) beads placed in a medium of known viscosity and attracted by a magnet. (B) Magnet force calibration: plot of the medium viscosity multiplied by the velocities of the beads placed in viscous media as a function of their distance to the magnet. The different data points colors account for different medium viscosities used. (C) Overlay of a fluorescent magnetic bead injected in a sea urchin embryo and attracted by a magnet. (D) Plot of the velocities of the beads injected in fertilized sea urchin eggs, as a function of their distance to the magnet. The magnet center was placed less than 100µm (dark blue) or less than 250µm (pale blue) from the sea urchins eggs. (E) Frequency diagram of the assessed viscosity, respectively in fertilized or unfertilized sea urchins eggs.

Figure 44 :

 44 Figure 44: The sea urchin egg centrifugation partitions its components as a function of their densities. (A) Scheme representing the sea urchin eggs centrifugation protocol. Eggs in artificial sea water are softly deposited above an equal volume of 0.85M sucrose in a microtube. The microtube is then tilted in order to establish a sucrose gradient, and centrifuged so as to partition the egg components as a function of their density. (B) Egg stratification profile after ~7min centrifugation. (C) Egg stratification profile after ~30min centrifugation. (B&C) The egg stratification is determined from DIC live images, as well as live imaging of labelled vesicles and organelles, and immunofixations, as shown in Figure 45.

Figure 45 :

 45 Figure 45: Sea urchin egg components stratification after egg centrifugation observed with live probes and immunofixation. (A) Observation of organelles and vesicles stratification after ~7min centrifugation. (A1) DIC image (A2) Endosomes (bright vesicles) and ER (blurred shadow) labelled with FM1-43 dye (A3) Mitochondria labelled with Mitotracker (A4) Lysosomes labelled with Lysotracker (A5) Yolk granules labelled with Nile Blue (B) Observation of organelles and vesicles stratification after ~30min centrifugation. (B1) DIC image (B2) ER stained with anti KDEL after immunofixation (B3) Mitochondria DNA and nucleus DNA labelled with Hoechst (B4) Yolk granules labelled with Nile Blue.

  ). The shift of the final MT aster position toward the centripetal pole was more important for the eggs centrifuged for ~30min than for the eggs centrifuged for ~7min (Figure46-B,C). In contrary, the average of the instantaneous velocities of the MT aster center were not different as a function of the sperm entry position, or the duration of the centrifugation (Figure46-D). The shift of the final MT aster position towards the centripetal pole after the partitioning of the egg components induced by centrifugation, suggests that lighter egg components enriched at the centripetal pole, such as ER, might be the main organelles, which, transported by dynein on the aster MTs, exert pulling forces in bulk to center asters after fertilization.

Figure 46 :

 46 Figure 46: MT aster trajectories in centrifuged sea urchins eggs. (A) Examples of MT aster trajectories after fertilization of eggs centrifuged for ~30 min. The end of the MT aster trajectory is shifted toward the centripetal pole, after sperm entry either at the centrifugal pole, middle or centripetal pole of the egg. (B) Scheme of the different sperm entry parts labelling, as well as the measure of the distance from the cell geometrical center to the sperm aster position at the end of its trajectory. If the MT aster position lies into the centripetal half, the distance value is negative. (C) Quantification of the distance from the cell geometrical center to the MT aster center at the end of its trajectory. A negative value accounts for a MT aster final position on the centripetal side. Values are expressed as a percentage of the egg long diameter (C) Quantification of the average of MT aster instantaneous velocity, expressed in µm/min.

Figure 47 :

 47 Figure 47: The sea urchin egg centrifugation causes the embryo to divide asymmetrically at the first cell stage (A) Timelapse of an embryo division after a ~30 min centrifugation of the egg. In this condition, the division plane, normally positioned at the cell mass center, is shifted toward the centripetal pole. It yields asymetric daughter cells: one smaller daughter cell, on the embryo side where lighter elements were enriched through egg centrifugation; and a bigger daughter cell, on the embryo side where denser elements were enriched through egg centrifugation. (B) Quantification of the relative surface area of the light daughter cells after the first embryonic cleavage in centrifuged eggs. The light daughter cell surface area is expressed as a percentage of the sum of the surface areas of the two daughter cells.

Figure 48 :

 48 Figure 48: DIC image of a centrifuged and cut egg. After centrifugation, eggs are deposited on a protamine coated dish, and cut in their middle with an elongated glass needle.

  -dependent forces are essential for various essential mechanisms, such as chromosome segregation, and positionning of MT asters and spindles. MTs can exert pushing forces based on MT polymerisation, as well as pulling forces generated by cortically anchored motors or by the capture of depolymerizing MTs, and pulling forces mediated by the transport of dynein cargoes on MTs in the viscous cytoplasm.The goal of this work was to better understand how dynein could indeed exert such pulling forces on MTs directly from the cytoplasm, without anchoring to the cell cortex. This process is of great interest, as it is notably at the core of the aster centration process after fertilization in large embryos.The in vitro reconstitution we developed allowed us to show that dynein coated objects moving on MTs in a viscous medium could indeed exert forces on MTs, displacing single MTs and minimal asters. The reconstitution of this force generation mechanism is of particular interest, as in vitro studies did not yet characterize it, in contrast to pulling forces generated by anchored motors or by MT depolymerisation, as well as pushing forces generated by MT polymerisation, which were already extensively studied in vitro.The centrifugation experiments we carried out in sea urchins eggs induced the partitioning of the egg components, and shifted the positionning of the MT aster toward the centripetal pole of the embryo, suggesting that lighter organelles, such as ER, might support an important part of the cytoplasmic pulling forces.Fascinating perspectives of this work include more complex in vitro reconstitution of dynein pulling on MT asters from the friction forces associated with dynein-dependent organelle transport, notably focusing on reconstituting an ER-like interconnected network. Perspectives also includes studies in the sea urchin embryo, where, for example, cytoplasmic flow analysis, as well as experiments affecting dynein-dependent organelle traffic, would continue to shed light on this captivating force generation mechanism, and participate in identifying the major cytoplasmic anchor.

  . The HFB380 380 kDa recombinant dynein was engineered from the Dictyostelium discoideum cytoplasmic dynein heavy chain gene. Affinity tags were introduced (His6 and FLAG) at the N-terminus as well as a N-terminal BioEase tag for in vivo biotinylation. Glutathione S-transferase (GST) could also be added for dynein dimerization. Finally, a MB35 plasmid (ID 44 -Dicty Stock Center) coding for a transcriptional activator controlled by tetracycline was added in order to control the recombinant protein overexpression. When cells are mentioned to be counted, it is done through the use of a Malassez cell. Transformation -Cells are cultivated at 21°C in culture dishes in HL-5 medium (with tetracycline (12µg/mL), G418 (10µg/mL)) until confluence is reached. -Transformation: o Cells are pelleted by centrifugation (500g, 5min, 4C°) o Cells are resuspended in H50 electroporation buffer (20mM HEPES, 10mM NaCl, 50mM KCl, 1mM NaH2PO4, 5mM NaHCO3, 1mM MgSO4, pH 7.0.) and pelleted again (500g, 5min, 4C°) o Cells are resuspended in 100µl of H50, and mixed with 1µg of MB38based plasmid containing the engineered dynein gene. o The mix is transferred in an electroporation cuvette (previously kept on ice) (2mm gap cuvette) o Cells are eletroporated in a Bio-rad gene pulser using "dicty" program (Fungi => Dic) and incubated on ice during 5min.

-

  Cells are pelleted (centrifugation 1320g -5 min -4°C) -The supernatant is removed, and cells are resuspended in half of the culture volume of cold KPi phosphate buffer.-Cells are pelleted again (centrifugation 1320g -7 min -4°C), and supernatant is removedCell lysis-The cell pellet is resuspended in the same volume of Lysis Buffer (100 mM PIPES-KOH, 0.1 mM EGTA, 4 mM MgCl2, 10 mM imidazole, 0.9 M glycerol, 50 mM leupeptin, 1mM mercaptoethanol, 10 mM chymostatin and pepstatin, 0,5mM PMSF, 0.1 mM ATP, pH 7.0) (V = volume of the cell pellet ≈ 40 mL).

TABLE Reagent or resource Source Identifier Bacterial and virus strains

  Sketch representing the bead surface gliding assay. Biotinamidocaproyl BSA is flowed in a microscopy chamber. Streptavidin beads coated with biotinylated dynein are then incubated in the chamber, and finally stabilized MTs are flowed in and can freely glide on dynein-coated beads anchored to the surface. (see STARMethods).

	MES (4-Morpholineethanesulfonic acid) PIPES (1,4-Piperazinediethanesulfonic acid) MgCl2 ATP (Adenosine 5′-triphosphate) GTP HEPES EGTA 1µm beads Dynabeads (streptavidin coated) ThermoFisher Sigma-Aldrich Sigma-Aldrich Sigma-Aldrich Sigma-Aldrich Sigma-Aldrich Sigma-Aldrich Sigma-Aldrich 1,9µm beads Bangs (streptavidin coated) Polysciences 2,2µm beads Bangs YG (biotin coated) Polysciences 2,8µm beads Dynabeads (streptavidin coated) ThermoFisher 3µm beads Bangs (streptavidin coated) Bangs laboratory HL5 medium Formedium LoBind eppendorf microcentrifuge tubes (A) (B) Time-lapse of a stabilized MT gliding on three anchored dynein-coated beads, in M8250 P6757 M2670 A2383 G8877 H3375 E3889 65001 24160 24173 65305 CP01N HLF2
	DTT 0,5mL a typical bead gliding assay experiment.	Euromedex Sigma	EU0006-D Z666491
	MgSO4 (Magnesium sulfate) CH3COOK (potassium acetate) (C) Average velocity of MTs gliding on a dynein-coated beads gliding assay Sigma-Aldrich 208094 Prolabo 26667.293
	Tris (Tris(hydroxymethyl)aminomethane) (Dynabeads, 1µm diameter) or a conventional dynein gliding assay, both in dynein Euromedex 200923-A
	EDTA Leupeptin assay buffer. N=42 MTs (bead gliding assay) and 29 MTs (gliding assay). Error bars Euromedex EU0007-C Sigma-Aldrich L2884 PMSF Roche 10837091001 represent standard deviation.
	Chymostatin (D) Table showing all viscous agents tried: sucrose, glycerol, PEG (molecular weight Sigma-Aldrich C7268
	Pepstatin Mercaptoethanol 20000 g/mol) and MC (molecular weight 41000 g/mol), and their potential inhibition Sigma-Aldrich P5318 Sigma-Aldrich M6250
	ATP-Mg of dynein ATPase activity and motility (from our gliding assays MTs speeds, or from Sigma-Aldrich A9187
	Imidazole KH2PO4 the literature) and their tendency to generate MT bundles when supplemented to our Merck 1.04716.1000 Sigma-Aldrich P0662 K2HPO4 Sigma-Aldrich P3786 dynein assay buffer.
	NaCl	VWR Chemicals		27810.295
	KCl	VWR Chemicals		26668.296
	NaH2PO4	Sigma-Aldrich		71505
	NaHCO3	VWR Chemicals		27775
	Biotin	Sigma-Aldrich		B4501
	Blasticidin	Sigma-Aldrich		15205
	G418	Sigma-Aldrich		A1720
	Tetracycline	Sigma-Aldrich		87128
	Kanamicin	Euromedex		UK0010-D
	Rosetta (DE3)pLysS E.coli Chloramphenicol MB35-integrated Dictyostelium discoideum FLAG	Sigma-Aldrich Euromedex [39] Sigma-Aldrich	3886-C 71401 IK40 F3290
	Recombinant DNA Biological samples Pig brain tubulin HFB380 dynein Dictyostelium discoideum heavy HFB GST380 SNAP2 dynein heavy chain from chain from Carsten Janke lab, Curie Orsay [40]	N/A	N/A
	Dictyostelium discoideum Chemicals, Peptides, and recombinant proteins ATTO-565 NHS-ester Mouse kinesin (pET28-mKif5B_N1665)	[21] Carsten Janke lab, Curie ATTO-TEC GmbH AD 565-35 N/A Orsay Uniprot: Q61768
	ATTO-565-biotin Methylcellulose (MC41000) Others Glycerol Ni-NTA agarose beads Sucrose Anti-FLAG beads Polyethylene Glycol (PEG 20000) NAP-5 column Docetaxel 0,5µm beads Bangs (streptavidin coated) Glutamate 1µm beads Bangs (streptavidin coated)	Sigma-Aldrich Sigma-Aldrich VWR Chemicals Invitrogen VWR Chemicals Sigma Sigma-Aldrich GE Healthcare Sigma-Aldrich Bangs laboratory Sigma-Aldrich Bangs laboratory	92637 M0262 R901-15 24388.295 F2426 27480.294 17-0853-01 95172 CP01F 1885 G1251 CP01F

Table of

 of

	viscosity assessment through diffusion coefficient determination for 1µm
	and 2.8µm beads (Dynabeads), and 1.18% and 1.60% methylcellulose (MC) dynein
	assay buffers (thus elsewhere named 39cP and 124cP MC viscous buffers).

  EGTA, 4mM MgCl2, 0.9M glycerol, 0.1mM EDTA, 200mM NaCl, 50 mM leupeptin, 1mM DTT, 10 mM chymostatin and pepstatin, 0.5mM PMSF, 0.1 mM ATP, pH 7) and 10CV of PMEG30 buffer (30mM PIPES, 5mM EGTA, 4mM MgCl2, 0.9M glycerol, 0.1mM EDTA, 50 mM leupeptin, 1mM DTT, 10 mM chymostatin and pepstatin, 0.5mM PMSF, 0.1 mM ATP, pH 7).

ACKNOWLEDGMENTS

We gratefully thank Carsten Janke from Curie -Orsay for providing us with porcine brain tubulin, and the mouse kinesin plasmid. We also thank Bérengère Guichard for her valuable technical help, and Dmitry Ershov and Serge Dmitrieff for their help with software and statistical analysis, and all the members of the teams "Cellular spatial organization" and "Regulation of actin assembly dynamics" for fruitful discussions.

We also thank our colleagues A. Guichet, J. Heuvingh and O. Du Roure for carefully reading the manuscript. This work was supported by the Centre National de la Recherche Scientifique (CNRS), the European Research Council (ERC CoG Forcaster no. 647073) to N.M., (ERC StG BundleForce no. 679116) to A.J, and Japan Society for the Promotion of Science KAKENHI (18K06147 and 19H05379) to T.S. H.P. acknowledges the "École Doctorale FIRE -Program Bettencourt" and a fellowship from the "Fondation pour la Recherche Médicale" (FRM, FDT201904008226).

These data thus show that dynein transported objects can exert forces on MTs, away from any surfaces and fix anchoring point, causing subsequent MT motions. This MT-dynein force generation mechanism could be relevant in cells, in aster centration mechanism, as explained above, as well as other processes, such as MT transport to the periphery of the aster, supporting aster growth.

The results are presented below, in the form of the paper we submitted to Current Biology, and which is now in revision.

IN VITRO RECONSTITUTION OF DYNEIN FORCE EXERTION IN BULK CYTOPLASM

(in revision at Current Biology) Héliciane Palenzuela 1 , Benjamin Lacroix 1 , Jérémy Sallé 1 , Katsuhiko Minami 2 , Tomohiro Shima 2 , Antoine Jegou 1 , Guillaume Romet-Lemonne 1,# and Nicolas Minc 1,#, § 1 Université de Paris, CNRS, Institut Jacques Monod, 75013 Paris, France 2 Graduate School of Science, The University of Tokyo, 113-0033 Tokyo, Japan # Co-Corresponding authors: NM: nicolas.minc@ijm.fr; GR: romet@ijm.fr § Lead contact: nicolas.minc@ijm.fr

SUMMARY

The forces generated by Microtubules (MTs) and their associated motors orchestrate essential cellular processes ranging from vesicular trafficking to centrosome positioning [START_REF] Dogterom | Force generation by dynamic microtubules[END_REF][START_REF]Mechanics of Motor Proteins and the Cytoskeleton[END_REF]. To date, most studies have focused on force exertion from motors anchored on a static surface, such as the cell cortex in vivo or glass surfaces in vitro [START_REF]Mechanics of Motor Proteins and the Cytoskeleton[END_REF][START_REF] Laan | Cortical Dynein Controls Microtubule Dynamics to Generate Pulling Forces that Position Microtubule Asters[END_REF][START_REF] Grill | Polarity controls forces governing asymmetric spindle positioning in the Caenorhabditis elegans embryo[END_REF]. However, motors also transport large cargos and endomembrane networks, whose hydrodynamic interactions with the viscous cytoplasm should generate sizable forces in bulk. Such forces may contribute to MT aster centration, organization and orientation [5][START_REF] Kimura | Intracellular organelles mediate cytoplasmic pulling force for centrosome centration in the Caenorhabditis elegans early embryo[END_REF][START_REF] Minc | Influence of cell geometry on division-plane positioning[END_REF][START_REF] Tanimoto | Shape-motion relationships of centering microtubule asters[END_REF][START_REF] Wuhr | A model for cleavage plane determination in early amphibian and fish embryos[END_REF][START_REF] Ishihara | Microtubule nucleation remote from centrosomes may explain how asters span large cells[END_REF][START_REF] Cheng | Spontaneous emergence of cell-like organization in <em>Xenopus</em> egg extracts[END_REF][START_REF] Xie | Cytoskeleton Force Exertion in Bulk Cytoplasm[END_REF][START_REF] Barbosa | Dynactin binding to tyrosinated microtubules promotes centrosome centration in C. elegans by enhancing dynein-mediated organelle transport[END_REF][START_REF] De Simone | Uncovering the balance of forces driving microtubule aster migration in C. elegans zygotes[END_REF], but have yet to be evidenced and studied in a minimal reconstituted system. By developing a bulk motility assay, based on stabilized MTs and dynein-coated beads freely floating in a viscous medium away from any surface, we demonstrate that the motion of a cargo exerts a pulling force on the MT and propels it in opposite direction. Quantification of resulting MT movements for different motors, motor velocities, over a range of cargo size and medium viscosities, shows that the efficiency of this mechanism is primarily determined by cargo size and MT length. Forces exerted by cargos are additive, allowing us to recapitulate tug-of-war situations, or bi-dimensional motions of minimal asters. These data also reveal unappreciated effects of the nature of viscous crowders and hydrodynamic interactions between cargos and MTs, likely relevant to understand this mode of force exertion in living cells. This study places endomembrane transport as a significant mode of MT force exertion with far-reaching consequences for cellular organization.

AUTHOR CONTRIBUTIONS

Conceptualization, N.M., G.R.L. A.J. and H.P.; Methodology, T.S., J.S. and B.L. and H.P..; Investigation, N.M., G.R.L., A.J., K.M., T.S., J.S., B.L. and H.P.; Writing -Original Draft, N.M., G.R.L and H.P. Draft Editing N.M., G.R.L., A.J., K.M., T.S., J.S., B.L. and H.P.

DECLARATION OF INTERESTS

The authors declare no conflict of interest.

Acquisition

The microscopy chamber was placed on a Nikon EclipseTi or Nikon Eclipse TE2000-U inverted microscopes, using a 60x oil-immersion objective, with epifluorescence illumination. Microscopes were controlled through micromanager. The Nikon EclipseTi and Eclipse TE2000-U microscopes were illuminated with a Lumen Dynamics lamp (respectively X-Cite-Exacte and X-Cite-Series 120 Q) and movies were acquired by a Hamamatsu digital camera C11440 (respectively ORCA-Flash 4.0 and ORCA-Flash 4.0 LT plus)

QUANTIFICATION AND STATISTICAL ANALYSIS

Specific experiments and analysis

Measurements were done manually using ImageJ data, and were treated using Microsoft Office Excel, Matlab and GraphPad Prism.

Classical gliding assay and bead gliding assay

In order to measure the MTs velocity on the dynein gliding assay, or the dyneincoated beads gliding assay, kymographs were constructed based on broken lines drawn along gliding MTs. Angles of MTs displacement were measured on the kymograph, and velocities were deducted from them.

3D motility assay: motility of one or several beads on one MT

The analysis was carried on ImageJ: events were checked for persistent movements and constant velocity on kymographs, and distances travelled by the MT and the bead were measured on the movie (as shown in Figure S1). In addition to brownian motion, moderate local fluid flows in the microchamber added some noise to our measurements. MT length was measured as the length of a broken line following the MT from its minus end to its plus end, on one frame of the movie, or, when the MT was not in perfect focus trough the time-lapse, on a small z stack done at the end of the movie.

-Reconstitution of dynein pulling on MTs in bulk under confinement

In order to get even closer to a cellular context, this experiment could also be conducted under confinement, with dynamic MTs growing from an artificial MTOC, which would be placed in microchambers, or encapsulated into large lipid vesicles, and motors linked to an interconnected ER like network, which could pull on these MTs.

Very interestingly, when a MT aster would start growing on a MTOC at the periphery of the confined zone, MTs would be allowed to grow longer on one side of the aster, as observed after fertilization in vivo. Thus, more dynein would be recruited on these longer MTs, and would presumably displace the MT aster center towards the confined zone center.

However, a drawback of this reconstitution under confinement, would be the difficulty to assess if MT pushing through polymerisation against the borders of the confined zone was also involved in the aster motion.

Abbreviations list

Annexes

The annex section will detail three specific protocols used during this work: the purification of cytoplasmic dynein, the 3D motility assay, and the centrifugation of sea urchins eggs.

-Events where a bead encountered a MT, which was away from the surface (>10µm from the surface) and almost parallel to the surface are recorded.

Handling of beads with the optical trap, forcing bead and MTs encounters

-Beads can be handled with the optical trap, in order to force the encounter of one bead on a MT, place two beads on a MT, or even build minimal asters -When placing the bead on the MT, care can be taken in order to place the bead on the MT in a way that positions the MT horizontally in the chamber, leading to the positionning of the MT and the bead at the same focal plane.

-It is possible to pull on a bead that had reached the MT minus end, in order to use it again on the same or another MT.

Centrifugation of sea urchins eggs

The protocol consisted in the centrifugation of the sea urchin eggs on a sucrose gradient, and then their imaging on protamine coated dishes, or channels on coverslips topped with PDMS chambers.

Establishing the centrifugation sucrose gradient

All solutions have to be at ~15°C.

-A 0.85M sucrose solution is made with 29.1g sucrose made up to 100mL distilled water.

-Put 1mL of the sucrose solution in a 2mL microtube.

-In this microtube, on top of the sucrose, place very gently 1mL of dejellied and labelled (Hoechst 1/1000, 10min incubation) eggs in artificial sea water with a 1mL pipet.

-Tilt the tube several times so as to generate a sucrose gradient.

Centrifugation of the eggs

-Centrifuge the eppendorf containing the eggs at ~10000g, for ~7 or ~30min, at 15°C. The egg components partitioning will depend on the length and force of the centrifugation, as well as the batch of eggs.

-After centrifugation, collect ~20µL of the centrifuged eggs, trying to collect as many eggs as possible, in the sucrose gradient layer where they were concentrated by the centrifugation.

-Centrifuged eggs are then placed in PDMS chambers, or layered on protamine coated dishes in order to immobilize them. The PDMS chamber making is explained below.

-It is possible to rinse the egg with artificial sea water, as sucrose can decreases the sperm activity.

-Fertilize the eggs by flowing ASW with activated sperm in one of the two wells of the PDMS chamber, or place a couple of drops of the ASW with activated sperm on top of the egg placed on the dish. Fertilize as soon as possible after centrifugation, in order to minimize the rehomogeneisation of the egg component stratification.