Prof Natalia Prevarskaya

Dr Giulia Bertolin

Dr Sébastien Roger

Prof Morad Roudbaraki

Mathieu Le Prof

Gautier

Dr Dmitrii Grodienko

' Knockout ORAI3 KO gRNA1 Fw: 5'-CACC GGGCCAGTCAGCACTCGCTG -3' Rv: 5'-AAAC CAGCGAGTGCTGACTGGCCC -3' Knockout ORAI3 KO gRNA2 Fw: 5'-CACC GCCGAGAGCAAGGCAGACGTG -3' Rv: 5'-AAAC CACGTCTGCCTTGCTCTCGGC -3' Knockout ORAI3 KO gRNA3 Fw: 5'-CACC GTGGCCCGGCTGAGGTAGAGG -3' Rv: 5'-AAAC CCTCTACCTCAGCCGGGCCAC -3' Knockout Act ORAI1 gRNA1 Fw: 5'-ACAC GCTTCCAGGAAAAGTGGCGGGG-3' Rv: 5'-AAAA CCCCGCCACTTTTCCTGGAAGC-3' Expression enhancement Act ORAI1 gRNA2 Fw: 5'-ACAC GAGTGACCAGAGACTGCGCGCG-3' Rv: 5'-AAAA CGCGCGCAGTCTCTGGTCACTC-3' Expression enhancement Act ORAI1 gRNA13 Fw: 5'-ACAC GACGTGACCCGCCCGCTCCGAG-3' Rv: 5'-AAAA CTCGGAGCGGGCGGGTCACGTC-3' Expression enhancement Act ORAI3 gRNA1 Fw: 5'-ACAC GAGTTCGTGTGTGTATCTGCGG-3' Rv: 5'-AAAA CCGCAGATACACACACGAACTC-3' Expression enhancement Act ORAI3 gRNA2 Fw: 5'-ACAC GCAAGCGCCCGGACCCCACCGG-3' Rv: 5'-AAAA CCGGTGGGGTCCGGGCGCTTGC-3' Expression enhancement

pour avoir accepté d'évaluer mes travaux de thèse. Je souhaite également remercier mon tuteur de thèse Dr Fabien Vanden Abeele, pour les conseils qu'il m'a prodigué pendant ces années de thèse. Je tiens à remercier particulièrement le Prof Prevarskaya de m'avoir autorisé à réaliser une thèse en parallèle à mes activités d'ingénieur au sein du laboratoire, sans votre accord et votre compréhension, je n'en serais pas là aujourd'hui. Je me souviens du jour ou vous m'aviez indiqué de la potentielle ouverture d'un poste d'ingénieur au sein du laboratoire auquel je pourrais postuler, vous m'aviez dit qu'il y avait le temps de faire la thèse plus tard. Vous aviez raison ! Merci encore. Je tiens également à remercier spécifiquement certains membres du laboratoire qui ont su me faire grandir en tant que scientifique de par leur conseil : Je souhaite adresser un grand merci au Dr Dimitra Gkika (Myrto), que de chemin parcouru depuis mon premier stage sous ta direction au sein de l'unité 1003 il y 10 ans ! Merci de m'avoir appris les bases des techniques de biologie moléculaire de m'avoir soutenu durant mon Master2, de m'avoir permis d'obtenir mon poste d'ingénieur au sein du laboratoire, je te suis extrêmement redevable, et comme pour Natacha, je n'en serais pas la ou j'en suis aujourd'hui sans ton aide, merci. Je tiens à remercier les étudiantes en thèse lors de mon arrivée dans le laboratoire : Dr Marine Warnier et Dr Sandra Derouiche, nous n'avons pas toujours été sérieux, mais vos conseils sur la manière de réaliser mes expériences m'ont grandement aidé. Merci également de m'avoir aidé à m'intégrer au sein de laboratoire et merci pour toutes les parties de rigolade que nous avons partagées. Je remercie également Mr Etienne Dewailly et le Dr Christian Slomianny, vous m'avez apporté un aide immense dès mon arrivée dans le laboratoire. Etienne, merci de m'avoir appris les secrets des techniques de biochimie ! Christian, merci pour l'aide que tu m'apporté lorsque j'apprenais à maitriser les microscopes confocaux du laboratoire. Merci également à vous deux pour votre bonne humeur. Merci à Elodie Richard, pour son aide sur la plateforme

Merci à Lucile, Valério, Emilie, Pauline, Guillaume, Michela, Eric, Abigael, Antoine, Katya, Artem, pour les moments passés en votre présence dans la laboratoire et l'ambiance chaleureuse que vous avais apporté à l'unité.

Un grand merci à mes deux acolytes Laurent Allart et Nathalie Ziental-Gelus, vous avez su m'aider et m'assister dans mes nombreuses manips de clonage et de screening, je vous en remercie grandement.

Je souhaite adresser un merci tout particulier aux Dr Gabriel Bidaux et Laurent Heliot. Gabriel, je ne pense pas te l'avoir dit mais je t'ai toujours considéré comme un modèle scientifique. Je me souviens de ta présentation d'HDR lorsque tu as présenté tes travaux de FLIM réalisés dans le laboratoire de Laurent, j'avais été particulièrement impressionné, et je me disais que j'aimerais être capable de maitriser des techniques aussi pointues ! Laurent, merci beaucoup de m'avoir laissé accéder aux microscopes, et de m'avoir « intégré » dans ton équipe au P5. Merci également de m'avoir permis de participer à MiFoBio et de m'avoir permis d'entrer dans le monde de microscopie. Merci à vous deux pour votre aide lors de mes comités de suivi de thèse, je vous considère comme mes co-encadrants, un grand merci.

Je tiens également à remercier les anciens membres du laboratoire du Dr Laurent Héliot : Dr Dorian Champelovier, Dr Marie Fournier, et Dr Alessandro Furlan, merci de m'avoir accueilli au sein du laboratoire et merci de m'avoir chaque jour de manip chez vous ! Vos conseils ont été plus que précieux.

Finalement, merci aux personnes qui ont été à mes côtés toutes ces années : Merci à ma « famille Ukrainienne », merci Yourii, Andrii, Luda, Sasha, Vika et Yulia pour votre soutien. Un merci spécial pour Viktoriia, sans votre présence pendant ces deux derniers mois, je n'aurais jamais réussi à rédiger cette thèse, je vous dois énormément.

Merci à mes parents de m'avoir toujours soutenu, Papa et Maman, merci de m'avoir toujours permis d'être dans les meilleures dispositions pour que je progresse dans mes études. Merci à ma soeur Laurane et à Morgan, pour leur soutien. Merci à mamie Jojo, Héléna, Nathalie, Alexandra, Valentin pour votre soutien. Merci à Guy pour avoir su me motiver à terminer ma thèse. Merci à Stéphanie et Patrick (et les lapins) de nous avoir aidé à nous installer en Suisse ! Merci également aux membres de la famille Dumoulin. Merci à mon parrain Gilles Grégoire pour ses encouragements. Vous tous m'avez permis de décompresser à un moment ou un autre durant mes travaux.

Enfin, merci à ma femme le Dr Oksana Iamshanova, tu as été là dès le début pour me soutenir, me motiver et me pousser à terminer cette thèse. Je n'aurais jamais pu accomplir tout cela sans toi. Tu es un modèle scientifique pour moi et tu es certainement la personne qui a eu la plus grande importance dans la conduite de ces travaux, merci ! Thank you for your wisest advice day after day: Start writing up your PhD Alex! Merci à ma fille Mila Bokhobza, merci pour le bonheur que tu me procure chaque jour. J'ai maintenant une longue histoire à te raconter pour t'endormir le soir !

TITRE DE LA THESE EN FRANÇAIS :

Etude du remodelage des protéines ORAI grâce à l'utilisation de la technique CRISPR/Cas9 et de la microscopie quantitative.

RÉSUMÉ DE LA THÈSE EN FRANÇAIS :

L'entrée capacitive de calcium (Ca2+), appelée SOCE en anglais (store operated Ca2+ entry) représente une entrée d'ions Ca2+ dans la cellule consécutive à la vidange des stocks calcique réticulaires. Ce processus constitue l'un des mécanismes d'entré majeure de Ca2+ dans les cellules non-excitables.

L'importance physiologique de ce processus est soulignée par la gravité des syndromes induits par des mutations des canaux responsables du SOCE : le syndrome sévère d'immunodéficience combinée induit par des mutations de types perte de fonction du SOCE et les syndromes d'agrégation tubulaire myopathique (TAM) et de Stormorken induit pas des mutations de type gain de fonction du SOCE. Le SOCE résulte de l'interaction de deux familles de protéines appelées STIM (Stromal interaction protein, 1,2) localisées dans la membrane du réticulum endoplasmique (RE) et ORAI (1-3) situées dans la membrane plasmique. Le processus classique d'apparition du SOCE dans les cellules peut être décrit de la sorte : La protéine STIM1, qui possède des motifs EF-hand sensible au Ca2+ dans la lumière du RE, détecte une diminution de la concentration en Ca2+. En réponse, la protéine STIM subit un changement conformationnel qui conduit à son oligomérisation et sa translocation au niveau des jonctions membrane plasmique -RE. Les protéines STIM vont ensuite interagir, regrouper et activer les protéines ORAI1 qui forment le canal appelé Ca2+-release activated Ca2+ (CRAC) aboutissant à la production du SOCE.

Cependant, de nombreuses publications ont démontré l'implication des autres isoformes des protéines STIM et ORAI dans ce processus. De manière intéressante, l'intervention des protéines STIM2 et ORAI2/3 dans le mécanisme du SOCE permet de moduler le signal produit et ainsi de réguler finement les effets physiologiques de l'entrée de Ca2+ dans les cellules. En particulier, notre laboratoire a démontré que les canaux hétéromériques formés par les protéines ORAI1 et ORAI3 définissent un "interrupteur" oncogénique dans les cellules cancéreuses prostatiques permettant l'apparition d'un phénotype plus agressif. L'étude des mécanismes amenant à la formation de ces canaux hétéromériques est complexe en raison des limites des techniques à disposition. Par exemple, la plupart des moyens d'études reposent sur des systèmes de surexpression ou de sous expression, qui ne permettent pas de supprimer totalement l'expression des protéines endogènes. La présence de ces protéines endogènes rend difficile l'interprétation des résultats obtenus. Le but de cette thèse était donc d'utiliser des techniques de pointe pour étudier les mécanismes d'association des protéines ORAI. Ainsi, nous avons utilisé la technique CRISPR/Cas9 afin de générer des cellules doubles knockout (KO) pour les protéines ORAI1 et ORAI3. Ces cellules, KO pour ORAI1 et ORAI3 ont été utilisées pour réaliser des expériences de microscopie quantitative. Nous avons notamment ré-exprimé des versions fluorescentes des protéines ORAI1 et ORAI3 dans ces cellules avant de réaliser des expériences de mesure de temps de vie de fluorescence via la technique de FLIM-FRET (fluorescence lifetime imaging microscopy -Förster resonance energy transfer). Cette technique nous a permis de suivre l'évolution des interactions entre les protéines ORAI1 et ORAI3 lors de différentes stimulations cellulaires. De la sorte nous avons pu montrer que l'interaction entre les protéines ORAI1 et ORAI3 est dynamique en fonction de la stimulation appliquée. De plus, nous avons tiré profit des cellules KO générées afin d'étudier le rôle des protéines ORAI1/3 et du SOCE dans les lignées HEK-293 et PC3. Nous avons ainsi démontré que, dans les cellules HEK, la protéine ORAI1 et le SOCE jouent un rôle limité dans le maintien de leur physiologie, alors que dans les cellules PC3, ORAI1 et ORAI3 sont importantes pour le maintien du phénotype migratoire de ces cellules.

TITLE OF THE THESIS IN ENGLISH:

Study of ORAI protein remodeling with CRISPR and quantitative microscopy.

ABSTRACT OF THE THESIS IN ENGLISH:

The store operated calcium (Ca2+) entry (SOCE) represents the entry of Ca2+ through the cell's plasma membrane consecutive to an endoplasmic reticulum (ER) Ca2+ store depletion. This process is described as one of the main calcium (Ca2+) pathway in the cells. Its importance is highlighted by the severe syndromes induced by loss or gain of function mutation of its constituent named severe combined immunodeficiency (SCID) and tubular aggregate myopathy (TAM)/Stormorken syndrome (STRMK) respectively. SOCE is the result of interaction between two families of proteins, the ER residing protein family Stromal interaction molecule (STIM 1&2) and the plasmalemmal proteins called ORAI (1-3).

The classic molecular choreography of SOCE activation is described as follow: a drop in ER-Ca2+ content is detected by the EF-hand domain present in the STIM protein. Following ER Ca2+ depletion, STIM protein oligomerize and translocate to ER-plasma membrane (PM) junctions where they bind and activate ORAI1 composed channel called Ca2+ release activated Ca2+ channel providing SOCE.

Interestingly, it appears that this choreography is much more complex than initially thought with the involvement of other STIM and ORAI isoforms (STIM2 and ORAI2,3). The involvement of these isoforms affects the properties of SOCE by modulating its Ca2+ signature resulting in different cellular answers. Especially, it was shown that heteromeric channels composed of ORAI1 and ORAI3 protein are defining an oncogenic switch in prostate cancer cell lines that lead to the more aggressive phenotype.

However, the study of the mechanisms leading to the creation of ORAI1/3 channels at the expanse of ORAI1-only channels is problematic due to technical limitations. For example, most of investigations are performed in overexpression or downregulation system where endogenous protein are still present and might blur the results of experiments. The goal of this PhD was to use high end techniques in order to study the mechanisms of ORAI1/3 interactions without facing the limitations mentioned above. Specifically, we implemented and used CRISPR/Cas9 technique to generate double knockout (KO) cell lines for ORAI1 and 3 proteins. We used these "ORAI1/3 free cells" to perform quantitative microscopy experiments. Specifically, we expressed fluorescently tagged ORAI1 and ORAI3 proteins and performed FLIM-FRET (fluorescence lifetime imaging microscopy -Förster resonance energy transfer) experiments enabling us to follow their association process in answer to different stimulations. We thus demonstrated that the association between ORAI1 and ORAI3 is a dynamic process in the cells.

Additionally, we took advantage of these KO cell lines to study the role of ORAI1/3 protein and SOCE in HEK-293 and PC3 cells physiology. We thus demonstrated that ORAI1 protein and SOCE only presented a limited role in the maintenance of HEK-293 physiology, while ORAI1 and ORAI3 are important to maintain migrative properties of the cancerous PC3 cells. Living organisms are embedded in a structure isolating them from the external medium. Except from some viruses this structure consists in lipidic membrane which provide an impermeable barrier allowing the organism to retain the vital molecules required for its growth. Nonetheless, in order to develop, all organisms need to excrete their waste products and import valuable elements. From all the proteins responsible for these exchanges, ion channels are the one responsible for ion flux across the membrane. The importance of this protein family is highlighted by the fact that ion channels were identified in virtually all living cells, from Bacteria and Archaea to Eukaryote and [START_REF] Jiang | X-ray structure of a voltage-dependent K+ channel[END_REF] even in some Viruses [START_REF] Moran | Evolution of voltage-gated ion channels at the emergence of Metazoa[END_REF]. Nonetheless, from an evolutionary point of view, there is no evidence that ion channels evolved from a unique ancestor as no sequence homology could be detected within the myriad of ion channel protein identified [START_REF] Nayak | Evolution of the Human Ion Channel Set[END_REF]. Nevertheless, all ion channels are sharing a number of common features that defines them as members of the ion channel protein family [START_REF] Anderson | Phylogeny of ion channels: Clues to structure and function[END_REF][START_REF] Nayak | Evolution of the Human Ion Channel Set[END_REF]. These features can be described as follow, ion channels:

(1) are integral membrane proteins and thus possess hydrophobic segments.

(2) allow passive transport of ion through membranes, meaning that ion fluxes diffuse according to the electrochemical gradient.

(3) main structural property is to form ion-permeable tunnel across the membrane, named pore.

(4) minimal structural constituent is represented by two transmembrane domains (TM) surrounding the pore-forming loop.

(5) possess a high rate of ion diffusion.

Functional diversity

Ion channels seems to have appeared several times throughout evolution depending on the specific needs of the organisms [START_REF] Nayak | Evolution of the Human Ion Channel Set[END_REF]. This is probably one of the reasons of their important diversity. Due to this diversity, several systems of classifications for ion channels co-exist. For example, ion channels can be separated in different families depending on their ion selectivity, and thus define the Potassium (K + ), Sodium (Na + ), Chloride (Cl -), Calcium (Ca 2+ ), Proton (H + ), and non-selective ion channel families. Alternatively, ion channel families can also be distinguished through their gating mechanisms. Of note, the term 'gating' refers to the mechanism by which the ion-permeable pore is opening (activation) or closing (by deactivation or inactivation). Apart from constitutively open leak channels, the activation of an ion channel results from a conformational change and thus requires an energetic input to allow their unlocking. The gating mechanism classification of ion channels includes three main families. The first group comprises ligand-gated channels, they can be activated by the binding of a specific molecule (e.g. acetylcholine for acetylcholine receptor), an ion (e.g. Ca 2+ for Ca 2+activated K + channels), or another protein (e.g. ORAI channels during the store-operated Ca 2+ entry [SOCE] mechanism). The second group represent pH-gated channels for which the protonation state of specific amino acid determines the opening or closing of the channel (e.g. Acid-sensing ion channels [ASIC]). The third group comprises channels which are gated by a physical process and includes the super-families of voltage-, mechano-, and temperature-gated ion channels (Figure 1). Importantly, several ion channels fall into multiple groups. For example, the transient receptor potential vanilloid 1 (TRPV1) is a ligand-gated ion channel that also displays voltage-gating properties [START_REF] Gunthorpe | Voltage-and time-dependent properties of the recombinant rat vanilloid receptor (rVR1)[END_REF]. Additionally, the channel Hv1 is a voltage-gated channel but is at the same time strongly regulated by pH, and sensitive to mechanical stimuli [START_REF] Pathak | The Hv1 proton channel responds to mechanical stimuli[END_REF][START_REF] Decoursey | Voltage-gated proton channels: Molecular biology, physiology, and pathophysiology of the HV family[END_REF]. Therefore, there is no unique and formal classification system for ion channels, and in fact, many classification systems coexist.

Ion channel structure diversity

Protein structure is characterized by four levels organization (Figure 2). The primary level is simply the linear chain of amino acid, the secondary level is represented the three-dimensional (3D) shape adopted by groups of amino acid, the tertiary structure of protein represents the 3D organization of the whole protein including its different domains, and the quaternary structure, which is not observed for all proteins, consists of the organization of multiple units of proteins into macromolecules, this process being called oligomerization. In addition to the existing diversity in their ion selectivity and gating mechanisms, ion channel also present heterogeneity at each of these structural levels:

1.1.3.1.

Primary structure

From a phylogenetic point of view, ion channel primary structure diversity is evident as no homology in their amino acid sequence could be identified and linked to single ancestor. A good example of ion channel diversity in their primary structure is the large difference in amino acid length observed between the smallest identified ion channel: Kcv (a potassium ion channel identified in virus constituted of 94 amino acid) and some of the longest ion channels such as ryanodine receptor (RyR) which can be composed of more than 5000 amino acids [START_REF] Kang | Small potassium ion channel proteins encoded by chlorella viruses[END_REF]) (Figure 3 A andB).

1.1.3.2. Secondary structure

Originally, three types of protein secondary structures were described: α-helix, β-strands, and random coil (Pauling et al, 1951;Pauling & Corey, 1951). However, several other non-repetitive secondary structures were later identified (such as turns and loops, for example) [START_REF] Fetrow | Omega loops; nonregular secondary structures significant in protein function and stability[END_REF]. Yet, no consensus about the description of all secondary (or super secondary) structures organization was reached as demonstrated by the classifications heterogeneity found between different structural databases [START_REF] Banach | Secondary and Supersecondary Structure of Proteins in Light of the Structure of Hydrophobic Cores[END_REF]. It is therefore, difficult to appreciate their representation level within the myriad of ion channel described. Nevertheless, it is interesting to note that TM domains, the structural basic element of ion channel structure, present diversity in their structure. Indeed, most of ion channel present TM domains composed of α-helix. However, some ion channels located in outer membranes of Gram-negative bacteria, chloroplasts or mitochondria such as the voltage-dependent anion channel (VDAC) present TM composed of β-barrel demonstrating that even in their most conserved part, ion channel also present structural heterogeneity (Figure 3 C) [START_REF] Cavalier-Smith | Membrane heredity and early chloroplast evolution[END_REF][START_REF] Moitra | The Biogenesis Process of VDAC -From Early Cytosolic Events to Its Final Membrane Integration[END_REF].

1.1.3.3. Tertiary structure

The tertiary structural organization level of proteins usually refers to the spatial arrangement of secondary and super secondary structures of proteins. While this term was initially used to describe the global 3D shape of proteins [START_REF] Moss | Basic Terminology of Stereochemistry[END_REF], tertiary structures element can also define protein regions that adopt a specific conformation independently from the rest of the protein structure. Such regions usually exhibit specific functions and are named protein domains (Skipper, 2005). Ion channels present a myriad of different domain depending on their mode of activation, or additional function (such as enzymatic functions). Nonetheless, the channel pore represents a domain which is evidently found in every ion channel. One of the best representations of tertiary structure diversity within ion channels is represented by their various gating mechanisms. Specifically, almost each gating mechanism is associated with its specific domains. For example, voltage-gated ion channel requires the presence of voltage sensing domain; ligand gated ion channel family entails the presence of specific ligand binding domain. In addition, several protein interaction domains are also found in ion channels due to the requirement of some ion channel protein to form oligomer to be active. For example, some ankyrin domains were found on TRPV5 channels and were shown to facilitate their tetramerization [START_REF] Chang | Molecular Determinants in TRPV5 Channel Assembly *[END_REF]. Similarly, coil-coiled domain present in Hv1/VSOP channel are important for their dimerization [START_REF] Fujiwara | The cytoplasmic coiled-coil mediates cooperative gating temperature sensitivity in the voltagegated H+ channel Hv1[END_REF]. Finally, some ion channels are also displaying domains bearing a secondary function, in addition to their ion conduction role, such as enzymatic activity. It is the case for the TRP melastatin 7 (TRPM7) channel which presents kinase activity [START_REF] Ryazanova | Characterization of the Protein Kinase Activity of TRPM7/ChaK1, a Protein Kinase Fused to the Transient Receptor Potential Ion Channel[END_REF].

1.1.3.4.

Quaternary structure

The last level of protein structure is called quaternary structure, it represents the association between multiple proteins called oligomerization and it enables the proteins to function properly [START_REF] Ouellette | Amino Acids, Peptides, and Proteins[END_REF]. Most ion channels are oligomeric assemblies of the same protein (homo-oligomers) or of different proteins (hereto oligomers). In fact, the specific oligomerization of an ion channel is usually related to its family and often represent a way of regulating the function or the properties of the channels

The oligomerization process of ion channels represents a way for the cell to tightly control and modulates their function. To appreciate the complexity in oligomeric assemblies of ion channels, some typical architecture of different ion channel families will be presented below:

• Voltage-gated cation ion channels superfamily.

It was postulated that all the members of this family are originating from a single ancestor ion channel. This prototypic ion channel ancestor underwent several rounds of gene duplication and gene fission leading to the multiplication of its TM domain and to the diversification of its architecture eventually leading to the apparition of a multitude of ion channel presenting distinct properties and different oligomerization states (Figure 4). Specifically, it is believed that the Inward-rectifier potassium channels family (Kir), whom members are found from Prokaryotes to Eukaryotes, are the closest example of the typical ion channel ancestor from this family [START_REF] Anderson | Phylogeny of ion channels: Clues to structure and function[END_REF][START_REF] Moran | Evolution of voltage-gated ion channels at the emergence of Metazoa[END_REF]. Kir channels are displaying the simplest ion channel structure with only 2 TM domains. All the members of the superfamily display this similar Kir-like domain but have acquired additional regulatory domain throughout evolution such as voltage-sensing, or ligand binding regions. Overall, due to successive rounds of gene duplication and fusion members of the superfamily can present 2, 4, 6, 12

or 24 TM domain (Figure 4) [START_REF] Nayak | Evolution of the Human Ion Channel Set[END_REF][START_REF] Moran | Evolution of voltage-gated ion channels at the emergence of Metazoa[END_REF]. Interestingly, despite their different numbers of TM, their pore architecture is somewhat conserved, and it is the number of protein units required to form a functional channel which differs. For example, 2TM, 4 TM and 6TM domain proteins such as Kir, K2P (Two-pore-domain potassium channel) or TRP need to tetramerize in order to form functional channels (Figure 5 A,C,D). Remarkably, channel from these sub-family are able to form hetero-or homo-tetramers therefore providing specific properties to the channel they form. At the opposite, 24 TM domain protein from the CaV or NaV family are strictly monomeric and are regulated by accessories subunits. Finally, the members of the two-pore channel (TPC) family present 12TM domain, they can form pseudo-tetramers which consists in the association of 2 homo-or hetero-dimers with each of the dimers forming one individual pore (Figure 5 E).

• Pentameric ligand-gated ion channels.

As indicated by their name, members of this family are gated by ligand binding. These channels are functional when assembled as pentamers. This group including the Cys-loop receptors family which are displaying extracellular C-and N-termini responsible for the binding of neurotransmitters that gate the channel. They possess 4TM domains and the pore is constituted primarily of the TM N°2 of each protein.

While they can associate as homo-pentamers, their preferred assembly consist of hetero-pentamers as the association of different subunit types confers different properties to the formed channel (Figure 5 G) [START_REF] Sine | Recent advances in Cys-loop receptor structure and function[END_REF][START_REF] Jaiteh | Evolution of pentameric ligand-gated ion channels: Pro-loop receptors[END_REF].

• Epithelial sodium channel (ENaC) /P2X family.

This superfamily includes members of the Enac, the P2X and Acid sensing ion channel (ASICs) families, these proteins present 2TM domain proteins with the pore mainly constituted by the TM N°2.

They associate as trimers to form functional channel. Similarly, to the group above, proteins can adopt hetero-or homo-trimers form in order to modulate the properties of the channel they form (Figure 5 H). [START_REF] Jasti | Structure of acid-sensing ion channel 1 at 1.9 Å resolution and low pH[END_REF][START_REF] Barrera | Atomic force microscopy imaging demonstrates that P2X2 receptors are trimers but that P2X6 receptor subunits do not oligomerize[END_REF][START_REF] Horikawa | Recent progress in the development of genetically encoded Ca<sup>2+ </sup>indicators[END_REF][START_REF] Kaczmarek-Hájek | Molecular and functional properties of P2X receptors-recent progress and persisting challenges[END_REF].

• Chloride Carrier/Channel family

The members of the chloride channel family display a very complex structure. They possess membrane domain which are not spanning the entire membrane and the number of 'real' TM is variable.

They assemble as dimers in an anti-parallel way and each individual protein bears its individual pore.

The functional channels are hetero-or homo-dimers(Figure 5 I) [START_REF] Jentsch | Molecular structure and physiological function of chloride channels[END_REF][START_REF] Poroca | ClC channels and transporters: Structure, physiological functions, and implications in human chloride channelopathies[END_REF].

• Ca 2+ release-activated Ca 2+ channel (CRAC-C) family

This unique family of ion channel protein count three members named ORAI1, 2 and 3. They show no sequence homology with any other known ion channels which explain their relatively recent discovery in 2006. The 3 human members of this family possess 4TM domains, and the pore-forming unit is constituted by their first TM1. They show a unique property as their stoichiometry can be different depending on the channel they form. As such, it was demonstrated that the Ca 2+ release activated Ca 2+

(CRAC) channel, responsible for the store operated Ca 2+ entry (SOCE), consists of a homo-hexameric assembly of ORAI1 protein whereas the Arachidonate-regulated channel (ARC) present a heteropentameric structure composed of ORAI1 and ORAI3 proteins (Figure 5J) (Mignen et al, 2008a;Lis et al, 2007a).

Conclusion on the diversity of ion channels

To conclude, ion channels are an extremely diverse protein family. Within this family, multimerization is a common process which is found across several ion channel families. Indeed, oligomerization process presents the advantage of allowing an adjustment of channel properties by simply modifying its subunit composition. Nonetheless, the modification of the channel composition usually does not affect their stoichiometry and thus leaves their pore architecture intact. Thus, within all ion channel identified, the ORAI protein family present a unique property since it was described that they can assemble in distinct channels of different stoichiometry bearing specific properties. The following part of the dissertation will be devoted to the description of ORAI proteins. Especially, ORAI proteins are essential component of one of the central mechanisms of Ca 2+ signaling in the cells called SOCE. Thus, we will start by describing the SOCE process and its specific molecular components before reporting the other types of channels that can be formed by ORAI proteins.

1.2. Store operated Ca 2+ entry (SOCE) and its components 1.2.1. Discovery of SOCE

The ORAI proteins were identified as the effector part of the cellular process called Store Operated Calcium Entry (SOCE). The SOCE correspond to an entry of Ca 2+ from the extracellular space into the cytoplasm in response to an endoplasmic reticulum (ER) Ca 2+ store depletion. The existence of a link between the Ca 2+ release from the ER, and the Ca 2+ entry through the plasma membrane (PM) was first postulated in 1977 by Dr Putney who named this process the capacitative entry. Indeed, Dr Putney predicted the existence of an intermediate step between the Ca 2+ release from the ER stores and the subsequent Ca 2+ entry across the PM. Thus, he used the term capacitative as an homology with the "electrical circuit where a charge must load a capacitor before current can flow through it" [START_REF] Putney | Recent breakthroughs in the molecular mechanism of capacitative calcium entry (with thoughts on how we got here)[END_REF]. Even though it was thought, at first, that the SOCE was allowing a direct refilling of the intracellular stock of Ca 2+ (i.e: the ER) from the exterior of the cells [START_REF] Casteels | Exchange characteristics of the noradrenaline-sensitive calcium store in vascular smooth muscle cells or rabbit ear artery[END_REF].

This hypothesis was later invalidated by a study conducted by Dr Kwan indicating that the refilling of the store was not a direct route [START_REF] Rosado | Discovering the mechanism of capacitative calcium entry[END_REF][START_REF] Kwan | Effects of MeCh, thapsigargin, and La3+ on plasmalemmal and intracellular Ca2+ transport in lacrimal acinar cells[END_REF]. In the following years, it was also demonstrated that the role of SOCE was not limited to the sole refilling of intracellular Ca 2+ store but that it was also of extreme importance for a variety of cellular processes such as differentiation, secretion, proliferation, migration in multiple cell types [START_REF] Darbellay | STIM1and Orai1-dependent store-operated calcium entry regulates human myoblast differentiation[END_REF][START_REF] Concepcion | Store-operated Ca2+ entry regulates Ca2+-activated chloride channels and eccrine sweat gland function[END_REF][START_REF] Abdullaev | Stim1 and orai1 mediate crac currents and store-operated calcium entry important for endothelial cell proliferation[END_REF][START_REF] Potier | Evidence for STIM1-and Orai1-dependent store-operated calcium influx through ICRAC in vascular smooth muscle cells: Role in proliferation and migration[END_REF]. Despite its physiological importance and the growing number of studies related to SOCE, the identification of its molecular component took almost 20 years [START_REF] Putney | A model for receptor-regulated calcium entry[END_REF][START_REF] Roos | STIM1, an essential and conserved component of store-operated Ca 2+ channel function[END_REF][START_REF] Liou | STIM is a Ca2+ sensor essential for Ca2+-store-depletion-triggered Ca2+ influx[END_REF][START_REF] Prakriya | Orai1 is an essential pore subunit of the CRAC channel[END_REF][START_REF] Vig | CRACM1 Multimers Form the Ion-Selective Pore of the CRAC Channel[END_REF]. Thus, in 2005 and2006, the discoveries and identification of STIM and ORAI proteins respectively enabled the description of the molecular choreography leading to the generation of SOCE. The simplified mechanism leading to SOCE generation could be described as follow: (1) The stimulation of a cell surface receptor results in the production of the second messenger inositol triphosphate (IP3). ( 2) The binding of IP3 on its ER receptor induces ER Ca 2+ release.

(3) The decrease in Ca 2+ concentration induces a conformational change in the ER resident protein STIM. (4) The STIM protein starts diffusing to in ER-PM junction where it binds the Ca 2+ channel composed of ORAI1 protein named Ca 2+ release-activated Ca 2+ channel (CRAC). ( 5) Binding of STIM1 to CRAC channel induce a conformational change which opens the channel and thus allows the Ca 2+ entry in the cytoplasm through the PM in a process called the SOCE (Figure 6). The precise molecular mechanisms leading to the activation of STIM and ORAI proteins have been extensively studied. In order to detail this mechanism, the structural elements present in ORAI and STIM proteins will be detailed thereafter.

1.2.1. STIM proteins 1.2.1.1.

Discovery of STIM proteins

The STIM gene was cloned in 1996 by two independent teams [START_REF] Oritani | Identification of stromal cell products that interact with pre-B cells[END_REF][START_REF] Parker | Molecular cloning of a novel human gene (D11S4896E) at chromosomal region 11p15.5[END_REF]. If Parker's team cloned the gene and named it GOK, they did not find any function for the newly discovered protein as it was not showing any sequence homology with other known proteins at the time. On contrary, Oritani & Kincade were the first to identify a role for the protein coded by GOK gene. They found that this protein played a role in the interaction process between stromal and pre-B cells, it is the reason why they initially named this protein SIM (Stromal Interaction Molecule) which was later renamed STIM (STromal Interaction Molecule). Even though the exact role of STIM protein was not identified by these teams, they both correctly identified important topological domain of the STIM protein, such as its transmembrane domain, its coil-coiled interaction domain and a signal peptide sequence. In 2001, a gene database screening revealed the existence of a homologue gene to STIM contributing to the creation of the STIM family including STIM1 and STIM2 genes [START_REF] Williams | Identification and characterization of the STIM (stromal interaction molecule) gene family: Coding for a novel class of transmembrane proteins[END_REF]. It is only, in 2005 that two teams have independently discovered the essential role of STIM proteins in the SOCE process [START_REF] Roos | STIM1, an essential and conserved component of store-operated Ca 2+ channel function[END_REF][START_REF] Liou | STIM is a Ca2+ sensor essential for Ca2+-store-depletion-triggered Ca2+ influx[END_REF]. Since this date, the STIM proteins (1&2) have been extensively studied (with more almost 200 publications including STIM in their title since 2005), and the structure of these proteins is well described, and will be recapitulated below. [START_REF] Darbellay | STIM1L is a new actin-binding splice variant involved in fast repetitive Ca2+ release[END_REF][START_REF] Ramesh | A short isoform of STIM1 confers frequency-dependent synaptic enhancement[END_REF][START_REF] Miederer | A STIM2 splice variant negatively regulates store-operated calcium entry[END_REF][START_REF] Yen | Alternative splicing converts STIM2 from an activator to an inhibitor of store-operated calcium channels[END_REF]. All STIM isoforms present the same overall structure constituted of a short N-terminus facing the ER lumen, a single-pass transmembrane domain, and a long C-terminus region facing the cytosol. In human, the predominant form of STIM1 and STIM2 (STIM1 and STIM2.2 respectively) are ubiquitously expressed.

These protein are mainly found in the ER membrane even though few publications have identified STIM proteins in PM [START_REF] Spassova | STIM1 has a plasma membrane role in the activation of store-operated Ca2+ channels[END_REF] and acidic stores [START_REF] Zbidi | STIM1 and STIM2 Are Located in the Acidic Ca2+ Stores and Associates with Orai1 upon Depletion of the Acidic Stores in Human Platelets[END_REF]. STIM proteins possess a dual role of sensing the Ca 2+ concentration in the ER and activating the ORAI1 channel in the PM. In addition, STIM proteins present several domains including (Figure 7):

• On the N-terminal extremity, facing the ER lumen:

o A canonical and a non-canonical EF hand motif (cEF and nEF respectively) associated with a sterile alpha motif (SAM). This domain called EF-SAM allows the STIM protein to detect variation in the ER Ca 2+ concentration. Of note, these domains are not identical for all the STIM isoforms. As a consequence, the o differences in EF hands sequence confers different sensitivities of the STIM isoforms to ER Ca 2+ concentration and thus allow for their selective activation depending on the level of Ca 2+ depletion.

• A transmembrane domain o A polybasic rich domain (PBD) (absent in STIM2.3) in the end of the C-terminal extremity which enables the interaction of STIM with the PIP2 from the PM and thus promotes its localization to ER-PM junctions.

In addition to these prevalent domains, the different STIM isoforms also present structural features which are specific to each isoform.

STIM1 isoforms

The classically described STIM1 protein possesses two isoforms. Both isoforms are displaying their own specific additional domains:

• STIM1L possesses an additional actin-binding domain (ABD), leading to the permanent formation of STIM1L clusters, which enable an almost instantaneous SOCE activation [START_REF] Darbellay | STIM1L is a new actin-binding splice variant involved in fast repetitive Ca2+ release[END_REF]. Of note, this isoform has been exclusively found in skeletal muscle.

Interestingly, it was demonstrated that the STIM1L isoform was able to interact with other proteins than ORAI, namely TRP canonical channels (TRPC1,3,4 and 6) [START_REF] Horinouchi | Different binding property of STIM1 and its novel splice variant STIM1L to Orai1, TRPC3, and TRPC6 channels[END_REF][START_REF] Antigny | TRPC1 and TRPC4 channels functionally interact with STIM1L to promote myogenesis and maintain fast repetitive Ca2+ release in human myotubes[END_REF][START_REF] Dyrda | STIM1 long and STIM1 gate differently TRPC1 during storeoperated calcium entry[END_REF].

• STIM1B, the latest STIM1 isoform identified, displays an additional domain named domain B. This isoform is the result of the incorporation of an alternative exon (13B) which leads to the insertion of this domain just after the ID domain of STIM1. Of note this insertion induces a shift in the open reading frame of STIM1, thereby inducing the apparition of an early STOP codon resulting in the production of a shorter isoform lacking the EB and PBD domain. This isoform in mainly found in brain tissue were it slows SOCE activation [START_REF] Ramesh | A short isoform of STIM1 confers frequency-dependent synaptic enhancement[END_REF].

STIM2 isoforms

The STIM2 protein (STIM2.2) presents a strong sequence homology with STIM1.Specifically, the main difference between STIM1 and 2 lies in their Ca 2+ affinity. In fact, STIM2 is more sensitive to changes in ER-Ca 2+ concentration since its EF-hand domain displays a lower affinity for Ca 2+ than the one of STIM1 [START_REF] Zheng | Biophysical characterization of the EF-hand and SAM domain containing Ca2+ sensory region of STIM1 and STIM2[END_REF]. In addition, it was shown that the CAD/SOAR domain of STIM2 also diverges from the one of STIM1 leading to a weaker coupling with ORAI1 [START_REF] Zheng | Identification of molecular determinants that govern distinct STIM2 activation dynamics[END_REF]. This reduced affinity for Ca 2+ and lower gating to ORAI1 enable a fine-tuning of SOCE which is even accentuated by the fact that STIM1 and STIM2 are able to form heterodimers [START_REF] Subedi | STIM2 Induces Activated Conformation of STIM1 to Control Orai1 Function in ER-PM Junctions[END_REF]. In addition to the classic STIM2, two alternatively spliced STIM2 protein have also been described:

• The STIM2.1 variant presents an additional exon resulting in an 8 amino acid insertion localized in the CC2 domain of the CAD/SOAR region of STIM2. This insertion prevents the association of STIM2.1 with ORAI [START_REF] Miederer | A STIM2 splice variant negatively regulates store-operated calcium entry[END_REF][START_REF] Yen | Alternative splicing converts STIM2 from an activator to an inhibitor of store-operated calcium channels[END_REF]. Thus, STIM2.1 possesses an inhibitory effect on SOCE but the mechanisms leading to this inactivation are not totally understood. Of note, as STIM2.2, the STIM2.1 protein variant is ubiquitously expressed in human but their relative expression levels are tissue specific [START_REF] Miederer | A STIM2 splice variant negatively regulates store-operated calcium entry[END_REF].

• STIM2.3 also includes an alternative exon expression. Nonetheless, the insertion of this additional exon induces the apparition of an early STOP codon resulting in the shortest STIM isoform identified. Of note, this isoform have only been detected on mRNA level [START_REF] Miederer | A STIM2 splice variant negatively regulates store-operated calcium entry[END_REF].

Overall, the 6 identified STIM isoforms are presenting a strong homology both in their sequences and their domains. While some isoforms possess opposite effects on the SOCE, their global mechanism of activation is believed to be comparable within all STIM isoforms. Since STIM1 represents the most studied of STIM isoforms, the next paragraph will focus on the description of its specific activation mechanism.

STIM activation during SOCE

The STIM1 activation processes leading to the generation of SOCE have been extensively studied, and while all the details are not yet understood, a global consensus on the STIM activation process has been reached and will be described thereafter (Figure 8). In resting state, STIM proteins are dimers diffusing along the ER membrane. The dimerization of STIM proteins is stabilized by the interaction between their adjacent cytosolic domains CAD/SOAR [START_REF] Yang | Structural and mechanistic insights into the activation of Stromal interaction molecule 1 (STIM1)[END_REF][START_REF] Muik | A Cytosolic Homomerization and a Modulatory Domain within STIM1 C Terminus Determine Coupling to ORAI1 Channels[END_REF]. The interaction between CAD/SOAR domain of STIM proteins maintain the C-terminus extremities of STIM in a compacted form sequestering the C-terminal ends near the ER membrane [START_REF] Yang | Structural and mechanistic insights into the activation of Stromal interaction molecule 1 (STIM1)[END_REF][START_REF] Muik | A Cytosolic Homomerization and a Modulatory Domain within STIM1 C Terminus Determine Coupling to ORAI1 Channels[END_REF]. Of note, in this resting state the N-terminal EF/SAM domain of adjacent domain are not interacting. This absence of interaction is due to the presence of Ca 2+ bound in the EF hand domains [START_REF] Li | Mapping the Interacting Domains of STIM1 and Orai1 in Ca2+ Release-activated Ca2+ Channel Activation[END_REF][START_REF] Korzeniowski | Molecular anatomy of the early events in STIM1 activation; oligomerization or conformational change?[END_REF]. Following drop in ER Ca 2+ concentration, Ca 2+ is unbound from EF hand. The Ca 2+ liberation from the EF hand motif allows the whole EF-SAM domains from adjacent STIM proteins to interact together. This conformational change is further propagated through the PM to the cytoplasmic region of STIM [START_REF] Hirve | Coiled-Coil Formation Conveys a STIM1 Signal from ER Lumen to Cytoplasm[END_REF][START_REF] Ma | Inside-out Ca2+ signalling prompted by STIM1 conformational switch[END_REF]. Due to the conformational change propagation, the interacting CC1 domain of adjacent STIM proteins are modifying their orientation and allowing the liberation of the CAD/SOAR domain. This whole process induces the deployment of STIM C-terminal extremities [START_REF] Korzeniowski | Molecular anatomy of the early events in STIM1 activation; oligomerization or conformational change?[END_REF][START_REF] Hirve | Coiled-Coil Formation Conveys a STIM1 Signal from ER Lumen to Cytoplasm[END_REF]. As a results, PBD are exposed to the phospholipids from the PM which allows the clustering of STIM proteins in higher oligomeric stated in the ER-PM junction [START_REF] Liou | Live-cell imaging reveals sequential oligomerization and local plasma membrane targeting of stromal interaction molecule 1 after Ca2+ store depletion[END_REF][START_REF] Walsh | Evidence for an interaction between Golli and STIM1 in store-operated calcium entry[END_REF][START_REF] Korzeniowski | Molecular anatomy of the early events in STIM1 activation; oligomerization or conformational change?[END_REF]Park et al, 2009a). Of note, this higher oligomeric state is thought to be promoted due to the extension of C-terminal extremities. Specifically, the new accessibility of the CC3 domains induced by the conformational change is thought to mediates the interaction between STIM protein to form high oligomeric states [START_REF] Fahrner | A Coiled-coil Clamp Controls Both Conformation and Clustering of Stromal Interaction Molecule 1 (STIM1)[END_REF]. The STIM protein clustering together with the availability of CAD/SOAR domain to interact with ORAI protein will lead to the opening of ORAI channel and ultimately to the induction of SOCE.

1.2.2. ORAI proteins and their associated channels 1.2.2.1. Discovery of ORAI proteins ORAI proteins were discovered in 2006 in an effort to identify the molecular component of the SOCE. Specifically, the ORAI1 gene was cloned during a genome-wide RNA interference experiment performed in Drosophila aiming to identify the proteins responsible for SOCE [START_REF] Feske | A mutation in Orai1 causes immune deficiency by abrogating CRAC channel function[END_REF][START_REF] Vig | CRACM1 Is a Plasma Membrane Protein Essential for Store-Operated Ca2+ Entry[END_REF]. Three homolog genes for ORAI were later identified and named ORAI1, ORAI2 and ORAI3 as a reference to the three keepers of the gate leading to heaven in Greek mythologic. Analysis of their protein sequence revealed the existence of 4 putative TM domains making them good candidates for being the channel accountable for SOCE. However, due to the absence of homology between the newly discovered ORAI proteins and any other ion channels, it was not clear whether they were the channels providing SOCE or auxiliary proteins important for the channel activation. During the year of their discovery, mutagenesis experiments demonstrated that ORAI1 proteins were forming the so-called Ca 2+

Release-Activated Ca 2+ (CRAC) channel [START_REF] Yeromin | Molecular identification of the CRAC channel by altered ion selectivity in a mutant of Orai[END_REF] and were the effector part of the SOCE.

It was further demonstrated that when overexpressed, the three homologues were able to generate different level of SOCE, with ORAI1 providing the larger Ca 2+ entry, ORAI3 the lowest and ORAI2

being intermediate (Mercer et al, 2006b). Over the years, studies revealed that the three ORAI homologs are expressed in all human tissues at the mRNA and protein level (Figure 9) [START_REF] Hoth | The Neglected CRAC Proteins: Orai2, Orai3, and STIM2 1st ed[END_REF][START_REF] Chalmers | ORAI channels and cancer[END_REF].

Phylogenetic origin of ORAI proteins

From a phylogenetic perspective, the ORAI protein family is unrelated to any other known ion channel. In fact, it is supposed that the ORAI protein family appeared following an unusual round of retro-evolution. Specifically, it has been shown that the ORAI protein sequences derive from the cation diffusion facilitator (CDF) carrier protein family [START_REF] Matias | Animal Ca2+ release-activated Ca2+ (CRAC) channels appear to be homologous to and derived from the ubiquitous cation diffusion facilitators[END_REF]. CDF carrier protein are 6TM domain protein involved in transport of metallic ions. Sequence homology was identified between the 4 ORAI TMs and the TMs N°3 to N°6 of CDF carrier. Based on this sequence homology it was proposed that the ORAI proteins originate from the loss, by the CDF carrier protein, of its two first TM domain [START_REF] Matias | Animal Ca2+ release-activated Ca2+ (CRAC) channels appear to be homologous to and derived from the ubiquitous cation diffusion facilitators[END_REF]. Of note, the emergence of the ORAI2 and ORAI3 genes are the result of ORAI1 gene duplication with ORAI2 appearing earlier than ORAI3, and with the latter found only in mammals while ORAI2 is found in lower vertebrates. Interestingly, it was shown that ORAI3 is evolving faster than its two homologs, and therefore has acquired distinct features compared to the two other ORAI homologues. This led to the hypothesis that ORAI3 could present distinct functions and properties compared to the two other homologues [START_REF] Cai | Molecular Evolution and Structural Analysis of the Ca2+ Release-Activated Ca2+ Channel Subunit, Orai[END_REF][START_REF] Shuttleworth | Orai3 -the 'exceptional' Orai?[END_REF].

ORAI proteins structure

The three ORAI proteins are sharing the same global topology: they possess 4 TM domain with both N-and C-termini facing the cytosol. Thus, they present two extracellular and one intracellular loop.

Overall, the amino acid sequence homology between the three protein is situated around 60%.

Interestingly, this homology shows large discrepancies amongst the different topological domains of ORAI proteins. These variations in sequence homology indicates on the crucial role of the specific conserved domains in the maintenance of ion channel function, but also highlight the existence of unique roles taken by each gene due to their non-conserved domains. Of note, ORAI1 is largely the most studied of all ORAI proteins. Thus, some of its important domains which present high sequence homology with the other homologues are reputed being present in all ORAI. Especially, the three ORAI protein present in their N-terminal extremity a CaM binding domain, responsible for the inactivation of the channels formed by ORAI proteins [START_REF] Mullins | STIM1 and calmodulin interact with Orai1 to induce Ca2+-dependent inactivation of CRAC channels[END_REF][START_REF] Frischauf | Cooperativeness of Orai cytosolic domains tunes subtype-specific gating[END_REF]. In addition, the ORAI1 domain named extended TM ORAI1 NH2-terminal (ETON), is conserved in the three homologues. This domain consists, as indicated by its name, in an extension of the ORAI first TM into the cytosol and is crucial for the maintenance of the CRAC channel properties. Finally, the C-terminal extremity of all three ORAI present coil-coiled interaction domain (Figure 10). Importantly, both the coil-coiled domains and ETON region were shown to play a role in the STIM-ORAI interaction [START_REF] Derler | The extended transmembrane orai1 N-terminal (ETON) region combines binding interface and gate for orai1 activation by STIM1[END_REF]Park et al, 2009b;Frischauf et al, 2009c). . Overall, the most conserved regions are represented by the TM domains of ORAI proteins: the TM1 is totally conserved among the three human isoforms while TM2 to 4 are overall displaying 81 % and 87 % homology between ORAI1/2 and ORAI1/3 respectively [START_REF] Hogan | Store-operated calcium entry: Mechanisms and modulation[END_REF] (Figure 11). The strong homology found in TM domain of ORAI protein helped to understand some of the main features of the channel they form. For example, two glutamic acid residues localized in the 1 st and 3 rd TM domain of ORAI homologs (E106 and E190 of ORAI1) were identified as crucial for conferring Ca 2+ selectivity to the CRAC channel. Thus, it was shown that the mutation of one of these residues leads to the expression of negative dominant form of ORAI which abolishes the SOCE (Gwack et al, 2007a). Aside from these conserved regions, each of the ORAI proteins also present ORAI1 ORAI2 ORAI3 some unique features. Specifically, the N-and C-termini domain present the lowest homology among the three homologs and these differences are providing specific characteristics to the channels they might form.

1.2.2.3.1. ORAI1

ORAI1 is the longest of the three ORAI proteins with 301 aa compared to the 254 aa of ORAI2 and the 295 aa of ORAI3. This difference in length is due to the presence at the beginning of its N-termini of a unique proline/arginine-rich region. These domains were shown to be responsible for the slow reactivation displayed CRAC channel [START_REF] Frischauf | Cooperativeness of Orai cytosolic domains tunes subtype-specific gating[END_REF]). In addition, some studies revealed that these domains were important for providing strong Ca 2+ current specific to the CRAC channel [START_REF] Takahashi | Essential role of the N-terminus of murine Orai1 in store-operated Ca2+ entry[END_REF][START_REF] Fahrner | Mechanistic view on domains mediating STIM1-Orai coupling[END_REF]. ORAI1 protein is also the only homologue presenting a site for N-glycosylation localized in the 2nd extracellular loop, between the TM3 and the TM4 (Figure 10) (Gwack et al, 2007b). Glycosylation at this residue seem to be cell type dependent, and while it is not strictly required for SOCE activity, some studies indicates that the glycosylation status of ORAI1 can affect the SOCE intensity (Gwack et al, 2007b;[START_REF] Dörr | Cell typespecific glycosylation of Orai1 modulates store-operated Ca2+ entry[END_REF] . Finally, as mentioned above, the three ORAI members possess a coil-coiled interaction domain allowing the interaction between ORAI and STIM proteins (Figure 12 A). Nonetheless, it was demonstrated that ORAI1 possess the "weakest" coilcoiled domain. (Frischauf et al, 2009a). Indeed, mutagenesis experiments showed that a single point mutation (L273S) is sufficient to suppress ORAI1-STIM1 interaction while ORAI2 and ORAI3 require two mutations (L237S + L244S and L285S + L292S respectively) to produce the same effect (Frischauf et al, 2009a). If this coil-coiled region was first demonstrated to be important for ORAI-STIM interaction, the solving of the crystal structure of the CRAC channel formed by ORAI1 in Drosophila melanogaster also indicates that this domain is involved in the interaction between ORAI proteins themselves (Figure 12 B,C) (Xiaowei Hou, Leanne Pedi, Melinda M. Diver, 2012;[START_REF] Tirado-Lee | Conformational changes in the Orai1 C-terminus evoked by STIM1 binding[END_REF]Hou et al, 2018b). Finally, one shorter isoform of the ORAI1 protein was identified named ORAI1 β. This isoform results from the existence of alternative translation initiation codon in the from the ORAI1 gene (Figure 10). This isoform, lacking the proline/arginine rich domain of classic ORAI1, is able to generate classic SOCE but its current present a stronger Ca 2+ dependent inhibition than the classic one. Of note, it was also demonstrated that ORAI1 β diffusion rate in the PM is higher than the classic ORAI1 protein [START_REF] Desai | Multiple types of calcium channels arising from alternative translation initiation of the Orai1 message[END_REF][START_REF] Fukushima | Alternative translation initiation gives rise to two isoforms of Orai1 with distinct plasma membrane mobilities[END_REF][START_REF] Zhang | A calcium/cAMP signaling loop at the ORAI1 mouth drives channel inactivation to shape NFAT induction[END_REF].

1.2.2.3.2. ORAI2
ORAI2 is the shortest of the ORAI isoform and the less studied of ORAI proteins. From the sequence homology, it is assumed that this homologue contains all the following elements from the ORAI1 proteins: The CaM binding domain and the ETON domain in the N-terminal extremity, and the coilcoiled region in its C-termini. As mentioned earlier, ORAI2 does not present the N-terminal prolin/arginin rich region (Figure 11). Overall, ORAI1 and ORAI2 protein present the strongest sequence homology. As a consequence, the biophysical properties of ORAI2 and ORAI1 composed channel are closely related compared to the channel formed by ORAI3 proteins (Gwack et al, 2007a;Lis et al, 2007a).

1.2.2.3.3. ORAI3
The ORAI3 protein is almost as long as ORAI1 (295 aa and 301 aa respectively) but both proteins are showing differences in their structure. As for ORAI2, it lacks the proline/arginin domain present in ORAI1 but conserves the most important elements displayed across the three proteins (ETON domain;

CaM binding domain; coil-coiled region). However, the C and N-termini of ORAI3 only show 34% and 46% sequence homology respectively to ORAI1 [START_REF] Shuttleworth | Orai3 -the 'exceptional' Orai?[END_REF]. In addition, the 2 nd loop, between TM2 and TM3, present non conserved amino acids and simulations demonstrated that this sequence difference was sufficient to alter the structural properties of the channel formed by ORAI3 proteins [START_REF] Fahrner | Communication between N terminus and loop2 tunes Orai activation[END_REF]. Finally, the 3 rd loop, between the TM domain 3 and 4, is sensibly elongated in ORAI1 compared to ORAI3 (38 amino acids versus 72 respectively) but the role of this difference elongated loop remains elusive (Figure 11 ) [START_REF] Shuttleworth | Orai3 -the 'exceptional' Orai?[END_REF]. Of note, since the sequence variation between ORAI3 and ORAI1 are mainly present in the non-pore forming region, it was hypothesized that ORAI3 channels possessed different gating properties than ORAI1 and ORAI2 channels. entry called SOCE. Indeed, SOCE was described long before the identification of its molecular components, and the electrophysiological characteristics of the current it provides were revealed by Hoth and Penner thanks to whole cell patch-clamp recording [START_REF] Hoth | Depletion of intracellular calcium stores activates a calcium current in mast cells[END_REF]. Following its identification, the current responsible for the SOCE was called ICRAC (Ca 2+ release activated Ca 2+ ) and its characteristics includes:

• An inward rectification (non-voltage-activated) [START_REF] Hoth | Depletion of intracellular calcium stores activates a calcium current in mast cells[END_REF] • A high Ca 2+ selectivity and low single channel conductance in the range of femtosiemens [START_REF] Zweifach | Mitogen-regulated Ca2+ current of[END_REF][START_REF] Hoth | Calcium and barium permeation through calcium release-activated calcium (CRAC) channels[END_REF].

• A Ca 2+ dependent inactivation (CDI) which can be separated in two distinct phases: a fast CDI (FCDI) occurring within milliseconds, and visualized during hyperpolarizing voltage steps, and a slow CDI (SCDI) completed within minutes (Zweifach & Lewis, 1995a, 1995b).

Of note, I CRAC first recording were acquired in mast cells and T lymphocytes. However, in different cell types, the currents evoked by SOCE activation presented alternative characteristics (especially different ionic selectivity) ,and were termed ISOC [START_REF] Liu | Distinct Ca2+-permeable cation currents are activated by internal Ca2+-store depletion in RBL-2H3 cells and human salivary gland cells, HSG and HSY[END_REF]. It was demonstrated later that TRP channels were participating in the ISOC currents. This is especially true for the TRPC channels family as it was shown that their activation mechanisms were relying on the Ca 2+ entry induced by ICRAC [START_REF] Ong | Role of TRPC channels in store-operated calcium entry[END_REF][START_REF] Shin | The TRPCs, Orais and STIMs in ER/PM junctions[END_REF]. As a consequence, before the ORAI identification, TRPC channels were thought to be responsible for CRAC [START_REF] Zhu | trp[END_REF][START_REF] Zitt | Cloning and Functional Expression of a Human Ca2+-Permeable Cation Channel Activated by Calcium Store Depletion[END_REF]. Nonetheless, following the identification of the three ORAI homologs, it was demonstrated that each ORAI protein was able to produce ICRAC-like currents when overexpressed together with STIM, indicating that ORAI proteins are forming homo-oligomers supporting SOCE entry (Mercer et al, 2006a). These studies also revealed that it was the ORAI1 protein who was the main responsible of SOCE due to the increased level of Ca 2+

entry it was able to provide. Thus, it is the ORAI1 channel structure that was the most studied.

CRAC channel detailed structure

Early studies aiming to determine the structure of the CRAC channel were using overexpression of concatemers composed of variable number of ORAI1, or single-molecule imaging techniques in combination with biochemical methods. These data pointed out that the CRAC channel was constituted of 4 units of ORAI1 (Mignen et al, 2008b;[START_REF] Penna | The CRAC channel consists of a tetramer formed by Stim-induced dimerization of Orai dimers[END_REF]. In addition, Dr Penna indicated that in resting cells, ORAI1 were forming dimers and were further assembled in tetramers due t the channel activation. During the following year it remained controversial whether ORAI1 channel were indeed dimers, tetramers or could even possess higher oligomeric states depending on their activation status.

While a variety of techniques, such as electron microscopy [START_REF] Maruyama | Tetrameric Orai1 is a teardrop-shaped molecule with a long, tapered cytoplasmic domain[END_REF], biochemical analysis (Gwack et al, 2007a;[START_REF] Zhou | Pore architecture of the ORAI1 store-operated calcium channel[END_REF], imaging methods like single molecule photobleaching (Ji et al, 2008a;[START_REF] Penna | The CRAC channel consists of a tetramer formed by Stim-induced dimerization of Orai dimers[END_REF] or brightness analysis (Madl et al, 2010a) were used to elucidate the composition of CRAC channels, its definitive structure was established in 2012 with the crystallization of the drosophila CRAC channel (Xiaowei Hou, Leanne Pedi, Melinda M. Diver, 2012) (Figure 13).

The 1 st crystal structure of the CRAC was obtained while the channel was in its close-state ORAI channel, it revealed the CRAC channel was a hexamer of 6 ORAI1 proteins. Since the human and drosophila ORAI1 are sharing a strong homology (≈ 78%) it was quickly admitted that the human CRAC channel composition possessed a similar structure. Later, the crystal structure of the open-state CRAC channel was obtained by the same group thanks to constitutively active mutant of drosophila ORAI1 (Hou et al, 2018a). Finally, a high resolution (3,3 Å versus 6,7 Å previously) picture of the CRAC channel in its open-state structure was obtained with Cryo-EM technique and revealed new structural details [START_REF] Hou | Cryo-EM structure of the calcium release-activated calcium channel Orai in an open conformation[END_REF]. In addition, these new structural data allowed the author to propose a model for the CRAC channel opening involving two strong conformational changes in the middle of the 4 th TM and in between the 4 th TM and the TM4 extension allowing the unlatching of the channel preceding its opening (Figure 13). Taken together, these data allowed a clearer depiction of the CRAC channel and of its important domain. Hence, unlike any other channel the pore of the CRAC channel is formed only by the 1 st TM of the six ORAI1 units. The three other TM are forming a shell that surrounds the pore with the 4 th TM being at the periphery of the channel. The importance of each TM domain, suggested by their significant degree of conservation across ORAI homologs, was highlighted by their specific structure as they are all presenting a helical extension in the cytoplasm (Figure 13). Especially, it is admitted that dilatation of the 1 st TM especially its ETON extension is responsible for pore opening (Tiffner & Derler, 2021;Tiffner et al, 2021). The specific configuration of the 4 th TM and its cytosolic extension was also shown to be essential for STIM binding, conformational change, and pore opening.

Specifically, the 4 th TM present a bend (at the position P261 for the human ORAI1) in its structure dividing it in two regions: TM4a and TM4b. Following the 4 th TM, a hinge region consisting of SHK (Serine -Histidine -Lysine) motif links the TM with the TM4 helical extension (Figure 11 and 13).

This specific structure was suggested to be important for the transmission of the conformational changes inducing pore opening to the whole protein through modification of its coil-coiled interaction domain (Hou et al, 2012a(Hou et al, , 2018b)). Additionally, the intracellular loop (loop2) was also shown to be particularly important for the maintenance of ORAI1 channel properties through a specific interaction with their Nterminal extremity [START_REF] Fahrner | Communication between N terminus and loop2 tunes Orai activation[END_REF]. Importantly, despite the solving of the CRAC channel structure, the exact structural modification leading to pore opening are not yet totally defined. The unveiling of the CRAC channel structure allowed a better comprehension of its gating by STIM1. Even though some mechanisms are still debated, a global picture of the CRAC activation through STIM1 gating has now emerged. Specifically, following their translocation to ER-PM junction due to Ca 2+ store depletion, STIM proteins trap ORAI through interaction between their respective coilcoiled domains. Importantly, several mutation of specific amino acids in the CAD/SOAR domain (CC2 and CC3) of STIM1 as well as in the C-terminal coil-coil domain of ORAI1 were shown to impact ORAI1-STIM1 interaction and gating (Park et al, 2009b;[START_REF] Muik | Dynamic coupling of the putative coiled-coil domain of ORAI1 with STIM1 mediates ORAI1 channel activation[END_REF][START_REF] Stathopulos | STIM1/Orai1 coiled-coil interplay in the regulation of store-operated calcium entry[END_REF][START_REF] Navarro-Borelly | STIM1-Orai1 interactions and Orai1 conformational changes revealed by live-cell FRET microscopy[END_REF]. In addition, N-termini and loop 2 of ORAI were also shown to participate in STIM interaction although to a lesser extent. The current hypothesis point-out the possibility that, following the establishment of a coil-coiled interaction between ORAI1 and STIM1 C-termini, a conformational change is propagated through the ORAI1 loop2 until the 1 st TM and the ETON domain leading to pore opening [START_REF] Palty | Sequential Steps of CRAC Channel Activation[END_REF][START_REF] Liu | Molecular understanding of calcium permeation through the open Orai channel[END_REF][START_REF] Niu | STIM1 interacts with termini of Orai channels in a sequential manner[END_REF] . This hypothesis proposes an explanation to the observations indicating that ORAI1 N-termini and STIM1 are interacting, but that mutation in ORAI1 N-terminal region is not abolishing SOCE [START_REF] Fahrner | Communication between N terminus and loop2 tunes Orai activation[END_REF][START_REF] Li | Mapping the Interacting Domains of STIM1 and Orai1 in Ca2+ Release-activated Ca2+ Channel Activation[END_REF][START_REF] Mcnally | The C-and Nterminal STIM1 binding sites on Orai1 are required for both trapping and gating CRAC channels[END_REF][START_REF] Butorac | A novel STIM1-Orai1 gating interface essential for CRAC channel activation[END_REF]. In addition, interactions between all cytosolic parts of ORAI1

(N-t, loop2 and C-t) and STIM1 were identified, but their respective contribution to the gating of the channel remains elusive. This can be partially attributed to the "weak" level of interaction between ORAI and STIM preventing the purification of the associated structures and compelling interaction studies to be conducted using fragments of each proteins [START_REF] Qiu | Structural features of STIM and Orai underlying store-operated calcium entry[END_REF]). An additional question arising from the ORAI1-STIM1 gating mechanism is the number of STIM1 and ORAI1 protein required to gate the channel. It is admitted that the trapping of CRAC channels by STIM1 is preceding its activation. For example, it was demonstrated that a single STIM1 dimer was sufficient to trap, but not to activate the CRAC channel [START_REF] Hoover | Stoichiometric requirements for trapping and gating of Ca 2+ releaseactivated Ca 2+ (CRAC) channels by stromal interaction molecule 1 (STIM1)[END_REF]. However, the exact STIM1:ORAI1 stoichiometry required to activate the CRAC channel remain subject to discussion, and two main hypothesis co-exists named the bimolecular and unimolecular models.

• The dimeric model proposes that SOAR/CAD domain of STIM1 dimers are interacting with two ORAI1 protein from the same CRAC channel, leading to a 1:1 stoichiometry (1 STIM protein for 1 ORAI1 in 1 CRAC channel) (Figure 14). This hypothesis is supported by NMR and FRET data [START_REF] Stathopulos | STIM1/Orai1 coiled-coil interplay in the regulation of store-operated calcium entry[END_REF][START_REF] Yen | Physiological CRAC channel activation and pore properties require STIM1 binding to all six Orai1 subunits[END_REF] but it does contradicts the structural evidence from the published CRAC open-state crystal structure. However, one cannot exclude that this contradiction is imputable to the utilization of constitutively active mutant that do not display STIM interaction for the crystallization experiments.

• The alternative unimolecular model proposes that STIM1 dimers are interacting with ORAI1 protein from different CRAC channel assemblies (Figure 14). Such model leads to a 2:1 stoichiometry (STIM1:ORAI1) and to clustering of ORAI channel which classically observed in overexpression models (Park et al, 2009b;[START_REF] Perni | Nanoscale patterning of STIM1 and Orai1 during store-operated Ca 2+ entry[END_REF]Zhou et al, 2018b;[START_REF] Baraniak | Orai channel C-terminal peptides are key modulators of STIM-Orai coupling and calcium signal generation[END_REF]. Interestingly, both model could actually co-exist and their specific proportion be dependent on the activation level of SOCE as proposed by Palty and al [START_REF] Palty | Sequential Steps of CRAC Channel Activation[END_REF].

ISOC

As mentioned earlier, some cell types, are displaying SOCE current presenting different characteristics than the classic I CRAC. Especially different Ca 2+ selectivity was observed. This alternative currents were named ISOC [START_REF] Liu | Distinct Ca2+-permeable cation currents are activated by internal Ca2+-store depletion in RBL-2H3 cells and human salivary gland cells, HSG and HSY[END_REF][START_REF] Liu | Ca2+-dependent Inactivation of a Store-operated Ca2+ Current in Human Submandibular Gland Cells[END_REF]. In fact, depending on the cell types, Ca 2+ store depletion induces a variety of different Ca 2+ current suggesting that several channels are participating in SOCE [START_REF] Parekh | Store-Operated Calcium Channels[END_REF][START_REF] Cheng | Contribution and Regulation of TRPC Channels in Store-Operated Ca2+ Entry[END_REF]. For this reason, it was early suggested that TRPC1

was the channel responsible for SOCE. For example, early data on SOCE showed that absence or downregulation of TRPC1 was leading to impaired SOCE while its overexpression was leading to an increased SOCE in different cell models such as salivary glands, or endothelial cells [START_REF] Sun | TRPC1 regulates calcium-activated chloride channels in salivary gland cells[END_REF][START_REF] Ma | Heteromeric TRPV4-C1 channels contribute to store-operated Ca2+ entry in vascular endothelial cells[END_REF][START_REF] Liu | Attenuation of store-operated Ca2+ current impairs salivary gland fluid secretion in TRPC1( / ) mice[END_REF][START_REF] Brough | Contribution of endogenously expressed Trp1 to a Ca2+-selective, store-operated Ca2+ entry pathway[END_REF][START_REF] Liu | Erratum: Trp1, a candidate protein for the store-operated Ca2+ influx mechanism in salivary gland cells[END_REF][START_REF] Rosado | Endogenously expressed Trp1 is involved in storemediated Ca2+ entry by conformational coupling in human platelets[END_REF]. The identification of ORAI proteins together with the decrease in SOCE observed while altering TRPC1 expression led to the hypothesis that both proteins could form a heteromeric channel [START_REF] Liao | Functional interactions among Orai1, TRPCs, and STIM1 suggest a STIM-regulated heteromeric Orai/TRPC model for SOCE/Icrac channels[END_REF][START_REF] Kwong | Functional requirement for Orai1 in storeoperated TRPC1-STIM1 channels[END_REF][START_REF] Huang | STIM1 carboxylterminus activates native SOC, Icrac and TRPC1 channels[END_REF][START_REF] Kim | Native Store-operated Ca2+ Influx Requires the Channel Function of Orai1 and TRPC1[END_REF], or could form dynamic assemblies all together with STIM1 [START_REF] Hwei | Dynamic assembly of TRPC1-STIM1-Orai1 ternary complex is involved in store-operated calcium influx: Evidence for similarities in store-operated and calcium release-activated calcium channel components[END_REF]. In fact, various microscopy techniques such as FRET and TIRF, demonstrated that STIM1 interacts with TRPC1 [START_REF] Huang | STIM1 carboxylterminus activates native SOC, Icrac and TRPC1 channels[END_REF][START_REF] Hwei | Dynamic assembly of TRPC1-STIM1-Orai1 ternary complex is involved in store-operated calcium influx: Evidence for similarities in store-operated and calcium release-activated calcium channel components[END_REF][START_REF] Alicia | STIM1 converts TRPC1 from a receptoroperated to a store-operated channel: Moving TRPC1 in and out of lipid rafts[END_REF][START_REF] Lee | Molecular determinants mediating gating of transient receptor potential canonical (TRPC) channels by stromal interaction molecule 1 (STIM1)[END_REF]. Additionally, it was demonstrated that the Ca 2+ entry induced by CRAC channel activation leads to the addressing of TRPC1 to the plasma membrane, indicating that the gating of TRPC1 is strictly dependent on the presence of both STIM1 and ORAI1 [START_REF] Lee | An endoplasmic reticulum/plasma membrane junction: STIM1/Orai1/TRPCs[END_REF][START_REF] Cheng | Local Ca2+ entry via Orai1 regulates plasma membrane recruitment of TRPC1 and controls cytosolic Ca2+ signals required for specific cell functions[END_REF]. In addition, it was later demonstrated that ORAI1 and TRPC1 channel are found in close vicinity, indicating on the existence of dynamic assemblies of protein complexes including ORAI1, TRPC1 and STIM1. Nonetheless, it was demonstrated that ORAI and TRPC1 proteins, both form two different channels ruling-out the existence of heteromeric ORAI1-TRPC1 channels (Ambudkar et al, 2017 (Lis et al, 2007a). These data have allowed to define the biophysical characteristics of the currents provided by these homomeric channels (Table 1) (Lis et al, 2007b;[START_REF] Dehaven | Calcium inhibition and calcium potentiation of Orai1, Orai2, and Orai3 calcium release-activated calcium channels[END_REF]DeHaven et al, , 2008)). Thus, while ORAI1-only channels (i.e the prototypic CRAC channel) are displaying the greatest current amplitude, ORAI2-only channels present an intermediate level of current and ORAI3-only channel the smallest. The activation time also differs within these channels with ORAI2-only channels presenting the fastest activation time while ORAI3-only are displaying the slowest. Additionally, the Ca 2+ inactivation features of these channel are also different. Of note, ORAI1 channel display a Ca 2+ -dependent inactivation (CDI) that is be decomposed in fast inactivation (FCDI) and slow inactivation (SCDI), interspersed by a small reactivation phase. The reactivation phase is absent in both ORAI2-and ORAI3-only channels as well as the SCDI. On the other hand, while the FCDI of ORAI2 is similar to the one observed for ORAI1, FCDI of ORAI3 is significantly stronger. In term of selectivity, all channels are highly selective for Ca 2+ over other ions. Nonetheless, ORAI3-only channels display larger Na + permeation in DVF solution [START_REF] Dehaven | Calcium inhibition and calcium potentiation of Orai1, Orai2, and Orai3 calcium release-activated calcium channels[END_REF]. Finally, all these ORAI channels present different sensitivities to the chemical compound 2-APB (2-aminoethyldiphenyl borate). This compound potentiate ICRAC in ORAI1 channels at low concentration and blocks it at high concentration, ORAI2 channels were shown to be less sensitive to its inhibition, while ORAI3 channels are never inhibited by 2-APB but rather activated (Lis et al, 2007b). 2-APB mediating its effect via binding on the extracellular motifs of ORAI channels, it was suggested that its specific effect on the ORAI3 channel could be attributed to the significantly longer 2 nd extracellular loop (between TM3 and TM4). In addition, it was shown that Orai3-channel activation by 2-APB was modifying its selectivity by allowing entry of monovalent ions probably through widening its pore [START_REF] Schindl | 2-Aminoethoxydiphenyl borate alters selectivity of Orai3 channels by increasing their pore size[END_REF]. Nonetheless, the physiological relevance of these differences remains under question since ORAI2-only and ORAI3-only channels current were never observed endogenously. In addition, these studies were conducted in HEK-293 cells expressing endogenous ORAI1 protein, and thus allowing the integration of ORAI1 proteins in channels supposedly composed of only ORAI2 or ORAI3 units. Nonetheless, Orai2 was reported to be present into intracellular compartment including the ER [START_REF] Ikeya | Orai-2 is localized on secretory granules and regulates antigen-evoked Ca2+ mobilization and exocytosis in mast cells[END_REF]. Moreover, another study have reported a potential role for endogenous Orai2 channel as ER Ca 2+ leak channel by using siRNA [START_REF] Bandara | Regulators of Calcium Homeostasis Identified by Inference of Kinetic Model Parameters from Live Single Cells Perturbed by siRNA[END_REF]. Thus ORAI2-only channels might represent a specific type of ER channels. On a similar note, it has been hypothesized that Orai3 channels exist on the ER membrane. Indeed, few publications described intracellular localization when overexpressing fluorescently labeled Orai3 proteins [START_REF] Varadarajan | Endoplasmic Reticulum Membrane Reorganization Is Regulated by Ionic Homeostasis[END_REF][START_REF] Dickson | Orai1 and Orai2 mediate store-operated calcium entry that regulates HL60 cell migration and FAK phosphorylation[END_REF]. Additionally, it was shown that the absence of the tripeptide EFA in Orai3

C-terminus prevent its addressing to the PM which is dependent of the presence of Orai1 (Alansary et al, 2015). Following these clues, Leon-Aparicio and colleagues demonstrated in 2017 that in HeLa cells, Orai3 channels are located on the ER and plays a role in preventing ER Ca 2+ overloading (Leon-Aparicio et al, 2017). On the other hand, the existence of ORAI3-only channels was suggested by Motiani and al., who demonstrated that in estrogen receptor-negative breast cancer cells, ORAI3 was alone responsible for the native SOCE [START_REF] Motiani | A novel native store-operated calcium channel encoded by Orai3: Selective requirement of Orai3 versus Orai1 in estrogen receptor-positive versus estrogen receptor-negative breast cancer cells[END_REF]. Few other publication also demonstrates that ORAI3 was responsible for SOCE in some cancer indicating on a potential existence of ORAI3 channels [START_REF] Faouzi | Downregulation of Orai3 arrests cell-cycle progression and induces apoptosis in breast cancer cells but not in normal breast epithelial cells[END_REF][START_REF] Ay | Erratum: Orai3 Constitutes a Native Store-Operated Calcium Entry That Regulates Non Small Cell Lung Adenocarcinoma Cell Proliferation[END_REF].

Heteromeric ORAI channels in SOCE

The first studies assessing the physiological properties of ORAI composed channels suggested the existence of ORAI heteromeric channels (Lis et al, 2007b;[START_REF] Dehaven | Calcium inhibition and calcium potentiation of Orai1, Orai2, and Orai3 calcium release-activated calcium channels[END_REF]Gwack et al, 2007a).

Nonetheless, physiological evidence of the existence of such channels were lacking for a long time.

Recently, several works have identified heteromeric ORAI1-ORAI2 and ORAI1-ORAI3 channels involved in SOCE.

• ORAI1-ORAI2

In 2015, Inayama and al., demonstrated that ORAI2 is abundantly expressed in the human chondrocyte cell-line OUMS-27. They showed that ORAI2 down-regulation or overexpression was affecting the SOCE amplitude, indicating on a role of ORAI2 in controlling SOCE. They further confirmed their hypothesis that ORAI2 and ORAI1 were forming a functional channel, by using a combination of microscopy techniques such as TIRF, single molecule photobleaching assays and bifunctional complementation assays (BiFC) (Inayama et al, 2015a). On a similar note, in 2017 Vaeth and al., demonstrated that ORAI1 and ORAI2 can form heteromeric channel that modulate the SOCE amplitude in mice T-cells. In fact, by overexpressing pore-dead mutant of ORAI1 or ORAI2 protein in knock-out models they were demonstrated that the function of the wild-type remaining ORAI was suppressed. Further patch-clamp analysis demonstrated that ORAI1-ORAI2 channels maintain the essential CRAC channel properties while presenting few differences in their ICRAC current compared to the ORAI1-only channel. Finally, they also show that the inhibitory effect of ORAI2 on the CRAC function was present in multiple human immune cells and that the ratio of expression between ORAI1 and ORAI2 was evolving during maturation process of T-cells. Thus, they hypothesized that the dynamic variation in the CRAC channel stoichiometry could be a mechanism to modulate SOCE (Vaeth et al, 2017a).

• ORAI1-ORAI3

The existence of ORAI1-ORAI3 channels responsible for SOCE was mainly proposed in the pathological process of cancer. For example, Motiani et al., were the first to demonstrate that SOCE, in estrogen-receptor positive (ER + ) breast cancer cell, was mediated by ORAI3, while estrogen-negative (ER -) cells were displaying classic ORAI1-mediated SOCE [START_REF] Motiani | A novel native store-operated calcium channel encoded by Orai3: Selective requirement of Orai3 versus Orai1 in estrogen receptor-positive versus estrogen receptor-negative breast cancer cells[END_REF]. A role of ORAI3 in SOCE induction was further demonstrated for other cancer cell lines such as non-small cell lung adenocarcinoma and pancreatic ductal adenocarcinoma (PDAC) cell lines [START_REF] Ay | Erratum: Orai3 Constitutes a Native Store-Operated Calcium Entry That Regulates Non Small Cell Lung Adenocarcinoma Cell Proliferation[END_REF][START_REF] Dubois | ORAI3 silencing alters cell proliferation and promotes mitotic catastrophe and apoptosis in pancreatic adenocarcinoma[END_REF]. Additionally, Dubois et al., also demonstrated a role for ORAI3 in SOCE in normal prostatic cell-line [START_REF] Dubois | ORAI3 silencing alters cell proliferation and promotes mitotic catastrophe and apoptosis in pancreatic adenocarcinoma[END_REF](Dubois et al, , 2014a)). Finally, Yoast and al., presented the strongest evidence of the existence of native heteromers between ORAI1, 2 and 3 and their role in physiology. By creating single, double and triple knockout cells for these proteins and using agonist leading to incomplete store depletion (mimicking physiological activation), they demonstrated that native CRAC channel are heteromers bearing different sensitivities to Ca 2+ store depletion. The proportion of these channels in specific cell-line would lead to different physiological answer depending on the intensity of the stimulation they receive. Of note, ORAI1 and ORAI3 are also participating in the formation of a second channel bearing different properties and being non-Store-Operated named arachidonate-regulated Ca 2+

(ARC) channel that will be describe thereafter.

1.2.2.5.3. Heteromeric non-SOCE channel, ARC/LTR Following the identification of ORAI protein, and the discovery that ORAI1 form the CRAC channel responsible for the SOCE, it was suggested that ORAI1 and ORAI3 can form another channel responsible for a store independent Ca 2+ entry (SICE). This channel was named arachidonate-regulated Ca 2+ (ARC) channel as it could be activated by low concentration of arachidonic acid (AA) (Mignen et al, 2008a). The ARC channel was shown to possess different properties compared to the CRAC channel (Table 2). Especially, ARC channels do not show Ca 2+ -dependent inhibition neither display sensitivity to the pharmacological agent 2-aminoethoxydiphenyl borate (2-APB) [START_REF] Mignen | I(ARC), a novel arachidonate-regulated, noncapacitative Ca2+ entry channel[END_REF], 2001). Additionally, ARC channels can exhibit a monovalent cation permeability in the absence of divalent cations [START_REF] Zhang | Complex role of STIM1 in the activation of store-independent Orai1/3 channels[END_REF]. The composition of this channel was established in 2009, with the usage of concatemeric constructs with different proportion of ORAI1 and ORAI3 subunits. This approach allowed the authors to demonstrate that the properties of the native IARC current were restored with the expression of hetero-pentameric assemblies of ORAI1 and ORAI3 proteins organized as follow:

31113 or 31311 (where 3 indicates ORAI3 proteins, and 1 ORAI1 proteins) [START_REF] Mignen | The molecular architecture of the arachidonateregulated Ca2+ -selective ARC channel is a pentameric assembly of Orai1 and Orai3 subunits[END_REF]. It was further demonstrated that this channel could be more robustly activated by leukotriene C4 (LTC4), a metabolite product of AA. Therefore, this channel could be referred in literature as ARC, LTC4regulated Ca2+ (LRC), or ARC/LRC [START_REF] González-Cobos | Store-independent Orai1/3 channels activated by intracrine leukotrienec4: Role in neointimal hyperplasia[END_REF][START_REF] Zhang | Complex role of STIM1 in the activation of store-independent Orai1/3 channels[END_REF]. While this channel present distinct feature from CRAC channel, several studies indicated that its activation was dependent on the presence of STIM1 protein although the activation of the channel was shown to be Ca 2+ store independent. Thus, early studies on ARC channel performed by Mignen and al., claimed that a small pool of STIM1 localized at the PM was required to provide functional ARC current [START_REF] Mignen | STIM1 regulates Ca2+ entry via arachidonateregulated Ca2+ -selective (ARC) channels without store depletion or translocation to the plasma membrane[END_REF].

Additionally, Trebbak's team indicated that ER-STIM was sufficient to activate ARC/LRC channel and that PM-STIM was simply playing a facilitator role in ARC/LRC activation [START_REF] Zhang | Complex role of STIM1 in the activation of store-independent Orai1/3 channels[END_REF]. In any way, interaction between ORAI3 and STIM was shown to be mediated through their CC domain and to be mandatory for the ARC-LRC activation by LTC4 [START_REF] Zhang | Mechanisms of STIM1 Activation of Store-Independent Leukotriene C4-Regulated Ca2+ Channels[END_REF]. SOCE is a ubiquitous process, and its main function is to induce Ca 2+ store refilling following depletion [START_REF] Putney | C4D3FB725CBAF80E0E0911B1F0B37AA65DC3A14A180AFF2E1E02310[END_REF]. Even though this function of store refilling was initially thought to be fundamental, the importance of this process can be questioned since absence of SOCE does not prevent fetal development until birth. Nonetheless, humans presenting SOCE deregulation display strong deficiencies resulting different syndrome ultimately leading to a premature death (Lacruz & Feske, 2015a). ORAI1 representing the principal SOCE effector, its normal functionality is crucial for several cell types. Specifically, ORAI1 is particularly important for the maintenance of normal of immune cell function [START_REF] Vig | Defective mast cell effector functions in mice lacking the CRACM1 pore subunit of store-operated calcium release-activated calcium channels[END_REF][START_REF] Feske | ORAI1 and STIM1 deficiency in human and mice: roles of store-operated Ca 2+ entry in the immune system and beyond[END_REF]. Aside from immune cells, ORAI1 is also a fundamental actor of the normal physiology of several cell types including cardiomyocytes, endothelial cells, and skeletal muscle (Tiffner & Derler, 2021;[START_REF] Michelucci | Role of STIM1/ORAI1mediated store-operated Ca2+ entry in skeletal muscle physiology and disease[END_REF]. The physiological functions of ORAI2 and ORAI3 are less clearly define because their expression pattern overlaps with ORAI1, and they are usually expressed at a lower level than the later. than. In fact, it was suggested that the lack of one homologues could be compensated by affecting the expression of another [START_REF] Feske | CRAC channelopathies Stefan[END_REF]. Nonetheless, the strongest indication of a physiological role of ORAI2 is related to the maturation of innates immune cells. Indeed, depending on their maturation level, innate cells present different expression ratio of the different ORAI proteins homologues. It was further demonstrated in murine and human cell line that the inclusion of ORAI2 in the CRAC channel was leading to modification of SOCE and thus modulating the immune responses provided by these cells [START_REF] Vaeth | ORAI2 modulates store-operated calcium entry and T cell-mediated immunity[END_REF]Inayama et al, 2015a;[START_REF] Gross | Murine ORAI2 splice variants form functional Ca2+ release-activated Ca2+ (CRAC) channels[END_REF]. As for a role of ORAI3 in physiology, this homologue has been identified as the main actor of SOCE in rat cardiac hypertrophy model (Saliba et al, 2015a). Overall, the main function of ORAI2 and ORAI3 seem to consist in the fine-tuning of Ca 2+ signals of various cell types such as cardiac and skeletal muscle cells (Tiffner & Derler, 2021). Of note, this fine-tuning mostly results in SOCE impairment, as it was demonstrated in dental enamel, T cells and mast cells (Yoast et al, 2020a;[START_REF] Vaeth | ORAI2 modulates store-operated calcium entry and T cell-mediated immunity[END_REF]Eckstein et al, 2019a;[START_REF] Tsvilovskyy | Deletion of Orai2 augments endogenous CRAC currents and degranulation in mast cells leading to enhanced anaphylaxis[END_REF].

1.2.3.1.2.
Role of ORAI and store independent Ca 2+ entry (SICE) in physiology

The physiological role of the ARC/LRC channel is not clearly defined as it was less studied than CRAC channel. Nonetheless, few physiological roles for these channels were identified. For example, AAinduced Ca 2+ entry was shown to promote proliferation of bovine endothelial cells [START_REF] Pla | Calcium influx, arachidonic acid,and control of endothelialcell proliferation[END_REF]. In human, it was shown that vascular endothelial growth factor (VEGF) induces ORAI3 expression at the cell surface leading cell remodeling [START_REF] Li | Orai3 Surface Accumulation and Calcium Entry Evoked by Vascular Endothelial Growth Factor[END_REF] Additionally, it was proposed that the remodeling of ORAI1/ORAI3 participate in the neointima formation after the vascular injury suggesting the existence of a remodeling mechanism [START_REF] González-Cobos | Store-independent Orai1/3 channels activated by intracrine leukotrienec4: Role in neointimal hyperplasia[END_REF]. Finally, ORAI3 knockdowns were shown to impairs the migration of vascular smooth muscle cells [START_REF] Zhang | Complex role of STIM1 in the activation of store-independent Orai1/3 channels[END_REF]) (Zhang et al, 2015a) along with non-syndromic tubular aggregate myopathy (TAM) consisting of myopathy, thrombocytopenia, thrombopathy and bleeding diathesis (Lacruz & Feske, 2015a). Aside from genetic pathologies, expression deregulation was also shown to be the cause of pathologies. For example, in skeletal muscle, enhanced SOCE level attributed to ORAI1/STIM1 overexpression were linked to promotion of dystrophic phenotype in muscular dystrophia. Additionally, SOCE impairment caused by diminution of STIM/ORAI expression was also associated to muscle aging and fatigue susceptibility [START_REF] Michelucci | Role of STIM1/ORAI1mediated store-operated Ca2+ entry in skeletal muscle physiology and disease[END_REF]. Moreover, changes in ORAI channels expression including the relative expression level of the different homologs was also suggested to participate in cancer development [START_REF] Chalmers | ORAI channels and cancer[END_REF]) (Table 3). For example, deregulations in ORAI1 expression were identified in liver, stomach, renal cancer [START_REF] Tang | Inhibition of Orai1-mediated Ca2+ entry enhances chemosensitivity of HepG2 hepatocarcinoma cells to 5-fluorouracil[END_REF][START_REF] Zhu | Elevated Orai1 expression mediates tumor-promoting intracellular Ca2+ oscillations in human esophageal squamous cell carcinoma[END_REF][START_REF] Xia | Elevated Orai1 and STIM1 expressions upregulate MACC1 expression to promote tumor cell proliferation, metabolism, migration, and invasion in human gastric cancer[END_REF][START_REF] Kim | Orai1 and STIM1 are critical for cell migration and proliferation of clear cell renal cell carcinoma[END_REF]. Additionally, ORAI2 was also proposed to undergo expression alteration in acute myeloid leukemia cell line (Diez-Bello et al, 2017). Finally, ORAI3 expression has been shown to be increased in estrogen positive breast cancer cells as well as in lung and pancreatic cancer [START_REF] Ay | Erratum: Orai3 Constitutes a Native Store-Operated Calcium Entry That Regulates Non Small Cell Lung Adenocarcinoma Cell Proliferation[END_REF][START_REF] Benzerdjeb | Orai3 is a predictive marker of metastasis and survival in resectable lung adenocarcinoma[END_REF][START_REF] Dubois | ORAI3 silencing alters cell proliferation and promotes mitotic catastrophe and apoptosis in pancreatic adenocarcinoma[END_REF]. Importantly, it was also shown that, by modifying Ca 2+ homeostasis, alteration in ORAI expression was affecting cancer cell properties including proliferation, migration, apoptosis [START_REF] Chalmers | ORAI channels and cancer[END_REF].

1.2.3.2.2. Role of ORAI and store independent Ca 2+ entry (SICE) in pathology

Few studies indicated on a potential involvement of ARC/LRC channels in pathologies. For example, it was suggested that ARC/LRC channels participate in asthma disease by dysregulating the Ca 2+ signalization [START_REF] Thompson | Arachidonate-regulated Ca2+ influx in human airway smooth muscle[END_REF]. Additionally, it was shown that cardiomyocytes display a Ca 2+ permeance when exposed to AA which was increased during cardiac hypertrophy (Saliba et al, 2015b).

Interestingly, it was shown that ORAI3 was upregulated after vascular injury in vascular smooth muscle cells (VSMC), and it was proposed that the remodeling of ORAI1/ORAI3 participate in the neointima formation after the vascular injury [START_REF] González-Cobos | Store-independent Orai1/3 channels activated by intracrine leukotrienec4: Role in neointimal hyperplasia[END_REF]. Finally, remodeling in ORAI1/ORAI3 channel composition was also shown to be implicated in prostate cancer. The authors proposed that the increased expression of ORAI3 observed in prostate cancer cell line favors the formation of heteromeric ORAI1/ORAI3 channels at the expense of the classic CRAC channel therefore providing a new store ARC-like independent Ca 2+ entry promoting cancer cell proliferation (Dubois et al, 2014b).

Evidence for an ORAI proteins dynamic associations

Since their identification 15 years ago, the importance of ORAI proteins in physiology and pathology has been demonstrated several times. Their ability to form channels of different stoichiometry is somewhat unique and studies exploring the functions and mechanisms of ORAI proteins association when forming channels are just emerging. In the recent years, our laboratory suggested that ORAI1 and ORAI3 proteins were responsible for the existence of a heteromeric channel bearing distinct properties than the classically described ARC channel. In addition, this publication highlighted that ORAI channels composition was susceptible to modification during cancerization process, but also depending on the stimulation received by the cell (Dubois et al, 2014b). Their conclusions led them to propose the existence of a dynamic remodeling in the ORAI channel composition. A similar hypothesis was latter suggested by another group who suggested that ORAI channels could be assembled "on site" via the α-SNAP protein (Li et al, 2016a). Finally, the recent demonstration by Trebak's team that native ORAI channels are heteromeric and that their compositions modulates the properties of SOCE also point towards the existence of mechanisms regulating the composition of ORAI channels (Yoast et al, 2020b).

Because structural studies on ORAI channels were focusing on the activation of the SOC channel and its specific gating by STIM, the mode of association within ORAI proteins in the formation of functional channels remain elusive. As a consequence, the modifications of interactions between ORAI proteins within a channel are not well documented. While biochemical studies were proven powerful in the study of protein-protein interactions, the identification of modulation of interaction is not straightforward and usually relies on the use of highly specific antibodies which are lacking for OAI homologues. On the other hand, microscopy also represent a powerful tool to study protein-protein interactions. In addition, the development of high-end microscopy techniques and their increased accessibility even for non-specialist user have made them a powerful alternative strategy for studying ORAI channel specificities.

Thus, the main aim of this PhD was to study the interaction between ORAI proteins with microscopy techniques. Especially, we wanted to implement the fluorescence lifetime imaging of Förster resonance energy transfer (FLIM-FRET) for studying the interaction between the ORAI1 and ORAI3 proteins.

Before developing the different objectives of this work, we will introduce the main microscopy techniques which were already used to study ORAI protein. In addition, a specific focus on the FLIM FRET technique principle and application will be described. Finally, because some of the limitation in the use of microscopy techniques could be overcome through the use of genome editing techniques, a brief introduction of the CRISPR-Cas9 mechanism will be presented thereafter.

1.3. Microscopy to study ion channels 1.3.1. Invention and popularization of the microscopes.

Allegedly, ancient Chinese texts are reporting the use of water microscope, a tube filled with different level of water depending on the magnification level required, more than 4000 years ago (History of Microscopes | Microscope.com). Nonetheless, the first reference indicating on the existence of microscopic device is dated from the 8th century BC as hieroglyphs dated from this age are thought to be representing glass lenses [START_REF] Enoch | Remarkable lenses and eye units in statues from the Egyptian Old Kingdom (ca. 4500 years ago): properties, timeline, questions requiring resolution[END_REF]. The physical proof suggesting the existence of microscopes is dated to 750 BC, and is represented by a polished piece of rock crystal found in the Assyrian palace of Nimrud (Figure 15A) [START_REF] Kriss | History of the Operating Microscope: From Magnifying Glass to Microneurosurgery[END_REF]. Its use as a magnifying glass, a burning glass or simply as ornaments is debated, however it appears evident that this object was deliberately manufactured as a lens. More formal proofs on the usage of magnifying devices are dated from Greeks and Romans writing. For instance, scriptures from the 1st century attributed to Seneca, the Nero emperor's tutor, are reporting the usage of a globe of glass filled with water used to enlarge letters [START_REF] Kriss | History of the Operating Microscope: From Magnifying Glass to Microneurosurgery[END_REF][START_REF] Bardell | The Biologists' Forum: The invention of the microscope[END_REF]. However, it is only in 10th century that the Arab mathematician and physicist Alhazen investigate, in the Book of Optics, the principles leading to magnification of object seen through a lens (Figure 15B) [START_REF] Deek | Complex actions of 2aminoethyldiphenyl borate on store-operated calcium entry[END_REF]. Three centuries later, a Florentine manuscript describing the use of spectacles represent the first practical application of the magnifying power of lenses [START_REF] Kriss | History of the Operating Microscope: From Magnifying Glass to Microneurosurgery[END_REF]. Around the same period, Roger Bacon, deepening the work of Alhazen, designed an instrument aiming to "help the aged and those with weak eyes to see enlarged letters" [START_REF] Bacon | Opus majus[END_REF] and named it eyeglass. Shortly after, the basic magnifying constituents of the eyeglasses were called lens [START_REF] Bardell | The Biologists' Forum: The invention of the microscope[END_REF]. It took three more centuries to appreciate the full potential of lenses for scientific purposes with the development of the first telescopes and microscopes around 1600. Their inventions are closely related since both instruments are relying on the alignment of two lenses to induce image magnification. The discovery of this principle is usually credited either to Hans Lippershey (known as inventor of telescopes) or to the Janssen family (father and son's Hans and Zacharias) who were all Dutch spectacle-maker from Middelburg [START_REF] Ball | The Early History of the Compound Microscope[END_REF]. Specifically, Hans and Zacharias Janssen will use their knowledge to create the first known compound microscope (i.e., a microscope composed by two lenses, as opposed to single lens microscopes) which was able to magnify object around 10 times (Figure 15 C). Their instrument consisted in a three tubes device, where the central tube was a sleeve in which the two other tubes (carrying the ocular lens and the objective lens) could be pushed to adjust the focus [START_REF] Ball | The Early History of the Compound Microscope[END_REF]. Because of its relatively simple composition, this new instrument will be reproduced several times in the following years. Thus, a letter from 1625 written by Giovanni Faber, mentions an instrument designed by Galileo that allows "to look at small things". Following this description, Faber will coin the term microscope by contracting the Greek words "micro" (meaning small) and "skopein" (meaning to look at). Quite surprisingly, the firsts microscopes were mostly seen as new toys rather than as scientific instruments allowing new discoveries. It is only in 1661, that Marcello Malpighi used a compound microscope for a scientific purpose, and thus discovered the blood capillaries [START_REF] West | Marcello Malpighi and the discovery of the pulmonary capillaries and alveoli[END_REF]. Four year later, Robert Hooke published his famous book Micrographia where, using microscopes, he could observe and describes with his drawing new details of insects and plant anatomy that were invisible to the eye. His discoveries led to the coining of the word "cell" which he used to describe the "plant basic structure". However, in the preface of his book, Robert Hooke indicates that his observations were mostly made using a single lens microscope. Indeed, at that time compound microscopes were suffering from many flaws such as chromatic & spherical aberrations making their usage laborious and preventing their magnification power to overcome the one of single lens microscopes. Nonetheless, Dr. Hooke made many improvements on the compound microscope.

Especially, through the addition of a base and screws to hold and modify the position of both the microscope and the samples. Additionally, he thought about improving the illumination of the sample by using a spherical glass filled with water. Thus, he could concentrate the light on the object to magnify (Figure 15D) [START_REF] Bardell | The Biologists' Forum: The invention of the microscope[END_REF]. But even with these improvements the maximal magnification obtained at the time was around 20 to 30x. This probably explains why the major discoveries of the time were obtained with single lens microscope. Indeed, it is the single lens microscope designed by Anton van Leeuwenhoek who enabled not only to make bigger images of an already visible objects but rather discover a whole new world invisible by eyes. Thanks to his skills he produced the lens of his single lens microscope and thus could reach a magnification up to 300x (Figure 15E). With his device, he revealed the existence of invisible 'creatures' named animalcules and opened the new era of microbiology. He was indeed, the first to discover the existence of protists, bacteria or spermatozoids [START_REF] Kriss | History of the Operating Microscope: From Magnifying Glass to Microneurosurgery[END_REF]. These discoveries eventually popularized the usage of microscopes in biological studies. During the next century both the single-lens and compound microscopes co-existed and were used amongst their advantages and flaws, until the quality improvement of the lens brought better power of magnification and correction of aberrations leading to the progressive abandon of single lens microscopes in favor of compound microscopes.

Characteristics of optical microscopes.

Microscopes are instruments allowing to improve the resolution of the image of an object through its magnification. The resolution represents the ability to distinguish two points as being separated. For example, the resolution of a human eye is approximately of 150 µm, which means that two objects separated by this distance will be visualized as separated entities. Optical microscopes are able to reach the maximal resolution of 0.2µm, which is usually called the diffraction barrier. The diffraction barrier is the result of the properties of the light (behaving like a wavefront) and of the fundamental laws of physics indicating that wavefront passing through or around an obstacle (i.e., a lens in the case of microscopy) are spreading around this obstacle. To overcome this resolution limit, other types of microscopes were developed which are using alternative source of illumination such as electron microscopes. Thus, three main categories of microscopes can be defined depending on their illumination sources: Optical microscopes using light (photons) as illumination source; electron microscopes using electron as illumination source; and scanning-probes microscopes using metallic tip to scan the surface of an object and measure its electronic and magnetic forces. One the one hand, electron and scanningprobes microscopes display the best resolution (0.1nm and 10nm respectively), but the high level of specialization required to master their use as well as their high cost limits their use in biology. On the other hand, optical microscopes present a lower resolution (0.2µm) but are more reasonably priced, and their use is more accessible to newcomers. Thus, they are widely used in biology. The elements constituting modern optical microscope are relatively simple, and includes four main components: a light source, a condenser lens, plus two lenses magnifying the desired object (Mualla et al, 2018) (Figure 16). The lens located the closer to the object is the objective lens, and the one closer to the observer's eye is the eyepiece also called ocular. The objective lens is characterized by its magnification factor, and its numerical aperture (NA). Of note, the NA represents the ability of the lens to gather light.

Specifically, it denotes the maximal angle of the incoming light that can be collected by the objective lens [START_REF] Kubitscheck | Principles of Light Microscopy[END_REF]. Of note, the resolution of an objective is directly dependent on the quantity of light it collects. Thus, to obtain the maximal resolution with a specific objective, the maximal light should be collected. This highlights the importance of the light source, which should be stable and powerful, and of the condenser lens which focuses the power light onto the object to be studied. Optical microscopes exist in two configurations according to the positions of the light source, and of the objective relative to the object to image. On the one hand, upright microscopes are imaging the sample from the top: the light source is situated below the object and the objective is placed above. On the other hand, in inverted microscopes the objective is placed below the sample and light is coming from above (Figure 17). For biological studies, inverted microscopes present a great advantage over upright microscopes as they allow to image samples from below. This specificity enables living samples present in liquid medium to be imaged while they lie on the bottom of Petri dishes or coverslips.

The simplest optical microscope is represented by the bright-field microscope, it is virtually present in every biological laboratory as a mean to observe cells. In brightfield microscopy, the incident light passes through the object and only structures opaques to photon are visible in gray level over a white background. Nonetheless, cells are relatively transparent to photon, thus they present a very low contrast making their observation arduous. Thus, element enabling phase contrast are often implemented in bright-field microscopes such as diaphragms. Alternatively, histological studies where samples are colored with specific dyes allow the visualization of the specifically colored structures (Figure 18) [START_REF] Davidson | Optical Microscopy[END_REF]. Despite these improvements, the evolution of biological studies towards the identification of molecular processes occurring within the cells has outdated brightfield microscopy techniques which were overwhelmed by fluorescence microscopy.

Fluorescence microscopy

The apparition of fluorescent microscopy results from a series of unrelated discoveries in the worlds of physics and biology. First, the identification of fluorescent materials, and the desire of physicists to apprehend the mechanism by which fluorescence occurs led the scientists to develop and use specific materials that will later be implemented in microscopes (such as quartz lenses for example). studies [START_REF] Ghiran | Light Microscopy[END_REF]. Later, the identification and cloning of the green fluorescent protein (GFP) was another breakthrough expending of the implementation of microscopes in biology laboratories.

Fluorescence discovery and principle

Several compounds naturally present fluorescence. The firsts reported observation of this phenomenon are dated from the 16 th century by a Spanish scientist, Nicolas Monardes, and a Franciscan missionary, Bernardino de Sahagun. They both reported their observation that infusion of wood (Lignum nephriticum , and Eysenhardtia polystachya respectively) gives to the water a bluish shade (Figure 19 A) [START_REF] Acuña | Structure and formation of the fluorescent compound of lignum nephriticum[END_REF] 19 B,C,&D). In 1852, the Irish physicist George Gabriel Stokes will coin the fluorescence in his treaty "on the change of refrangibility of light" [START_REF] Stokes | On the change of refrangibility of light[END_REF]. Stokes was also the first one to notice that the light emitted by fluorescent compound had a longer wavelength than the one from the excitation light [START_REF] Ghiran | Light Microscopy[END_REF].

Formally, fluorescence is defined as " the emission of electromagnetic radiation, usually visible light, caused by excitation of atoms in a material, which then reemit almost immediately (within about 10 -8 seconds)" (Britanica encyclopedia). In fluorescent microscopy, the material mentioned in the above definition is usually represented by fluorophores (i.e, inorganic dyes or genetically encoded fluorescent proteins), and the electromagnetic radiation used to excite the fluorophore correspond to the specific light wavelength allowing its excitation. The physical principle of fluorescence was described by Dr Jablonksi and is schematized in the Jablonski diagram (Figure 20) [START_REF] Jablonski | Efficiency of Anti-Stokes Fluorescence in Dyes[END_REF]. This principle stipulates that when a fluorophore is hit by a photon, it will absorb its energy. The acquisition of additional energy by the fluorescent molecule is characterized by the passage of its electrons from a ground state energy level (named S0) to the first excited singlet state (named S1). Of note, this first excited energy state is itself subdivided in several intermediates vibrational level, and when an electron enters in the excited energy, it reaches one of these intermediate vibrational electronic states. Due to vibrational relaxation the electron will release energy to reach the lowest vibrational energy level from S1. This state being by essence unstable, the electrons will eventually return to their ground energy level by dissipating the surplus energy through the emission of a photon carrying less energy than the one being absorbed (due to the loss of energy induced by the vibrational relaxation process mentioned earlier) (Figure 20). The difference in energy between the absorbed and emitted photon is called Stokes shift in the honor of Stokes who was to first to describe the phenomenon. Since the energy carried by a photon is associated to its wavelength, with the shortest wavelength representing the highest level of energy, during the fluorescence process the absorbed photon possess a shorter wavelength than the emitted and thus is red shifted (Figure 21).

1.3.3.2. Fluorescent proteins 1.3.3.2.1. GFP discovery
The discovery GFP is dated from 1955, when Davenport and al., identified a jellyfish presenting photogenic tissue. At the time the authors did not link this photogenicity to a protein (D. [START_REF] Davenport | Luminescence in Hydromedusae[END_REF], and it took almost ten years before the Dr Shimomura established that a protein was responsible for this fluorescence [START_REF] Shimomura | Extraction, Purification and Properties of Aequorin, a Bioluminescent Protein from the Luminous Hydromedusan[END_REF]. It took another 30 years, before the first cloning of the GFP gene realized by Dr Prasher in 1992 [START_REF] Prasher | Primary structure of the Aequorea victoria green-fluorescent protein[END_REF]. In addition, to cloning the gene of GFP, Dr Prasher will be the first to have the intuition that GFP could be used as a tag to track proteins in cells. However, unable to pursue his academic career, Dr Prasher will send the cloned GFP gene to Dr Chalfie who will be the first to express the GFP in Escherichia coli and Caenorhabditis elegans [START_REF] Chalfie | Green fluorescent protein as a marker for gene expression[END_REF]. One year later, Tsien and al., started genetically affecting the GFP and modifying its properties paving the way for its general use in microscopy [START_REF] Heim | Improved green fluorescence[END_REF]. Of note, the importance of the discovery of GFP was recognize as the Dr Shimomura, Dr Chalfie and Dr Tsien were awarded by the Nobel prize in 2008.

Structure and properties of GFP

The GFP present a cylinder-like structure organized in β-barrel. Of note, the wall of this cylinder is formed by 11 anti-parallel β-sheets with most of the amino acid side chains pointing to the cylinder center (Figure 22). This specific arrangement of the side amino acid pointing toward the center of the cylinder creates the adequate chemical conditions allowing the formation of the chromophore (i.e the part of the protein responsible for its fluorescence). Specifically, the side chains of 3 consecutive amino acids (Ser-65, Tyr-66, Gly-67) are reacting together to form the GFP chromophore (Figure 22) [START_REF] Niwa | Chemical nature of the light emitter of the Aequorea green fluorescent protein[END_REF]. This specific structure, in which the side chains of the amino acids are pointing inside of the β-barrel, provides the advantage of decreasing the possibility of the GFP to interact with other proteins. This characteristic allows the creation of chimeric proteins where the GFP is fused to virtually any protein of interest without affecting its functionality nor the fluorescence mechanism of the GFP. In addition, scientists quickly realized that the GFP structure could be engineered to meet criteria required for specific microscopic applications. Thus, a myriad of random mutations was induced in the original GFP sequence, leading to the generation of a multitude of GFP variants presenting different characteristics than the original one (Figure 23 ). Nonetheless, even though an impressive number of GFP variants was created, the color spectra of these GFP mutants are limited to the blue-yellow range.

Fortunately, several other species displaying fluorescent proteins were identified and thus expended the color palette of fluorescent proteins (FP) to the whole visible spectra (Figure 24) [START_REF] Wouters | The physics and biology of fluorescence microscopy in the life sciences[END_REF].

1.3.3.2.3. Properties of fluorescent proteins (FP)
Because of the variety of available FPs, one should carefully choose the most suited to its specific experiment. In fact, FP do not only differ by their "fluorescent color" but also possess several features that make them more or less suited for specific imaging techniques. These characteristics can be separated in two categories: optical properties and physicochemical properties.

Physicochemical properties

The physicochemical properties of FP include:

• Oligomerization state. Most of the FP derived from the original GFP were shown to be dimeric. As such, fusion of the non-monomeric GFP to a protein of interest might affect the experiment outcome. Fortunately, the propension to dimerization of FP can be abolished with a single mutation (A206K) [START_REF] Zacharias | Partitioning of Lipid-Modified Monomeric GFPs into Membrane Microdomains of Live Cells[END_REF]. This propension to dimerize is not restricted to FP derived from the GFP but is also displayed by most of the red-shifted FP that tend to form tetramer, nonetheless following round of genetic engineering, monomeric forms were made available [START_REF] Campbell | A monomeric red fluorescent protein[END_REF][START_REF] Bulina | Heterooligomeric tagging diminishes non-specific aggregation of target proteins fused with Anthozoa fluorescent proteins[END_REF][START_REF] Yg | A strategy for the generation of nonaggregating mutants of Anthozoa fluorescent proteins[END_REF]. Of note, the evaluation of the tendency of a specific FP to oligomerize can be tested by fusing a desired FP to an ER resident protein. In case the tested FP is oligomerizing, the pattern of ER will be distorted into a specific structure named organized smooth endoplasmic reticulum (OSER) (Figure 25). Thus, the OSER assay became the method of choice to determine the tendency of newly created or engineered FP to dimerize [START_REF] Costantini | Assessing the Tendency of Fluorescent Proteins to Oligomerize Under Physiologic Conditions[END_REF].

• Maturation process. Most of the FP were identified in jellyfishes. Thus, their optimal thermal stability and folding properties are adapted to the temperature of their living environment (usually below room temperature). Codon optimization procedure or mutation of few amino acids were introduced in the FP sequences in order to obtain thermostable and properly folded FP at 37°C [START_REF] Yang | Optimized Codon Usage and Chromophore Mutations Provide Enhanced Sensitivity with the Green Fluorescent Protein[END_REF][START_REF] Nagai | A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications[END_REF]. Nonetheless, disparities exist from a specific FP to another, and the speed of maturation can be an important factor to account for depending on the experiment performed. Of note, presence of oxygen (O2) is a pre-requisite to the chromophore formation within the GFP structure. Thus, experiments performed in anaerobic bacteria or in O2-deprived environment should be carefully designed before conducting fluorescent microscopy experiment.

• pH stability. The pH affects the properties of the FP [START_REF] Griesbeck | Reducing the Environmental Sensitivity of Yellow Fluorescent Protein: MECHANISM AND APPLICATIONS *[END_REF][START_REF] Shaner | Fluorescent proteins for quantitative microscopy[END_REF].

Thus, fusion protein targeting acidic compartment should be fused to FP resistant to low pH. Of note, the sensitivity of certain FP to pH was used to created pH sensors (M et al, 2011).

1.3.3.2.3.2. Optical properties
The optical properties of FP represent another set of important characteristics to be considered when one wants to perform fluorescence microscopy experiment. Several features are included within the optical properties of a given FP:

• The excitation/emission spectra. The excitation spectrum defines the set of wavelengths allowing the excitation a given FP. The emission spectrum represents the set of wavelengths from which a photon can be emitted by the FP. Of note, in databases it not unusual to see single values given to describe FP excitation, they represent the "peak" excitation/emission, meaning, the wavelength at which most of the photons will be absorbed/emitted. The choice of FP spectrum is dictated by the set filters equipped on the microscope where the experiment will be performed. In addition, in the case of multiple labelling, one should choose fluorophores with minimal crosstalk between their specific spectra. Thus, FP with narrow excitation and emission spectra are usually preferred. Additionally, because excitation and emission spectra of a given FP are usually overlapping, one would want to choose FP presenting a large Stokes shift. Finally, autofluorescence of the sample to be imaged should be taken into account in order to obtain the best contrast.

• The brightness. Brightness of a FP represents the efficiency with which it will converts the energy it absorbs into a fluorescent photon emission. On the one hand, the capacity of the FP to absorb a photon (and thus its energy) is represented by extinction coefficient (𝜀), expressed in M -1 .cm -1 . On the other hand, the quantum yield (𝜙) is the ratio between the number of photons absorbed and emitted by the FP (equation 1). The molecular brightness of a FP is calculated as follow (equation 2):

𝜙 = 𝐴𝑏𝑠𝑜𝑟𝑏𝑒𝑑 𝑝ℎ𝑜𝑡𝑜𝑛𝑠 𝐸𝑚𝑖𝑡𝑡𝑒𝑑 𝑝ℎ𝑜𝑡𝑜𝑛𝑠 (1) 𝑏𝑟𝑖𝑔ℎ𝑡𝑛𝑒𝑠𝑠 = 𝜀 × 𝜙 1000
(2)

Of note, the extinction coefficient is specific to a given chromophore. Nonetheless, because the maturation of the chromophore within the FP depends on its proper folding, the effective extinction coefficient is susceptible to variation within experiments [START_REF] Shaner | Fluorescent proteins for quantitative microscopy[END_REF].

Noteworthy, quantum yield (𝜙) is comprised between 0 and 1. This suggests that a photon absorption by a FP is not obligatory leading the emission of the fluorescent. In fact, the emission of a photon represent the radiative pathway of energy release and competes with non-emissive or nonradiative pathways which includes heat production, collision to another molecule (for example, quenching) or intersystem crossing (leading to delayed fluorescence of phosphorescence) (Figure 20) [START_REF] Shaner | Fluorescent proteins for quantitative microscopy[END_REF][START_REF] Wolf | Fundamentals of fluorescence and fluorescence microscopy[END_REF].

• The lifetime. The lifetime of a given FP is a constant value expressed in nanoseconds (ns).

In fact, fluorescence emission is a stochastic process. As such, considering a group of excited molecules, they will not return to their ground energy stated at the same time. The lifetime of a specific FP, represent the time for which 36.8% (1/e) of the excited population will have returned to their ground energy state. Noteworthy, we described above that nonradiative de-excitation pathways are competing with the fluorescence emission pathway of relaxation [START_REF] Wolf | Fundamentals of fluorescence and fluorescence microscopy[END_REF]. In fact, some specific microscopic applications (such as Fluorescence lifetime imaging -Förster resonance energy transfer [FLIM-FRET]) are specifically modifications in the lifetime of fluorescent proteins due to FRET in order to perform quantitative measurement of the distance between fluorescent molecules.

• Photostability. The photostability is a critical parameter of the FPs. It represents the amount of energy a FP can absorb before getting photobleached (i.e, being unable to produce fluorescence). Photostability depends both on the FP characteristics and on the imaging conditions such as the temperature, the pH, and the illumination intensity. Interestingly, photobleaching can occur in several pathways and not all of them are leading to permanent fluorescence deactivation. For example, physical breakage of the chromophore (cleavage of the peptide bonds within the chromophore) is irreversible, while other mechanisms (such as entry of the chromophore in a triplet state, or chromophore protonation) are reversible [START_REF] Shaner | Fluorescent proteins for quantitative microscopy[END_REF]. Because of the variability of photobleaching events, it is usually recommended to test different FPs in a given experimental condition before performing a series of experiment.

Of note, the FPBase online tool lists 711 different FP together with their optical properties, and probably represent the most complete database to date enabling to choose a FP [START_REF] Lambert | FPbase: a community-editable fluorescent protein database[END_REF]. To conclude, FP are extremely complex, and their properties are greatly affected by their environmental and experimental condition. Thus, the choice of the adequate FP for a given experiment should not only be dictated by properties from database but should also result from testing different FP in the experimental condition.

Types of fluorescence microscopy

The use of fluorescence to acquire images of biological samples presents several advantages over the classic brightfield microscopy such as a very high contrast, sensitivity, specificity, and selectivity.

Nonetheless, in order to perform fluorescence microscopy acquisitions, several adaptations need to be implemented in a classic light microscope. From the many types of fluorescent microscopes available, only the microscopy techniques used in this PhD will be described thereafter.

Widefield fluorescence microscopy

Widefield fluorescence microscopy, is performed on the simplest type of fluorescent microscope.

The term widefield signifies that the whole sample is illuminated by the microscope. In that regard, widefield fluorescence is similar to the classic brightfield microscope. Nonetheless, the major difference between the two configurations arises from the presence of a filter that will deviates most of the incoming light preventing it to reach the detector (Figure 26). Specifically, the filter allows exclusively the passage of the wavelength corresponding to the excited fluorochrome. Additionally, one should mention that most fluorescent microscopes are using epi-illumination. This mode of illumination is opposed to the classic trans-illumination, where the incoming light is transmitted through the sample, as it is instead reflected by the sample. Specifically, the incident light passes through the observation objective, and is reflected by the sample (Figure 26). The reflected light is subsequently collected back the objective. The fluorescent epi-illumination mode dictates the implementation of an additional element in the microscope: the dichroic mirror.

Dichroic mirrors and filters

The dichroic mirror also called beam splitter is reflecting a specific set of wavelengths onto the sample and allows the transmission of a different set of wavelengths into to microscope. Classically, in epi-fluorescent microscopes, the light produced by the source is reflected by the dichroic mirror, enters the objective through its back rear aperture, and is focused on the sample where it excites the fluorochromes. This configuration implies that only an extremely reduced fraction of the incident light is reflected by the sample back to the objective lens, thus providing a better contrast. The subsequent fluorescent light emitted by the sample enters the objective, passes through the dichroic mirror to reach the detector. In addition, two supplementary filters are usually located up-and down-stream the dichroic mirror to refine the incoming and outgoing light wavelength (Figure 26). This allows the light to be specifically fitted to the emission and excitation spectra of the desired FP [START_REF] Lichtman | Fluorescence microscopy[END_REF]. Of note, the dichroic mirrors and the filters are usually assembled in a single element named the cube which can be changed depending on the specific fluorochrome being illuminated (Figure 27). • The other major type of camera is represented by the complementary-metal-oxide semiconductor (CMOS) camera. While the principle of light detection is similar between CCD and CMOS camera (i.e the photoelectric effect), they differ by the specific architecture of their structural element (pixel or photodiode). In CMOS, each photodiode possesses its own amplifiers that converts photoelectron into voltage signal and this specific element is called the active pixel sensor (Figure 30). This element allows a very fast frame rate due to the absence of readout time. In addition, CMOS allows the control of individual pixels or group of pixels (imaging of a specific region of interest). The downside is that a big part of the array consists of electronic elements which do not detect incident light. Nonetheless, improvement in technology and implementation of micro lenses in scientific CMOS (sCMOS) made them a choice alternative to CCD camera.

Of note, the sensitivity of a camera is defined by its ability to detect faint signal (low number of photon). Importantly, the light-sensitive element of the camera is not able to produce an electron for every single photon hitting its surface. Thus, the ratio between the number of photons hitting the camera and the number of photons leading to the generation of an electron is called the quantum efficiency. Finally, cameras present the possibility of increasing the time during which the sensitive array is exposed to the incoming light thus, allowing the recording of weak signals.

Nonetheless, electrons may occur randomly during image acquisition leading to "false" light signal. As their apparition is stochastic, increasing the exposure time also increases the camera noise.

Confocal microscopy

In a widefield epi-fluorescent microscope, the sample is evenly illuminated by the incident light. In addition these papers demonstrated that confocal microcopy allows the observation of dynamic processes [START_REF] Amos | Use of confocal imaging in the study of biological structures[END_REF][START_REF] Van Meer | Sorting of sphingolipids in epithelial (Madin-Darby canine kidney) cells[END_REF].

Nowadays, technological development has led to the modification of the initial Minsk's prototype.

Especially, the use of laser as light source has made the use of illumination pinhole obsolete. In addition, galvanometric mirror which enabled to displace the incident light over the sample instead of moving the sample under the light beam were implemented in confocal microscopes (Figure 32). Finally, because the sample is scanned point-by-point, the image acquisition process of a confocal microscope also differs from widefield microscope. Thus, CCD cameras are usually replaced by a photomultiplier tube (PMT), avalanche photo diode (APD) or a mixture between these two technologies named hybrid detectors (HyD). Overall, the differences between a widefield microscope and a confocal microscope can be resumed by the addition of four elements:

• The use of laser as illuminating source. Lasers are producing a collimated light, meaning that all light rays are parallel. The use of additional focusing lenses, allows the incident light to be highly collimated at a specific diffraction limited spot. Of note, lasers are specific to a set of wavelengths, thus confocal microscope scopes are usually equipped with several laser lines to give them the ability to excite fluorophores with different wavelength.

• The pinhole implemented before the detector prevents the light not originating from the focal plane or from the diffraction limited illuminated spot to reach the detectors, resulting in optical sectioning of thin layer of the sample. Of note, pinhole size can be adjusted to increase or reduce the optical sectioning (Figure 32 A).

• The galvanometer mirrors are a pair of mirrors which are concertedly vibrating at high speed due to galvanometer motors. These vibrations allow the creation of a scanning pattern leading to the point-by-point scanning of the desired region's sample (Figure 32 B).

• The detectors of a confocal microscope possess different characteristic than the camera present on widefield epifluorescence microscopes. Indeed, because the sample is scanned point-by-point, the detector does not require to spatially discriminate the signal but rather respond very quickly to changes light intensity. In addition, since the pinhole restrict the passage of the light originating only from the focal plane, the recorded signals are usually of low intensity. Thus, highly sensitive photon detectors are required in confocal microscope.

o On the one hand, PMT are amplifying the signal produced when a photon hits its photosensitive surface based on the photoelectric effect. The electrons produced in response to photons detection are accelerated in a vacuum chamber and amplified by a succession of electrodes (called dynodes). The current measured on the last dynode being proportional to the initial light intensity (Figure 33A). The advantages of PMT are their rapidity of detection (typical response time of a PMT is in the order of nanosecond) and their ability to detect single photons. However, they tend to be noisy because electrons are produced in the PMT even in the absence of light. In addition, they present a low gain between each multiplication step. This technique allows break of the diffraction limit of the light, and to significantly improve the SNR of widefield microscopes. Specifically, in biological application a light beam is shined at a specific angle toward two media possessing different refractive indices represented by the glass bottom petri dish and the cell's media. The specific angle allowing total reflection is called the critical angle. Importantly, the reflection of the light beam is accompanied by the creation of a very thin evanescent wave that diffuses towards the cell medium for 60 to 200nm (below the light's diffraction limit) (Figure 34A). Thus, in biological application, TIRFm allows the specific excitation of fluorescent molecules located right above the bottom of the dish and thus, this technique is especially suited for the observation of membranes proteins. The main difference between TIRF microscopes and classic widefield fluorescent microscope, is the obligation to shine the light on the sample through a specific angle. As for classic widefield microscopy, the excitation light passes through directly from the objective. However, critical angle of illumination is obtained by deviating the laser from the center to the sides of the objective back focal plane (Figure 34B). Thanks to this deviation, the incident angle of the light beam can be controlled and adjusted the critical angle. Of note, the modulation of the deviation level from the back focal plane, allows to adjust the illumination angle, and thus allow an easy transition from widefield to TIRF modality. In addition, surpassing the critical angle induces a lower penetration of the evanescent wave in the sample and thus reduced the depth of illumination. In order for the angle of incidence to be greater Additionally, the PM localization of ORAI channels as well as the clustering they undergo following SOCE activation has been assessed due to TIRF, fluorescence correlation spectroscopy (FCS) and single particle tracking (spt-) microscopy techniques. Finally, FRET microscopy as well as bimolecular fluorescence complementation (BiFC), were also used to study the interactions between ORAI and STIM proteins, but also the interactions within ORAI homologues (homo-and heteromeric channels formation).

Calcium entry

The main physiological function of the CRAC channel is to provide Ca 2+ entry in the cells following SOCE activation. Since Ca 2+ is a crucial second messenger involved in a myriad of cellular processes, several techniques were elaborated to measure its concentration inside the cell. Importantly, Ca 2+ concentration is tightly regulated in the cell and its concentration is modulated depending on the stimulation received by the cell. Thus, several dyes were developed to enable the measurement of Ca 2+ concentration in the cell with widefield fluorescence microscopy. On the one hand, measuring the absolute Ca 2+ concentration (or any other compound) using fluorescent dyes is an extremely complex task due to the existence of several factor that might affect the fluorescent properties of the dyes independently from the Ca 2+ concentration such as local dye concentration variation, dye leakage, changes in cell volume, and variations in illumination conditions between experiments. One the other hand, ratiometric dyes enable the evaluation of relative Ca 2+ concentration independently of the aforementioned limitations. As such they are commonly used for in Ca 2+ imaging studies. Structurally, chemical Ca 2+ dyes are derived from Ca 2+ chelators [START_REF] Paredes | Chemical calcium indicators[END_REF]. Especially, BAPTA (bis(2aminophenoxy) ethane tetraacetic acid) derived structures are the most common because of its high Ca 2+

affinity and selectivity over Mg 2+ , and fast kinetic of Ca 2+ binding and release. Importantly, excitation or emission spectra of ratiometric Ca 2+ dyes are modified upon Ca 2+ binding. Several dyes exists but Fura-2 is one of the most popular and thus its properties will be described below. In addition, several GECI were developed to study local Ca 2+ variation, they will be described thereafter.

1.3.4.1.1. Ca 2+ imaging with Fura-2 Fura-2 was developed by Tsien's team and shares common structural elements with BAPTA (Figure 35) [START_REF] Grynkiewicz | A new generation of Ca2+ indicators with greatly improved fluorescence properties[END_REF] . This compound is hydrophilic, and cannot naturally cross the cell's PM. Thus, to facilitate its incorporation in cells, the commercially available form of Fura-2 possesses an additional acetoxymethyl (AM) ester allowing its diffusion through the PM inside the cell.

Importantly, once internalized, the AM ester group bound to Fura-2 is cleaved by intracellular esterases and thus traps the dye inside the cell. Fura-2 affinity for Ca 2+ is similar to endogenous Ca 2+ levels (Kd 145mM). In addition, in its Ca 2+ free state Fura-2 is excited at 380nm while its Ca 2+ bound state excitation peak is at 340 nm. The fluorescence emission peak is around 500nm independently from its Ca 2+ bound status (Figure 36) [START_REF] Grynkiewicz | A new generation of Ca2+ indicators with greatly improved fluorescence properties[END_REF][START_REF] Paredes | Chemical calcium indicators[END_REF]. Finally, due to its ratiometric properties, a simple normalization of fluorescence intensity acquired in Ca 2+ bound state (340nm) versus Ca 2+ free state (380nm) allow comparison between cells and between experiments [START_REF] Paredes | Chemical calcium indicators[END_REF]. If the first calcium experiments conducted with the aim of identifying ORAI as effector of SOCE were performed with the non ratiometric dye Fura-5F (Mercer et al, 2006a), several subsequent publications successfully used the ratiometric Fura-2 dye to observe Ca 2+ entry induced by ORAI channel opening. For example, Forsell and al., performed Ca 2+ measurement using Fura-2 dye and demonstrated that D vitamin was inducing SOCE inhuman enterocytes [START_REF] Forsell | Rapid modulation of Ca2+ uptake in human jejunal enterocytes[END_REF]. In line with these results, Barrit et al., confirmed by using a combination of Ca 2+ imaging technique as well as patch clamp recording that SOCE in rat liver cell is provided by ORAI protein s [START_REF] Barritt | Store-Operated Ca 2+ Channels and Microdomains of Ca 2+ in Liver Cells[END_REF]. In addition, Ca 2+ imaging techniques were useful in the identification of pharmacological compounds altering SOCE entry. For instance, it was shown that pyrazole compounds enable the selective activation of either SOCE or receptor activated Ca 2+ entry (ROCE) [START_REF] Schleifer | Modulation of cellular Ca2+ handling by pyrazoles[END_REF]. Of note, evaluation of pharmacological activators of SOCE with Ca 2+ imaging using Fura-2 were also successfully performed in animal tissue such as retinal rat arteriolar smooth muscle [START_REF] Mcgahon | Pharmacological Profiling of Store-Operated Ca2+ Entry in Retinal Arteriolar Smooth Muscle[END_REF]. Finally, Ca 2+

imaging techniques were also used to demonstrate that the modulation of ORAI expression affects the SOCE level which in turns modify cell physiological properties [START_REF] Abdullaev | Stim1 and orai1 mediate crac currents and store-operated calcium entry important for endothelial cell proliferation[END_REF]Diez-Bello et al, 2017;Dubois et al, 2014c). To conclude, Ca 2+ imaging in general is a very versatile tool used routinely to measure Ca 2+ entries in cells or tissues. However, chemical dyes do not allow to target specific region since the dye is free in the cytoplasm. This is one of the reasons that lead to the creation of genetically encoded Ca 2+ sensor that could be specifically targeted to subcellular regions or compartments. structure where the formation of chromophore is allowed (Figure 37) [START_REF] Nakai | A high signal-to-noise ca2+ probe composed of a single green fluorescent protein[END_REF]. Thus, following Ca 2+ detection GFP fluorescence intensity increases drastically. Successive rounds of genetic engineering led to specific improvement of the properties of GCaMPs, such as increase of their dynamic range (the ratio of the lowest fluorescent signal to the highest one), or refinement of their calcium affinity and kinetics, leading to the generation of a whole palette of GCaMPs with different properties allowing their use in several different applications [START_REF] Redolfi | Lighting up ca2+ dynamics in animal models[END_REF]. Interestingly, fusion construct associating ORAI to GCaMP were used to monitor the localization and specific activity of channels formed by ORAI. Specifically, Dynes et al. revealed that optical recordings of calcium entry through single ORAI channels were possible (Dynes et al, 2016a[START_REF] Dynes | Cell-wide mapping of Orai1 channel activity reveals functional heterogeneity in STIM1-Orai1 puncta[END_REF]. This ability was further confirmed by other teams [START_REF] Maneshi | Orai1 Channels Are Essential for Amplification of Glutamate-Evoked Ca2+ Signals in Dendritic Spines to Regulate Working and Associative Memory[END_REF][START_REF] Son | STIM2 targets Orai1/STIM1 to the AKAP79 signaling complex and confers coupling of Ca2+ entry with NFAT1 activation[END_REF]. Additionally, constructs associating ORAI3 and GECO (redshifted equivalent of GCaMP) were also used to visualize specific Ca 2+ entry du to Orai3 channels in rat neonatal cardiomyocytes [START_REF] Gammons | Cardiac-specific deletion of orai3 leads to severe dilated cardiomyopathy and heart failure in mice[END_REF], and to demonstrate the absence of ARC channel in breast cancer cell despite the expression of all its components [START_REF] Cantonero | Arachidonic Acid Attenuates Cell Proliferation, Migration and Viability by a Mechanism Independent on Calcium Entry[END_REF].

1.3.4.2.
Localization and diffusion properties of ORAI proteins 1.3.4.2.1. Usage of TIRF microscopy TIRF microscopy (TIRFm) allows the specific illumination of extremely thin space above coverslip that correspond to the evanescent field, and which includes around 200 nanometers above the PM.

Therefore, this technique is particularly well suited for the study of membrane proteins. Thus, it is not surprising that ORAI channel properties were investigated using this method. In fact, following the identification of SOCE components, combination of TIRFm and patch-clamp techniques revealed that STIM and ORAI were in close proximity in during CRAC activation. The detection of ORAI1 through TIRFm was not a surprise since it was quickly identified as a PM protein. Nonetheless, the detection of both STIM and ORAI due to TIRFm confirmed the hypothesis that STIM was specifically located in the ER-PM junctions following Ca 2+ store depeltion. [START_REF] Luik | The elementary unit of store-operated Ca2+ entry: Local activation of CRAC channels by STIM1 at ER-plasma membrane junctions[END_REF]Mercer et al, 2006a). Later, other studies tried to use TIRFm microscopy to elucidate the stoichiometry of ORAI protein through single molecule photobleaching [START_REF] Demuro | Subunit stoichiometry of human Orai1 and Orai3 channels in closed and open states[END_REF]. Importantly, when fluorescent proteins are exposed to an exciting light of strong intensity, or for a long period of time, the atomic structure of the chromophore becomes altered resulting in the abolition of light emission called bleaching. This process was used in order to identify the number of "steps" required to fully extinct the fluorescence of a single CRAC channel and thus extrapolate the stoichiometry of the channel (Figure 38). While this technique has been used several times to determine stoichiometry of other ion channels [START_REF] Yamamura | New light on ion channel imaging by total internal reflection fluorescence (TIRF) microscopy[END_REF], its use to determine the stoichiometry of the CRAC channel did not allow to demonstrate a hexamer stoichiometry. In fact, studies concluded on the existence of dimers of ORAI1 in non-active channel and of tetramers in activated channels (Ji et al, 2008b;[START_REF] Penna | The CRAC channel consists of a tetramer formed by Stim-induced dimerization of Orai dimers[END_REF][START_REF] Demuro | Subunit stoichiometry of human Orai1 and Orai3 channels in closed and open states[END_REF]. These results argue with the hexameric stoichiometry observed in dORAI1 crystallography and cryo-EM. These divergences might be explained by the difficulty of analyzing photobleaching steps for high oligomeric structures due to the different types of photobleaching (reversible vs irreversible), and the presence of endogenous non-tagged ORAI protein in these studies. Nonetheless, it is interesting to note that transitions form dimeric to tetrameric states were observed following SOCE activation, suggesting the existence of dynamic associations between ORAI proteins. Noteworthy, TIRFm has also been used to study interaction within ORAI homologs [START_REF] Alansary | Facilitation of Orai3 targeting and store-operated function by Orai1[END_REF]Inayama et al, 2015a), as well as with STIM protein [START_REF] Mcnally | The C-and Nterminal STIM1 binding sites on Orai1 are required for both trapping and gating CRAC channels[END_REF]. Finally, TIRFm confirmed that ORAI proteins can be located in vesicles under PM [START_REF] Hodeify | A STIM1-dependent 'trafficking trap' mechanism regulates Orai1 plasma membrane residence and Ca2+ influx levels[END_REF][START_REF] Wu | Single-molecule analysis of diffusion and trapping of STIM1 and Orai1 at endoplasmic reticulum-plasma membrane junctions[END_REF].

Usage of FRAP and FCS

One of the specificities of ORAI channels is the clustering they undergo following their activation by STIM. Specifically, ORAI are trapped and gathered by STIM following SOCE activation, and this mechanism has been studied due to microscopy techniques. In fact, fluorescence recovery after photobleaching (FRAP) and FCS are two techniques aiming to study the diffusion properties of proteins.

In addition, they are widely used to study membrane proteins because these proteins present the advantage of diffusing in only 2 dimensions, while cytosolic protein are diffusing in 3 dimensions which complexify the analysis process of their diffusion properties. For these reasons, the mobilities of the SOCE components were studied with such techniques. The first studies using FRAP techniques were mainly focusing on STIM1 protein, and the results obtained led to the hypothesis that STIM activation was gradual and could be separated in several steps (i.e, oligomerization, diffusion to ER-PM junctions, binding on ORAI1 and activation of CRAC channel) [START_REF] Liou | Live-cell imaging reveals sequential oligomerization and local plasma membrane targeting of stromal interaction molecule 1 after Ca2+ store depletion[END_REF][START_REF] Covington | Essential Role for the CRAC Activation Domain in Store-dependent Oligomerization of STIM1[END_REF]. Further, studies focusing on the ORAI protein demonstrated that they were in majority diffusing in the PM and that the simple binding of STIM1 CAD/SOAR was decreasing their mobility. This result was at the basis of the hypothesis suggesting the bind and trap model for the STIM1-activation of CRAC channels (Figure 39) (Park et al, 2009a). Additionally, some FRAP experiments are performed in TIRF modality to obtain a better SNR. Thus, while studying ORAI diffusive properties with FRAP technique, Madl et al., took advantage of the TIRF modality to demonstrate that ORAI1 localization was not limited to PM but also existed in cytosolic vesicles that could be integrated in the PM (Madl et al, 2010a). In addition Following the initial studies using FRAP microscopy, new details on the ORAI-STIM coupling and motility were revealed using more advanced microscopy techniques such as super resolution microscopy. In fact, early TIRFm studies investigating ORAI oligomerization through photobleaching steps could be defined as super resolution microscopy as the enabled the imaging of single channels and single ORAI molecules. Nonetheless, more sophisticated super resolution techniques such as single particle tracking were applied to the SOCE components only recently. In fact, the identification and development of photoactivable and photoswitchable FP greatly contributed to the development of single particle tracking technique. Importantly, photoactivable FPs are able to undergo a switch from a dark state (unable to provide fluorescence) to a bright state due to UV illumination [START_REF] Henderson | Structure and mechanism of the photoactivatable green fluorescent protein[END_REF], and photoswitchable FP see their emission spectra switched after being illuminated by UV light [START_REF] Wiedenmann | EosFP, a fluorescent marker protein with UV-inducible green-to-red fluorescence conversion[END_REF]. In fact, single particle tracking techniques rely on the sequential illumination fluorophores within a sample to allow their individual localization. Specifically, if one manages to illuminate a single fluorescent particle, the observed result is a diffraction limited dot (roughly a 200nm

diameter dot) within the image. Nonetheless, the 3D shape of this diffraction limited spot is known and correspond to the point spread function (PSF). Mathematical operation performed on the PSF such as deconvolution and fitting of Gaussian equation according to the shape of the PSF enables to localize precisely the origin of the light signal (Figure 41). On the one hand, single particle modality can be achieved with classic FP, however it is limited to region where single molecules are located within a diffraction limited region. Of note, this configuration is usually hardly reachable because protein need to be expressed at a low level. Nonetheless, some strategies inducing photobleaching of molecule until reaching a low concentration of the fluorescent protein were successfully used. On the other hand, the proportion of detectable photoswitchable and/or photoactivated FP can be modulated though modification of the intensity of the switching light (UV light) and thus eases the obtention of images where single molecules are localized within a diffraction limited space [START_REF] Manley | Single-Particle Tracking Photoactivated Localization Microscopy for Mapping Single-Molecule Dynamics[END_REF]. The use of these specific FP allowed the development of the single-particle tracking photoactivated localization microscopy (sptPALM), which allows the obtention of information about the behavior of studied molecules [START_REF] Bayle | Single-particle tracking photoactivated localization microscopy of membrane proteins in living plant tissues[END_REF].

1.3.4.2.3.2. Single particle tracking of STIM and ORAI

Tracking of ORAI1 and STIM1 single molecules before and after ER Ca 2+ depletion enabled the refining the mechanisms describing their interactions and diffusion. For example, Wu and al., confirmed that STIM1-ORAI1 complexes are trapped in ER-PM junctions but also revealed that even after store depletion mobile fraction of ORAI1 and STIM1 are still present. Furthermore, they indicated that STIM1-ORAI1 complexes are dynamic assemblies and that ORAI1 and STIM1 molecule are able to diffuse between mobile and immobile pools [START_REF] Wu | Single-molecule analysis of diffusion and trapping of STIM1 and Orai1 at endoplasmic reticulum-plasma membrane junctions[END_REF]. In 2016, Li et al., used the photoswitchable FP mEOS fused to ORAI1 to study the oligomerization level of ORAI1 proteins in cells originating from ORAI1 KO mice. They showed that under resting condition ORAI1 are mainly dimeric and that following SOCE activation their stoichiometry evolves up to hexameric assemblies (Figure 42). In addition, they show that a cytosolic protein, α-SNAP, is modulating the assembly of ORAI proteins channel and the interaction between ORAI channel and STIM protein. Finally, they concluded that ORAI channels might be assembled on site due to a mechanism involving α-SNAP protein, and suggested that these assembly is dynamic depending on the stimuli received by the cell (Li et al, 2016b).

Protein-protein interactions

Another specificity of ORAI proteins is their mode of activation through the interaction with STIM.

In addition, ORAI proteins are also interacting within themselves to form functional channels and some data are suggesting the existence of dynamic processes in the assembly of the ORAI channels. Once (Figure 43) [START_REF] Kodama | Bimolecular fluorescence complementation (BiFC): A 5-year update and future perspectives[END_REF]. This technique presents the advantage to be relatively simple to implement and interpret compared to FRET. Nevertheless, the irreversibility of the reaction prevents its use to study dynamic interactions. Importantly, BiFC was successfully used in the context of SOCE studies. For example, it was used to demonstrated that STIM protein are oligomeric in their resting state and that their interaction was mediated by their C-terminal domain (Figure 44) [START_REF] He | Visualisation and identification of the interaction between STIM1s in resting cells[END_REF]. This technique was also used to invalidate the theory that in skeletal muscle ORAI and STIM are preassembled [START_REF] Wei-Lapierre | Orai1-dependent calcium entry promotes skeletal muscle growth and limits fatigue[END_REF]. Of note, this technique was also used to study ORAI-STIM interaction in the context of ARC channel. Specifically, the authors used the cytosolic part of STIM protein fused to the C-terminal extremity of the eGFP, and an ORAI3 protein fused to N-terminal part the eGFP. With these constructs, they demonstrate that ORAI3 and STIM are interacting in the cell PM [START_REF] Thompson | Molecular basis of activation of the arachidonate-regulated Ca 2 + ( ARC ) channel , a store-independent Orai channel , by plasma membrane[END_REF]. In addition, BiFC was used to demonstrate that ORAI1 and ORAI2 are forming a functional CRAC channel in chondrocyte cell line. Specifically, ORAI1 and ORAI2 were fused to YFP fragment and fluorescence resulting from YFP complementation was detectable in basal condition. Moreover, following Ca 2+ store depletion, YFP signal was overlapping with STIM-mCherry signal suggesting that ORAI1/2 heteromers were activated by STIM to provide SOCE in this cell line (Inayama et al, 2015b). The theoretical demonstration of the FRET phenomenon was proposed by Dr Förster, and the phenomenon was later named after him [START_REF] Piston | Fluorescent protein FRET: the good, the bad and the ugly[END_REF][START_REF] Forster | Energiewanderung und Fluoreszenz[END_REF][START_REF] Förster | Zwischenmolekulare Energiewanderung und Fluoreszenz[END_REF]. To detail the mechanism of FRET, one should assimilate the fluorophores to dipoles, and understand that the resonance transfer is occurring through dipole-dipole coupling. Specifically, when two dipoles are meeting some specific criteria (described further) a long-range dipole-dipole coupling can arise. In term of fluorescence, this coupling provides an additional non-radiative de-excitation pathway for the excited molecule to return to its ground state energy level. This additional way is represented by the nonradiative energy transfer to the non-excited dipole (i.e, the acceptor molecule) leading to its excitation and to the emission of a fluorescent photon. The specific criteria allowing the development of longrange dipole-dipole interaction are the distance between the dipoles, their respective orientation one to another, and the sharing of a similar resonance frequency. Of note, for fluorescent proteins, the resonance frequency is linked to the excitation and emission spectra of the donor and acceptor fluorophores. Therefore, it explains the requirement of overlapping between the emission and excitation spectra of the donor and acceptor molecules respectively, to observe FRET (Wouters, 2013a).

In biological terms, as described earlier, when a fluorophore is hit by a photon of an appropriate wavelength, it will enter in an excited state. Subsequently, the excited molecule will return to its ground state energy level. Of note, this de-excitation can occur, either by non-radiative process (such as intersystem crossing), or through radiative process leading to the emission of a fluorescent photon Importantly, FRET is not directly measured but it is rather deduced from the modification of the photophysical properties of the donor and acceptor molecules. Especially, the modifications of the donor and acceptor properties allow the calculation of the FRET efficiency (EFRET) which permit the quantification of FRET. Of note, the FRET efficiency is simply the ratio of the number of donor molecule that have transferred energy to the acceptor by the number of photons absorbed by the acceptor

(3):

𝐸 𝐹𝑅𝐸𝑇 = 𝑁𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑑𝑜𝑛𝑜𝑟 𝑚𝑜𝑙𝑒𝑐𝑢𝑙𝑒𝑠 𝑡𝑟𝑎𝑛𝑠𝑓𝑒𝑟𝑟𝑖𝑛𝑔 𝑒𝑛𝑒𝑟𝑔𝑦 𝑡𝑜 𝑡ℎ𝑒 𝑎𝑐𝑐𝑒𝑝𝑡𝑜𝑟 𝑁𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑝ℎ𝑜𝑡𝑜𝑛 𝑎𝑏𝑠𝑜𝑟𝑏𝑒𝑑 𝑏𝑦 𝑡ℎ𝑒 𝑑𝑜𝑛𝑜𝑟 (3)
The FRET theory stipulates that FRET efficiency varies with distance between the fluorophore according to the following equation [4, [START_REF] Piston | Fluorescent protein FRET: the good, the bad and the ugly[END_REF]], where R0 represent the Förster radius: the distance for which half of the excited donor molecule are transferring energy to the acceptor molecule (i.e 50% FRET efficiency, for a specific couple of fluorophore), and r represent the effective distance between the donor and the acceptor during the experiment.

𝐸 𝐹𝑅𝐸𝑇 = 𝑅 0 6 𝑅 0 6 + 𝑟 6 (4)
with:

𝑅 0 = [2.8 • 10 17 • 𝑘 2 • 𝑄 𝐷 • 𝜀 𝐴 • 𝐽(𝜆)] 1/6 (5)
Thus, from the equation (3), one can observe that EFRET allows to measure distance between donor and acceptor molecule. In addition, because of the 6 th order of magnitude applied to R0 and r, distances between donor and acceptor to allow FRET decreases extremely sharply as a function of the distance, in the order of few nm (Figure 46). Importantly, 𝑅 0 depends on several factors (equation 5) including the quantum yield of the donor (𝑄 𝐷 ), the extinction coefficient of the acceptor (𝜀 𝐴 ), and the overlap integral between the emission spectrum of the donor and excitation spectrum of the acceptor (𝐽(𝜆)). The aforementioned values are constant for a given couple of fluorophores, thus with a careful experiment design and fluorophore choice, one can expect to optimize FRET between two fluorophores. In addition, considering the strong dependency of FRET efficiency to the distance between the fluorophores one could hypothesize that the FRET efficiency value could be used to accurately measure distances between proteins in the cells. However, this is not the case because of the dependency between the FRET efficiency and the orientation factor 𝑘 2 .. Importantly, 𝑘 2 is not a constant and represents the angle between the two fluorophore dipoles. In fact, dipole orientation bears a major influence on FRET efficiency: a colinear orientation of the dipoles is the best orientation to induce FRET (value of k² = 4), a parallel orientation being less optimal (value of k² = 1), and a perpendicular orientation preventing FRET (k² = 0) (Figure 47). Of note, databases are usually indicating a 𝑅 0 of a typical FRET pair assuming the value of k² being 2/3 as the results of the average of all possible orientations taken by a fluorophore during the time of a FRET experiment [START_REF] Wouters | The physics and biology of fluorescence microscopy in the life sciences[END_REF]. While this is valid for small fluorophores in solution, FP are large structure where the chromophore is embedded within the protein structure preventing rapid movements of the chromophore on the time scale of FRET. Unfortunately, it is almost impossible to arbitrary fix the orientation of FP in biological systems. Therefore, the possibility to measure precise distances between proteins due to FRET efficiency variations is limited. Nonetheless, the strict presence or absence of FRET between two molecules is a valid indicator of the existence or the absence of interaction between two proteins. In addition, in the cases where two FP are linked on the same polypeptidic chain, changes in EFRET within this complex allows to identify conformational changes, this property led to the development of several biosensors based on FRET level modifications.

Finally, considering a hypothetical experiment, the calculated FRET efficiency, also called apparent efficiency, is the product between the real FRET efficiency between two individual fluorophores (strictly defined by the distance and orientation between the fluorophores), and the number of interacting molecules within the volume imaged (i.e, pixels). Thus, the FRET efficiency provides information about the localization of the interactions within the cell as well as an estimation of the number of molecules interacting together within each pixel. In addition, it allows the visualization of dynamic association between proteins which are not easily detectable by traditional biochemical techniques. For these reasons, FRET has been widely used in biological studies. To do so, images of the donor and of the acceptor alone needs to be recorded. Specifically, the donor alone should be excited at its specific excitation wavelength (Dex), and its fluorescence emission should be recorded at both its specific emission peak (Dem), and the emission peak of the acceptor (Aem).

Calculating the ratio of the fluorescence emission (DexAem /DexDem) provides a factor to correct for bleedthrough (β). On a similar principle, to compensate for the direct excitation of the acceptor, one need to record the emission of the acceptor (Aem) when excited both at its peak absorption wavelength (Aex), and at the peak absorption wavelength of the donor (Dex). The ratio of the emissions intensities (DexAem/AexAem) gives a factor to correct for direct excitation (δ). Therefore, the final calculation of EFRET for sensitized emission is resumed in the following equation ( 6): FRET technique has been widely used to study the interaction between ORAI and STIM during SOCE. Importantly, first studies using FRET techniques aimed to validate, localize and follow ORAI1-STIM1 interactions within cells were SOCE was activated [START_REF] Barr | Dynamic Movement of the Calcium Sensor STIM1 and the Calcium Channel Orai1 in Activated T-Cells: Puncta and Distal Caps[END_REF][START_REF] Navarro-Borelly | STIM1-Orai1 interactions and Orai1 conformational changes revealed by live-cell FRET microscopy[END_REF][START_REF] Jp | Molecular clustering of STIM1 with Orai1/CRACM1 at the plasma membrane depends dynamically on depletion of Ca2+ stores and on electrostatic interactions[END_REF]. In addition, Frischauf et al., used FRET to demonstrate that STIM1 was able to interact with the three ORAI homologues (Frischauf et al, 2009b). Later, fluorescently labeled mutant and/or truncated STIM1 proteins were used and their FRET level with ORAI1 protein were measured.

𝐸 𝐹𝑅𝐸𝑇 = 𝐷 𝑒𝑥 𝐴 𝑒𝑚 -𝛽•𝐷 𝑒𝑥 𝐷 𝑒𝑚 -𝛿•𝐴 𝑒𝑥 𝐴 𝑒𝑚 𝐴 𝑒𝑥 𝐴 𝑒𝑚 (
This allowed to define the specific regions and even to identify the specific amino acids involved in ORAI channel binding and gating [START_REF] Muik | A Cytosolic Homomerization and a Modulatory Domain within STIM1 C Terminus Determine Coupling to ORAI1 Channels[END_REF][START_REF] Muik | STIM1 couples to ORAI1 via an intramolecular transition into an extended conformation[END_REF]Park et al, 2009a;[START_REF] Covington | Essential Role for the CRAC Activation Domain in Store-dependent Oligomerization of STIM1[END_REF][START_REF] Mcnally | The C-and Nterminal STIM1 binding sites on Orai1 are required for both trapping and gating CRAC channels[END_REF]. Following the confirmation that ORAI1 and STIM1 are interacting during SOCE, and the definition of the specific domains involved in this interaction, FRET technique was used to study ORAI/STIM stoichiometry. While the specific mode of interaction and stoichiometry is still under debate, FRET data suggest that ORAI1 and STIM1 are interacting among the unimolecular coupling model where one STIM dimer binds two ORAI1 proteins belonging to two independent ORAI-channels.

Thus, Zhou et al., measured the FRET efficiency between dimers of STIM1 C-terminal extremities (bearing the SOAR domain) and ORAI1. By introducing a point-mutation preventing their association to ORAI1 in one or two of the units from the STIM construct they observed a reduction in the FRET efficiency, and an almost complete abolition of FRET when mutation was introduced in both units of the STIM1 dimer. This result suggest that each unit of STIM1 is interacting with different ORAI1 units and thus confirms an unimolecular binding model between ORAI1 and STIM1 [START_REF] Zhou | STIM1 dimers undergo unimolecular coupling to activate Orai1 channels[END_REF]. This hypothesis of unimolecular coupling was further confirmed by Baraniak et al., who used a similar strategy where the mutation were located in ORAI C-terminal fragments instead of STIM [START_REF] Baraniak | Orai channel C-terminal peptides are key modulators of STIM-Orai coupling and calcium signal generation[END_REF]. Finally, FRET experiments were performed to assess interaction level between all ORAI homologs and STIM1 during SOCE induction. For example, using C-terminally tagged version of the ORAI protein, Yoast et al., revealed that ORAI2 and ORAI3 present higher EFRET with STIM1 protein in basal level compared to ORAI1-STIM1. In addition, they showed that activation of SOCE lead to an increase of FRET for all ORAI homologs. Nonetheless, the extend of this increase was more pronounced for ORAI2-STIM1 than for ORAI1-STIM1, while ORAI3-STIM1 showed the smallest increase in FRET efficiency following SOCE induction. The same group performed similar experiments with STIM2, and revealed that STIM2 interaction with ORAI1 and ORAI2 is more pronounced than with ORAI3 in basal condition and that store depletion does not affect their interaction level [START_REF] Emrich | Omnitemporal choreographies of all five STIM/Orai and IP3Rs underlie the complexity of mammalian Ca2+ signaling[END_REF]. fusion proteins fluorescently labeled in their N-terminal extremities to perform FRET. They showed that ORAI1 and ORAI3 were displaying strong FRET level albeit lower than within ORAI3 proteins but higher than within ORAI1 proteins [START_REF] Schindl | Plasticity in Ca2+ selectivity of Orai1/Orai3 heteromeric channel[END_REF]. The specific interaction between ORAI1 and ORAI2 was also investigated by FRET and confirmed by BiFC technique in chondrocyte cell-line by Inyama et al., (Inayama et al, 2015a). Finally, Yoast et al., showed that all ORAI isoforms are interacting in basal conditions and that SOCE induction with carbachol or ionomycin as not modifying FRET levels (Yoast et al, 2020b).

Overall, FRET technique has been widely used to study ORAI proteins. Nonetheless, as explained earlier ratio imaging FRET and sensitized FRET experiments presents limitations in analysis of FRET intensities that might limit the conclusions that one could draw from experiments. Importantly, fluorescence lifetime imaging FRET enables to overcome these limitations and to quantify FRET between two proteins., as a consequence the specificity of this type of FRET measurement will be developed thereafter. As described earlier, fluorescence lifetime is specific for a considered fluorophore. Nonetheless, it is affected by pH, temperature, or FRET. Indeed, when a fluorophore is excited, it enters a higher energy level state, the return to the ground state energy level can occur through radiative (photon emission, Γ)

or non-radiative processes (kNR) (equation 7). The time an excited molecule will spend in the higher energy state is dependent on the possible ways of de-excitation. Thus, for an excited fluorophore, FRET represents an additional way of de-excitation (equation 5), leading to a decrease of lifetime.

𝜏 𝐷 = 1 Γ+𝑘 𝑁𝑅 ≥ 1 Γ+𝑘 𝑁𝑅 +𝑘 𝐹𝑅𝐸𝑇 = 𝜏 𝐹𝑅𝐸𝑇 (7)
The return of the fluorescent molecule to the non-excited state is a stochastic process. Thus, if one considers a single molecule excited by repeated laser pulses, the time this molecule spends in higher energy will vary between each measurement following an exponential decay. The specific average lifetime of a fluorophore represents the time for which a decrease in fluorescence intensity of 1/e of its initial intensity is observed (Figure 49). In case of FRET, the decrease in lifetime corresponds to an increased decay of the fluorophore lifetime. Importantly, the fluorescence lifetime is independent on, local concentration of fluorophores, excitation intensity, and fluorescence intensity. In addition, when measuring FRET by FLIM, one should carefully choose a donor molecule presenting a single exponential decay lifetime. Indeed, multiples exponential decay lifetime increases drastically the analysis processes. Finally, one would observe that during FLIM-FRET experiment, only the lifetime of the donor is measured. Thus, FLIM-FRET present the advantage of eliminating excitation and bleed through cross-contamination, as well as variation induced by changes in the fluorophores concentration [START_REF] Yasuda | Imaging spatiotemporal dynamics of neuronal signaling using fluorescence resonance energy transfer and fluorescence lifetime imaging microscopy[END_REF]Ishikawa-Ankerhold et al, 2012b).

FLIM-FRET measurements

Considering a system where the donor molecules are not interacting with the acceptor molecules, the lifetime of the chosen donor molecule follows a single exponential decay allowing the calculation of its lifetime that will corresponds to the lifetime indicated in databases. Considering the opposite system, where all the donor molecules are interacting with acceptor, the lifetime will still follow a single exponential decay, but will display a shorter characteristic decay time because the probability of the donor molecule de-excitation is increased due to the presence of the additional de-excitation pathway represented by the FRET. In a cellular context when a population of donor is excited, some of the donor molecules will be interacting, while other will not. In this case, the measured lifetime is not the average of not-interacting and interacting lifetimes, instead it follows a double exponential decay (equation 8).

Thus, the signal intensity for each pixel in time (𝐼 𝑖 (𝑡)) corresponds to the double exponential function relative to the decays of the donor alone (τD) and the decay of the donor interacting with the acceptor (τDA). Since the two lifetimes are constant, the only variable in the equation is represented by the number of interacting molecules within the cell (αi, also called local parameter) for each pixel.

𝐼 𝑖 (𝑡) = (1 -𝛼 𝑖 ) • 𝑒 -𝑡 𝜏 𝐷 ⁄ + 𝛼 𝑖 • 𝑒 -𝑡 𝜏 𝐷𝐴 ⁄ (8)
Thus, considering the FLIM efficiency equation ( 9):

𝐸 𝐹𝑅𝐸𝑇 = 1 - 𝜏 𝐷𝐴 𝜏 𝐷 = 𝑅 0 6 𝑅 0 6 + 𝑟 6 (9)
In a single FLIM measurement of FRET, one can obtain both the efficiency of FRET which is characteristic of the conformation of the complex, and the local parameter indicating for each pixel the proportion of interacting molecules. Of note, measurement of lifetime decay profiles can be performed in two ways named the time domain (TD) and the frequency domain (FD). Because TD-FLIM represents the easiest and more straight forward way to measure fluorescence lifetime, and because it was the measuring technique used throughout this PhD work, it will be the only method described thereafter. In order to measure the exponential decay in the TD, one needs to build a histogram of the number of photons detected as a function of the time. This type of measurement is performed with specific equipment. Especially a pulsed laser coupled to a time correlated single photon counting (TCSPC) board. In brief, laser pulses are exciting donor molecules, while single-emitted photon arrival times are recorded to build a histogram of time arrival (Figure 50 [START_REF] Sipieter | From FRET imaging to practical methodology for Kinase activity sensing in living cells Skipper L (2005) PROTEINS | Overview[END_REF]). From this histogram, the decay of the fluorescence lifetime can be extracted.

FLIM-FRET analysis

During a TD-FLIM experiment, the samples are excited by light pulses several times. As a result, histograms of the fluorescent intensity decay are established for each pixel. The classical way of extracting lifetimes from these histograms has been represented by fitting methods, where each the lifetimes are deduced by adjusting each histogram with an exponential model (Figure50) [START_REF] Verveer | Quantitative Imaging of Lateral ErbB1 Receptor Signal Propagation in the Plasma Membrane[END_REF][START_REF] Peter | Imaging molecular interactions by multiphoton FLIM[END_REF]. However, such method requires a high level of expertise and an important computational time. Because such methods are hardly exploitable for non-expert, non-fitting methods were developed, such as the phasor approach (also called polar representation approach). In brief, the lifetime image is converted into a two-dimensional histogram due to the application of sine On the other hand, the fraction of donor molecule interacting is not easily extracted from these data [START_REF] Sipieter | From FRET imaging to practical methodology for Kinase activity sensing in living cells Skipper L (2005) PROTEINS | Overview[END_REF]. Nonetheless, for most cases, the FRET efficiency estimated from the average lifetime, provides sufficient information to evaluates interaction level within proteins (Wouters, 2013b).

Of note, another non fitting approach was developed by Padilla-Parra et al., introducing the minimal fraction of interacting donor. This method can be applied to TD FLIM technique and provides an indicator of the minimal interacting proportion of the donor when the lifetime of the donor alone is determined in a separate experiment [START_REF] Padilla-Parra | Quantitative FRET analysis by fast acquisition time domain FLIM at high spatial resolution in living cells[END_REF].

To conclude, while powerful, FLIM-FRET was never applied for the study of ORAI and STIM.

This might be imputed to the specific equipment required to perform this type of experiment as well as the relative degree of specialization required to assimilate and perform analysis of the data obtained.

Nonetheless, it enables a more precise measurement of the interaction level between proteins than the classic intensity-based method FRET, and thus might allow to obtain better pictures of the mechanism of interaction between ORAI proteins. when Dr Ishino identified in Escherichia..coli arrays of interspaced and partially palindromic DNA repeats [START_REF] Ishino | Nucleotide sequence of the iap gene, responsible for alkaline phosphatase isozyme conversion in Escherichia coli, and identification of the gene product[END_REF]. Subsequently, several sequences sharing this pattern were observed in different bacteria species, and the acronym CRISPR, to refer to these sequences, was coined in 2002 [START_REF] Jansen | Identification of genes that are associated with DNA repeats in prokaryotes[END_REF]. In 2005, it was proposed, by three different groups, that the CRISPR sequences could represent a prokaryotic adaptative immunity system [START_REF] Bolotin | Clustered regularly interspaced short palindrome repeats (CRISPRs) have spacers of extrachromosomal origin[END_REF][START_REF] Pourcel | CRISPR elements in Yersinia pestis acquire new repeats by preferential uptake of bacteriophage DNA, and provide additional tools for evolutionary studies[END_REF][START_REF] Mojica | Intervening Sequences of Regularly Spaced Prokaryotic Repeats Derive from Foreign Genetic Elements[END_REF]. The confirmation, and identification of the role of CRISPR sequences together with the CRISPR-associated sequence (Cas) proteins in prokaryotic immunity was established in 2007 [START_REF] Barrangou | CRISPR Provides Acquired Resistance Against Viruses in Prokaryotes[END_REF]. Finally, the beginning of the CRISPR revolution is dated from 2012, with the repurposing of an engineer CRISPR/Cas9 system as a genome editing tool for eukaryotic cells [START_REF] Jinek | A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity[END_REF].

1.4.2. Mechanism of CRISPR-Cas9 system 1.4.2.1. Physiological mechanism of CRISPR-Cas9
In prokaryotes, the CRISPR-Cas system serves as an adaptive immunity system. CRISPR-Cas system are bacterial loci encompassing: an array of repetitive sequence interspaced by non-repetitive sequences (CRISPR), a set of Cas genes, and sometimes a trans-activating CRISPR RNA (tracerRNA) gene (Figure 52A) [START_REF] Amitai | CRISPR-Cas adaptation: Insights into the mechanism of action[END_REF]. The mechanism of action of this locus in bacterial immunity is divided in three stages: adaptation, expression and maturation, and interference.

• The adaptation stage (Figure 52B). In brief, when a phage infects a prokaryote (bacteria or archaea for instance), its DNA can be recognized as foreign by the Rec BCD nuclease which breaks it down into single-stranded DNA (ssDNA) pieces. It is suggested that these ssDNA pieces represent the material recognized by the Cas1-Cas2 protein complex which will integrate these sequences, called spacer sequences, into the CRISPR array. The initial "choice" of the sequences to be integrated is dictated by the presence of a specific short nucleotide sequences (2 to 5 base pairs [bp]), named the protospacer adjacent motif (PAM).

The spacer sequences acquired by the Cas1-Cas2 system are further integrated (without the PAM sequence) in the bacterial chromosome between the repetitive sequences of the CRISPR array. This process leads to the insertion of spacer sequences (foreign DNA pieces) every time a foreign DNA piece is identified by the bacteria. Therefore, the CRISPR array represent the memory of infections encountered by the bacteria [START_REF] Amitai | CRISPR-Cas adaptation: Insights into the mechanism of action[END_REF].

• The expression and maturation stage (Figure 52C). During this stage the whole CRISPR array is transcribed into a precursor CRISPR-RNA (pre-crRNA) which will be processed into several mature CRISPR-RNA (crRNA), each representing a single spacer motif (homologue to the initial foreign DNA element encountered), and a repeat sequence. These crRNA sequences are assembled with one or more Cas protein to form the Cas-crRNA complex [START_REF] Amitai | CRISPR-Cas adaptation: Insights into the mechanism of action[END_REF].

• The interference stage (Figure 52D). Following its formation, the Cas-crRNA complex will scan the cell for foreign DNA sequences presenting a PAM motif. Following a PAM detection, the protospacer aligns with the foreign DNA, and case of matching between both sequences, the DNA piece will be cleaved by the Cas protein, leading to its final destruction and thus stopping the infection process of the bacteria [START_REF] Amitai | CRISPR-Cas adaptation: Insights into the mechanism of action[END_REF].

Several types of CRISPR-Cas system have been identified since the initial discovery of CRISPR-Cas9. They are separated in two main classes, themselves divided in several subclasses (Types I, III, and IV for the class one; and Types II, V, VI for the Class 2, [Figure 53]). Of note, in the class one system the interference stage is performed by multiple Cas proteins associated together in a complex.

At the opposite, in the class two systems (from which the Cas9 system belongs), a single Cas protein is sufficient to perform the whole interference process [START_REF] Makarova | Evolutionary classification of CRISPR-Cas systems: a burst of class 2 and derived variants[END_REF]. Importantly, the existing diversity in the CRISPR-Cas systems allowed the identification of specific properties that were selected for improving the capacities CRISPR derived genome editing techniques (different double strand break (DSB) induction mechanism, change in the PAM sequence required for cleavage, etc.).

Usage of CRISPR-Cas9 for genome editing

As mentioned previously, the advent of CRISPR-Cas9 for genome editing is due to the demonstration in 2012 that the system could be repurposed and used in eukaryotic cells. In fact, in the native CRISPR-Cas9 system the Cas9-RNA complex is constituted of two RNA sequences: the crRNA, described above, and the transactivating crRNA (tracrRNA). The tracr RNA is specific to Class two CRISPR-Cas system. This tracr RNA possess a specific structure allowing, first, its association with the Cas protein, and second, its association with the crRNA due to homology to the repetitive sequence present in crRNA sequence [START_REF] Makarova | Evolutionary classification of CRISPR-Cas systems: a burst of class 2 and derived variants[END_REF]. Part of the engineering applied to the original CRISPR-Cas system consisted in generating a single RNA molecule that would serve as a guide to induce DSB at a specific location. This single RNA molecule is named single-guide RNA (sgRNA) or simply guide RNA (gRNA) (Figure 54A). Of note, the gRNA is designed by the user such as it is complementary to a (ideally) unique 20-nucleotides sequence (for the Cas9 system) targeting the genomic loci of interest to edit. Overall, the mechanism of genome edition in eukaryote cells greatly relates to the original interfering mechanism described in prokaryotes. Specifically, following the introduction of Cas protein and gRNA in the cell, the Cas9-gRNA complex is assembled and scans the cellular genome for PAM sequences. Subsequently, for every PAM sequence located by the Cas9-gRNA complex, the gRNA aligns to the genomic DNA (gDNA) sequence. If the gDNA sequence is homologous to the gRNA the Cas9 protein will cleave the double stranded DNA at the position -3 relative to the PAM (Figure 54B) (Zhang et al, 2015b).

Aim and scope of the PhD

As developed throughout the introduction, there is an accumulation of evidence indicating that CRAC channels are heteromeric in their native form. In addition, their composition modulates the level of SOCE displayed by the cells with several phenotypic consequences. Moreover, in addition to the CRAC channels, ORAI1 and ORAI3 proteins are able to form a specific channel, namely the ARC channel, presenting a different stoichiometry than the CRAC channel as well as a specific mode of activation. The study of the ORAI channel composition is still an emerging topic, and not much is yet known about the mechanisms of association between ORAI proteins to form functional channels. In this PhD we proposed to study the specific interaction between the ORAI1 and the ORAI3 proteins by using the quantitative microscopy technique FLIM-FRET. To do so, we decided to implement the genome editing technique CRISR-Cas9, in order to avoid the limitations induced by the overexpression of fluorescent protein of interest. Thus, our specific aims were:

• To implement the CRISPR/Cas9 technique to generate:

o Knokin (KI) cells in order to fluorescently label ORAI1 and ORAI3 proteins.

o Knockout (KO) cells for ORAI1 and ORAI3 proteins.

• Study the fundamental physiological role of ORAI1 protein in the KO cell line

• Study the interactions between ORAI1 and ORAI3 proteins with the FLIM-FRET technique in order to determine the mode of association of these protein in a functional channel., and to establish if this association is resulting from a dynamic process. Cloning experiments were performed using the NEBuilder cloning kit. This kit enables the cloning of multiple cDNA fragment in a single reaction due to the use of PCR primers presenting overlaps (15 to 80 bp) in their 3' and 5' extremities (Fig X). Specifically, the kit includes: an exonuclease creating 3' overhangs allowing annealing of fragments sharing sequence complementarity; a polymerase that will fill the gaps in the annealed 3' overhangs; a ligase that seals the nicks. Fragments used for the assemblies were PCR amplified with specific primers containing homology sequences one-to-another. Primer designed were performed using the NEB online tool (https://international.neb.com/externallinks/nebuilder-assembly-tool). The list of the primers used to generate these assemblies are presented in the Table X. Classic assembly mixes were set up as follow: 1-For assemblies of 2 to 3 fragment, the recommended amount of fragment per reaction should be included between 0.03 and 0.2 pmols, with an ideal ratio vector to fragment of 1:2. In addition the fragments, master mix containing enzymes required for assembly was added to the tube with its specific buffer, and reaction was performed by an incubation of 15 minutes at 50°C in a thermocycler. Following cool down of the tube, 2µl of the assembly were used to transform competent cells. 2-For assemblies of 4 or more fragments, the quantity of fragments was ranging from 0.2 to 0.5 pmols and the ration insert to vector was 1:1. In addition, reaction was carried-out at 50°C for 60 minutes.

Materiel and methods

3

Classic expression cloning

Bacterial transformation

Transformation of bacteria was performed with the heat shock method. In brief, 50µL of chemically competent bacteria were mixed with the appropriate quantity of plasmid (5 to 50 ng) and incubated on ice for 20 minutes. Heat-shock procedure included a 45seconds incubation at 42°C in a water bath, before a resting period of 2 minutes on ice. Next, 100µML of pre-warmed (37°C) Super Optimal Broth with Catabolite repression (SOC) media was added on the bacteria and left shaking in orbital shaker at 200rpm/minutes for 1 hours at 37°C. The mixture was then spread on agar plates and incubated at 37°C overnight or until apparition of bacteria colonies. Bacteria colonies were further inoculated in LB media for plasmid amplification and extraction procedures.

Plasmid isolation

Plasmid isolation was performed by using NucleoBond Xtra Midi Plasmid DNA purification kit (Macherey-Nagel) according to the manufacturer's instructions.

Sequencing

DNA sequencing were performed by Eurofins genomics company.

Restriction digest

The restriction digest was performed by using restriction enzymes from NEB according to the manufacturer's instructions. qPCR was performed using SsoFast TM EvaGreen ® Supermix polymerase (Bio-Rad) on CFX C1000 instrument (Bio-Rad). Primer sequences used in this study are indicated in the oligonucleotide section.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and TATA-box-binding protein (TBP) were used as housekeeping genes. Each experiment was repeated four times. RT-qPCR data were analyzed with the common base method, which accounts for the specific qPCR efficiency of each analyzed gene [START_REF] Ganger | A common base method for analysis of qPCR data and the application of simple blocking in qPCR experiments[END_REF].

CRISPR/Cas9 related techniques

gRNA design

The gRNAs used in this study were designed using the CRISPOR website (http://crispor.tefor.net/) [START_REF] Concordet | CRISPOR: intuitive guide selection for CRISPR/Cas9 genome editing experiments and screens[END_REF]. For the ORAI1 and ORAI3 gene knockin (KI) experiment, the input sequence consisted in a 100 base pair region surrounding the ORAI1 or ORAI3 start codon. The genome chosen for identification of potential off-target sequences was the human genome assembly (GRCh38/hg38). The protospacer motif (PAM) selected for identification gRNAs was NGG sequence (With N for any nucleotide, and G for guanine). From the selection of gRNAs proposed by the algorithm (53 gRNAs for ORAI1; 63 for ORAI3), the final choice of gRNAs was dictated by: 1-the localization of the double strand break site induction; 2-the specificity score; 3-the predicted efficiency score.

For the ORAI1 knockout (KO) experiment, input sequence corresponded to a 114 base pair sequence ranging from alternative start codon of ORAI1 until the end of the first exon (+189 to +303 from CDS).

For the ORAI3 (KO), input sequences corresponded to a 115 base pair sequence equivalent (+114 to +229 from CDS). From the selection of gRNAs proposed by the algorithm (14 gRNAs for ORAI1; 63 for ORAI3), the final choice of gRNAs was dictated by: 1-the specificity score; 2-the predicted efficiency score.

For the ORAI 1 and ORAI3 expression enhancement experiments, the putative promoting region were identified via e!Ensembl website (https://www.ensembl.org/index.html). For ORAI1, a 350 bp region located above the ORAI1 mRNA was used as input. For ORAI3, a 500 bp region located above the ORAI3 mRNA was used as input. From the potential gRNA obtained, criteria for selection were the specificity and the localization across the targeted regions.

Sequences of gRNAs used in the study are presented in the table below: N°71814, 79145, 48138, 62988 ,48873, 48140, 62987) or in MLM3636 (addgene N° 43860).

Name

Specifically, plasmids were digested with Type IIS restriction enzyme (BbsI and BsmBI, for pX330derived, and MLM3636 plasmids, respectively). Following digestion, plasmids were presenting the following overhangs: 5'-GTTT and 3'-GTGG, for pX330 derived plasmids; 5'-GTTT and 3'-TATC for MLM3636. As a consequence, complementary sequences of the overhangs were added to the gRNA sequences from above. Of note, because transcripts made under the control of the eukaryotic U6 promoters generally start with a guanine (G), this nucleotide was added in the beginning of the gRNA sequences when not naturally present. In addition, gRNA sequences were inspected for the absence of termination signal (TTTT).

To proceed to the cloning, a pair of oligonucleotides (sense and anti-sense) with appropriate overhangs (5'CACC or 5'-ATAG and 3'-CAAA) were ordered through Eurogentec.

Phosphorylation and annealing of the oligonucleotides

Prior to cloning, annealing and phosphorylation of the oligonucleotide was performed as follow:

100µM of each oligo were added in a PCR tube together with the T4 polynucleotide kinase (New England Biolabs [NEB]) and its buffer. In the thermocycler, phosphorylation step was performed due to a 30 min incubation at 37°C. The annealing step was performed due to a 5-minute incubation at 95°C

followed by a ramp down to 25°C at 5°C/minute. Annealed oligonucleotides were subsequently diluted 250-fold (1:250) prior to ligation in the desired plasmid.

Ligation of oligonucleotides into CRISPR/Cas plasmids

Ligation of annealed oligo into the desired plasmid was performed in a single digestion-ligation reaction prepared as follow: 100ng of the appropriate vector were added in a PCR tube with 2µl of the diluted annealed oligonucleotides, 0.5µl of T7DNA ligase, 1µl of the appropriate restriction enzyme (BbsI or BsmBI), 2 µl of 10X buffer corresponding to the enzyme used and 1µl of a 1mM solution of DTT and ATP. This mixture tube was submitted to 6 cycles of 5 minutes incubation at 37°C and 5 minutes incubation at 23°C. Finally, 1 µl of the ligation product was transformed in competent bacteria.

T7 Endonuclease 1 (T7E1) assay

The T7Endonuclease1 (T7E1, NEB) assay allows the identification of mismatches in double stranded DNA. It is commonly used to report the level of activity of CRISPR-cas9 DSB induction. The principle of this assay is based on the fact that cellular reparation mechanisms through non homologous end joining (NHEJ) is error prone. Thus, following CRISPR-Cas9 and gRNA transfection in the cells targeted gDNA region will display "random scares" of the DSB. The region of interest is amplified by PCR and the PCR products are denatures and re-annealed. This lead to the annealing of DNA fragments that present different scares and thus presenting mismatches which are detected and cleaved by the endonuclease (Figure 55). Specifically, two days after the transfection of cells with the CRISPR-Cas9 plasmid and gRNA, gDNA was extracted. The region of DSB induction was amplified by PCR with the use of primers surrounding the region of interest (See primer list for details). Of note, since the result of the assay is representative of the level of mismatches present in the sample, this assay requires the use of proof-reading taq polymerase to avoid introduction of polymerization errors. In addition, PCR need to be robust and clean (conditions reported in table ). PCR products are denatured by a two-minutes incubation at 95°C and subsequently re-annealed by a ramp down protocol as follow: a decrease from 95°C to 85°C at -2°C per second, followed by a decrease of 0.1°C per second until reaching 25°C. 1µg

of the denatured/re-annealed PCR product is then incubated with the 0.5µl of T7E1 enzyme and its buffer for 20 minutes at 37°C. Cleaved product are subsequently loaded on a 2% agarose gel and visualized under UV light.

3.3.4. CRISPR-Cas9 related plasmids construction 3.3.4.1.

CRISPR-Cas9 plasmids

Most of the CRISPR-cas9 plasmid used throughout thus study were derived from the pX330 plasmid created by Zhang's team [START_REF] Slaymaker | Rationally engineered Cas9 nucleases with improved specificity[END_REF]. This plasmid contains two expression cassettes: 1-the -One backbone plasmid. pUC19 backbone was chosen because of its small size and because it does not allow expression of the sequence in contains in eukaryotic cells.

-Two homology sequences. The homologue sequences were stretches of 800 bp flanking the site of DSB induction of ORAI1 or ORAI3 -The fluorescent protein tags. The fluorescent proteins chosen were mTurqoise2 and sYFP2.

To obtain the homology sequences for ORAI1 and ORAI3, a PCR on gDNA extracted from HEK-293 was performed. The sequence amplified corresponded to the region -1000 / +1000 relative the ATG of ORAI1 or ORAI3 gene. This PCR product was purified and NEBuilder PCR were performed using this template. The primer used for the creation of HR plasmid with the NEBuilder kit are shown in the primer section.

PITCh plasmids

The PITCh systems for generating KI requires the use of two plasmids (Figure 57 [START_REF] Sakuma | MMEJ-Assisted gene knock-in using TALENs and CRISPR-Cas9 with the PITCh systems[END_REF]):

-The CRISPR/Cas9 plasmid coding for the Cas9 protein and inducing the transcription of two gRNAs. The first gRNA sequence corresponding to the desired sequence of KI induction, and the second gRNAs named PITCh gRNA leading to the induction of DSB in the recombination plasmid (PITCh plasmid).

-The PITCh plasmid containing the following elements: the backbone plasmids, the specific gRNA sequences of the PITCh system, of the microhomology domains (25 nucleotides) corresponding to the 3' and 5' sides of the DSB induction site for ORAI1 or ORAI3 sequence, a cassette coding for the puromycin resistance gene followed by a self-cleavable T2A sequence followed by the desired fluorophore to insert.

Thus following, the transfection of both plasmids in the cell, DSB are induced at the location of desired insertion in the genomic DNA as well as in the donor PITCh plasmid leading. The KI generation is induced due to the presence of the microhomology domains, and the selection of the correctly edited cells is achieved via puromycin selection.

mCherry-eGFP Surrogate plasmids

In brief this method is based on the co-transfection of a Cas9-gRNA plasmid, and of a second plasmid designed as follow: a first expression cassette coding for mCherry is followed by the target gRNA sequence together with its PAM sequence. Further away an out-of-frame (relative to the initial mCherry) eGFP coding sequence is inserted after the PAM sequence (Figure 58). Thus, in case of successful induction of DSB in the cell, the plasmid will be cut. Since cellular reparation mechanisms aiming to repair DBS are error prone and are targeting all DSB, the plasmid might be repaired. In addition, due to error prone reparation mechanism, the repaired plasmid might display the eGFP cassette in frame with mCherry, as a consequence, successful DSB induction and reparation should results in cells displaying eGFP and mCherry fluorescence.

Genomic sequencing

Genomic sequencing was performed to validate the generation of KO cells. PCR region of the desired gene was amplified with proof-reading high-fidelity polymerases (Q5, NEB). PCR products were loaded on agarose gels and purified, before being cloned in pMiniT vector using the PCR cloning kit from NEB. In brief, the pMiniT vector allows blunt cloning in the open pMiniT vector. This vector is encoding a toxic minigene, positive insertion of genomic products in this sequence, breaks the reading frame and allow the bacteria to grow. The PCR products cloned into the vector were sequenced to verify the induction of KO from the CRISPR/Cas9 experiment.

3.4. Oligonucleotides

Primers

All oligonucleotides used in this work were purchased from Eurogentec.

PCR

Several couples of primers were designed and tested throughout this study. The following table recapitulates:

-The target gene and as well as the annealing area within the gene target.

-The forward (FW) and reverse (RV) sequences of the primers.

-The expected sizes of the PCR product in base pair (bp). When two sizes are indicated, the italicized corresponds the size expected for the specific experiment (i.e, recombination or KO screening)

-The type of experiment for which primers were used (T7E1 assay, Recombination assessment, knockout assessment). In case the primers were used for T7E1, the number between parenthesis indicates the expected size of cleaved bands following T7E1 cleavage. The following table display the primer couples that did not lead to robust PCR signal. Thus, they were tested but not used in the experiment due to, absence of PCR products, or high level of nonspecific amplifications.

Target

Target

Expression plasmid

The expression plasmids coding for ORAI1 and ORAI3 fused to mTurquoise2, and sYFP2

respectively, used in this study were a generous gift from Dr Bidaux. In brief, ORAI1 and ORAI3 coding sequence were cloned in frame with mTuruqoise2 or sYFP2 sequences into a pVLL vector [START_REF] Bidaux | FRET Image Correlation Spectroscopy Reveals RNAPII-Independent P-TEFb Recruitment on Chromatin[END_REF]. These vectors were designed in order to improve FRET occurrence by facilitating the modification of the linker size between the FP and GOI sequences. In addition, the linker sequence is enriched in glycine and valine residues to limit misfolding of the chimera and increase the flexibility.

Proliferation

Proliferation was assessed by using colorimetric assay. Initially, 1x10 3 cells per well were plated into 96-well plate and left to settle overnight. Cells were fixed with 50% trichloroacetate and total proteins were stained with 0.4% sulforhodamine B. Stain was fixed with 1% acetic acid and dye was released by applying 10mM unbuffered Tris-base solution (pH=10.5). Wells without cells were considered as blank. Absorbance was measured at 485 nm with TriStar 2 Multimodal Reader LB942 (Berthold Technologies).

Cell cycle analysis

For cell cycle analysis, cells were fixed with ice-cold 70% methanol, resuspended in 100 µL of PBS with 500 µg/mL Ribonuclease A and stained with 50 µg/mL of propidium iodide for 1 hour at 4°C. Afterwards, cells were analysed with CyAnTM ADP Analyser FACScan flow cytometer (Beckton-Dickinson).

Adhesion Assay

For the adhesion assays, 96-well plates were coated with fibronectin as follow: 1 mg of lyophilized fibronectin (Sigma-Aldrich) was reconstituted in 1 mL of ddH2O, this solution was diluted in PBS to a final concentration of 10µg/mL. 20 µL of this solution were distributed in the 96-weel plate and incubates for 5 minutes at room temperature. Next, fibronectin solution was removed by aspiration and remaining liquid was left to air-dry for 30 minutes under the hood. Following coating, 1x 10 3 cells were loaded in each well and incubated at 37°C for one hour. After one hour, medium was discarded and teh cells carefully washed twice with PBS before being fixed with ice-cold ethanol. The cell nuclei were stained with incubation in Hoechst solution (2.5µg/mL) fro15 minutes at room temperature. Fluorescent signal from the nuclei was recorded with Nikon Eclipse Ti-E microscope. 4X objective was used to record the image of the whole well. Cell nuclei were counted automatically with an ImageJ macro. Data were plotted by using the SuperPlotsOfData webapp; for each individual experiment, 6-8 wells were used, and the experiments were repeated 4 times.

Video microscopy migration assay (Wound healing)

For the wound healing assay, culture insert from Ibidi were used to generate wound of a constant size throughout experiments. The silicon inserts delimitate two cell-containing compartments separated by a 500 µm cell-free space (±100 µm). The inserts were placed in 12-well plates, 9 × 10 4 cells suspended in 70 µL of DMEM were added in each cell compartment, and 1mL of DMEM was added in the well. 48 hours after seeding, inserts were removed creating a cell-free gap between the two compartments. 12-well plates were placed on the automated stage of a Nikon Eclipse Ti-E microscope equipped with a chamber to maintain the temperature at 37 °C and 5% CO2. Pictures of the center of the wound were taken with the 4× objective lens every hour for a period of 48 hours. Wound closure was analyzed using a FiJi macro code modified from BioImage Informatics Index (https://biii.eu/). Time-series data representation was performed using PlotTwist webapp, quantitative date plotted using SuperPlotsOfData webapp 3.6.6. Boyden chamber migration assay (Transwell ®) Boyden chamber assay were performed using Transwell® inserts with 8 µm pore-size. 2 × 10 5 cells in 500µL of DMEM were added on the top part of the insert. 24 hours post-seeding, insert were collected and the cells from the upper part of the membrane were removed by scraping. Cells migrated on the lower part of the insert were fixed with ice-cold methanol bath, washed with ddH2O bathes and stained with crystal violet (1% crystal violet solution in 20% methanol). Counting of migrated cells was performed was performed using the 10× objective lens of a Nikon Eclipse TS100 microscope. Five randomly selected fields of the insert were captured, and cells were counted manually using ImageJ software. Data obtained were plotted using SuperPlotsOfData webapp.

Transfections

Transfections were performed using PEI Max 40X. A 1mg/mL stock solution was prepared in ddH2O and filtered-sterilized using 0.22µm pore-size filter. For transfections, the stock solution was diluted to obtain a 1µg/µL solution. Transfection solutions were prepared in FBS-free medium, and a ratio of plasmid quantity to PEI Max volume of 1µg for 2 µL was used. Transfection solutions were gently mixed by tube flicking and following a 15-minute incubation, the solution was added on the cells in a dropwise manner. Of note, the cell's medium was refreshed before addition of transfection mixes.

Macromolecule blotting

Protein extraction

The cells were lysed in 1x RIPA buffer supplemented with protease inhibitors. Next, the cell lysates were sonicated for 10 seconds, and centrifuged at 10000 g for 10 minutes to eliminate the nuclei fraction.

The protein lysate concentration was measured with Pierce® BCA Protein Assay (Thermo Fisher Scientific).

Immunoblotting

For sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), 50 µg of total protein lysate were loaded on polyacrylamide gels and submitted to electrophoresis. Next, separated proteins were transferred to methanol-activated polyvinylidene fluoride (PVDF) membranes with the Pierce G2 Fast Blotter (Thermo Fischer ScientificThermo Fisher Scientific). Subsequently, the membranes were incubated in 5% nonfat milk in TBS-T buffer (15 mM Tris-HCl, 140 mM NaCl, 0.05% Tween20®, pH 7.4) for 1 hour at room temperature to prevent nonspecific protein binding. The blocked membranes were then incubated with primary antibodies overnight at 4°C. The next day, the membranes were incubated for 1 hour with horseradish peroxidase (HRP)-conjugated anti-mouse or anti-rabbit secondary antibodies diluted at 1/100,000 (Chemicon). Chemiluminescent detection of bound secondary antibodies was captured with Amersham Imager 600 (GE Healthcare Life Sciences) using the SuperSignal™ West was used to measure cytosolic Ca 2+ concentration variations. Specifically, Fura-2-AM was diluted in the 2 mM Ca 2+ HBSS solution to a final concentration of 1µM. The cells were incubated in this solution for 45 minutes in the dark at 37°C. Following this incubation, the cells were washed twice with the 0 mM Ca 2+ HBSS solution. The experiments were performed in 1 ml of 0 mM Ca 2+ HBSS solution. At the desired time, 100 µL of 0 mM Ca 2+ HBSS containing 10 µM of thapsigargin (TG) was added (final TG concentration = 1 µM) to induce Ca 2+ store depletion. Next, 1 mL of 8 mM Ca2+ HBSS was added upon the previous solution into the dish.

Acquisitions were performed on a Superfluor Nikon Eclipse Ti-series inverted microscope equipped with a 510/84 emission filter (wavelength/bandwidth) and coupled to an Rolera EM-C2 (Qimaging), under the objective 20x. A xenon lamp light (300 W) was used to excite Fura-2 and fluorescence was recorded after wavelength filtering due to the following filter pairs (wavelength/bandwidth): 340/26 and 387/11 nm. The acquisition software was Metafluor; the data obtained were exported to Microsoft Excel.

Time-series graphics were generated by using the PlotTwist webapp for each individual experiment.

Integral measurements were performed with Microsoft Excel, and data were plotted by using the SuperPlotsOfData webapp.

FLIM-FRET

Fluorophore choice

For the FLIM-FRET experiments, the mTurquoise2 and sYFP2 fluorescent protein were used as donor and acceptor molecules, respectively. Of note, in order to conduct successful FRET experiment, the FP chosen should possess some specific characteristics such as, a good overlap between the donor emission spectrum, and the acceptor excitation spectra, an important quantum yield of donor molecule, and a good extinction coefficient of the acceptor molecule. All these characteristics are displayed by the chosen FP (Figure 59). In addition, because FRET efficiency was measured due to changes in donor lifetime, it is required that the donor present a mono exponential decay which the case of the mTurquoise2.

Experimental procedure

The microscope equipped for FLIM-FRET experiment was located in distant building. Because transportation of the cells from one building to another induced stress (changing of temperature, movements…) to the cells, optimization procedures for plating, transfection, transportation and acquisition were implemented. Thus, the cells plating in 30mm glass-bottom dishes pre-coated with

Poly-L-Lysin was performed in the resident building. Following cell spreading in the dishes, cells were directly brought to the building equipped for FLIM-FRET experiment. One day later, cells were transfected, and imaging experiments were performed two days after transfection. For HEK-293, 1.5 x 10 5 cells were plated per dish. For PC3, 1.2 X10 5 cells were plated per dish. Following their adhesion, the cells were transfected with different ratio of ORAI1-mTurquoise2, ORAI3-sYFP2, and STIM1-mCherry plasmids. Because the detector used during the FLIM measurements can be saturated by high levels of fluorescence, the quantity of plasmids transfected by condition was maintained low. The quantities of plasmid used for the different ratio are displayed in the table below. Because the statistical determination of the distribution of single photon arrival times requires a minimum of 100 photons per pixel that was reached in _120 frames, therefore 120 frames were acquired at 2 Hz for each TCSPC recording.

Statistical analysis and data presentation

Whenever possible, data were represented using superplots. Superplots, allow the visualization of all every data point performed within each experiment replicate and thus allow a better representation of the dispersion of the data. Super plots were generated using the SuperPlotsOfData webapp. Times series data were generated using the PlotTwist webapp and whenever possible traces corresponding to single data point were displayed. For all experiments and within technical replicates, median values were used whenever the distribution of the data was considered non-normal (evaluated with the Shapiro-Wilk test, p < 0.05); otherwise, mean values were used. For statistical analysis, the means of the biological replicates was used. Error bars represent the 95% confidence interval (CI), except when stated otherwise. Of note, the 95% CI represents the interval in which the real value for a specific experiment is included, thus giving a visual interpretation of the significance of a result. The 95% CI was calculated from the mean of biological replicates [27]. Statistical analysis was performed by using the differences between the means of the conditions, except if stated otherwise. T.test were used for statistical analysis except if stated otherwise.

Results

Consideration before performing CRISPR-Cas experiment

The first aim of this PhD work was to generate knock-in in the ORAI1 and ORAI3 genes sequences.

Specifically, it was decided to insert fluorescent tags at the N-terminal extremities of these genes because preliminary experiments and literature demonstrated that this location was not interfering with their functional roles. In order to generate KI one should: 1-create DSB in the genomic region where the foreign DNA piece wants to be inserted, and 2-provide a recombination template from which the DSB will be repaired by the cell that contain the foreign DNA piece to be inserted. Both mechanisms will be described in their specific section thereafter.

Model cell line to edit

The cell line to edit plays a crucial role in a successful CRISPR-Cas9 genome edition experiment.

Because plasmids coding for Cas9 and gRNA need to be transfected and its coding sequence transcribed and translated into protein, it is required to use a proliferative cell line. In addition, a cell line that possesses a good transfection rate represents a good choice. Finally, one should pay attention to the cell line ploidy. Indeed, the rate of successful KI in the cell line will depend on the number of recombination event that occur within one cell, in case of haploid cells, a single HDR event per cell is required, while in diploid cell line, two HDR event will be required and so on for cell lines displaying higher ploidy. As a consequence, the HAP-1 haploid cell line was tested, but their cultivation and transfection rates turned out to be not optimal. Therefore, we used the HEK-293 cell line because: 1-it was routinely cultivated in the laboratory; 2-it is easily transfected; 3-it is a classic model used in the study of ORAI protein properties; 4-they are hypotriploid with a modal chromosome number of 64 in 30% of cells, and higher ploidy occurring in 4.2% of cells, meaning that around 65% of the cells are diploid.

4.1.2. Choice of the Cas protein to induce double strand break (DSB)

Nowadays, performing a genome editing experiment with the CRISPR-Cas9 is relatively simple but requires a careful design. Especially, one should consider the presence and the type of PAM present next to the desired target sequence. In addition, the best way of introducing both the Cas9 protein and gRNA sequences in the cell should be evaluated depending on the cellular type that one wants to edit.

Moreover, the screening procedure for successful genome edition should also be taken into account.

Finally, the existence of potential off-target effect (i.e induction of DSB at a non-desired location) should be studied, and if possible, evaluated. As described in the introducion, several CRISPR-Cas system were identified and have expanded the possibilities offered by genome editing techniques. Nonetheless, at the starting time of this project (i.e 2015) the number of CRISPR-Cas system available were more scarce.

Thus, two different systems using different nucleases were available and considered:

-eSpCas9, engineered version of the classic Cas9 identified in Streptocccus pyogenes. Presenting an increased cutting specificity and efficiency developed by Zhang's lab [START_REF] Slaymaker | Rationally engineered Cas9 nucleases with improved specificity[END_REF]. Of note, this version uses the classic CRISPR-Cas9 PAM: NGG (where N denotes any nucleotide and G denotes guanine).

-hAsCpf1, humanized acidaminococcus sp. enzyme which generates staggered DSB and requires a TTT(A/C/G) PAM (where T, A, C, G denotes Thymine, Adenine, Cytosine, and Guanine respectively) [START_REF] Zetsche | Cpf1 Is a Single RNA-Guided Endonuclease of a Class 2 CRISPR-Cas System[END_REF]. Because the DSB induced is staggered with this enzyme, the risk of deleterious off-target effect is decreased compared to the classic Cas9.

Thus, we faced the choice to use eSpCas9 of hAsCpf1 to perform our experiments. The final choice between these two systems was dictated by the presence of the required PAM in the 5'UTR regions of ORAI1 and ORAI3 genes as well as the number of potential off-target sites. Of note, when one wants to perform recombination, the DSB should be located as close as possible from the desired insertion point.

Thus, the search for PAM motif was restricted to 100pb up-and down-stream of the start codon for both genes. Note that, at the time the search was performed, PAM sequence required for hAsCpf1 DSB induction was defined as: TTN, ORAI1 region of interest present a single location for this motif, while ORAI3 region of interest display six locations for this motif. At the opposite, more than 30 PAM motif corresponding to the eSpCas9 requirements were found next to our target region. Because of low number of potential gRNA to be used with the hAsCpf1 enzyme compared to the eSpCas9, the later was chosen for our experiments.

Delivery of the Cas9 protein in the cells

Following the choice of using eSpCas9 to induce the DSB, the best method for introducing the Cas9 protein and gRNA was considered. Currently, three main ways co-exist: the direct introduction of the complex Cas9-gRNA in the cells via microinjection; the transfection of two plasmids leading the expression of the Cas9 and the sgRNA independently; the transfection of a single plasmid coding for both the Cas9 and the sgRNA. The first solution is mainly proposed by specialized companies (selling lyophilized Cas9 proteins to be injected in the cells) and was marginal at the time of this work and thus was not considered. We choose to use the single plasmid method because it presents the advantage of performing a single transfection per experiment. Of note, the first experiments (i.e, cutting efficiency assessment of the gRNAs) were conducted using a plasmid coding for eSpCas9. Nonetheless, the rapid development of CRISPR plasmids led us to use different plasmids throughout the PhD. Especially, plasmids encoding Cas9 protein linked via a self-cleavable T2A-peptide to a GFP or puromycin resistance cassette were used for genome editing experiments.

Generating gene knockin (KI) with CRISPR-Cas9

Design of gRNA

Design of the gRNA is probably the most crucial point of the genome editing experiment. Indeed, inattentive design might lead to low cutting efficiency preventing or delaying the obtention of edited genome, or to generation of off-target cuts that might affect the outcome of the totality of the experiment performed on edited cells. Thus, two crucial points for design of gRNA to consider are the cutting efficiency and the off-target probability. The design of gRNA used were performed with the CRISPOR website (http://crispor.tefor.net/crispor.py) [START_REF] Concordet | CRISPOR: intuitive guide selection for CRISPR/Cas9 genome editing experiments and screens[END_REF].

Cutting efficiency

The cutting efficiency of a given gRNA is a predicted value (ranging from 0 to 100) relative to the probability of DSB induction at the specific site. This score is susceptible to variation for reasons not clearly defined. Secondary structure of chromatin is suggested to play a role as well as method of gRNA introduction in the cell (transfection of the plasmid and transcription inside the cell, versus direct modified gRNA injection). Of note, the predicted efficiency is not very accurate and constitute a simple indication [START_REF] Haeussler | Evaluation of off-target and on-target scoring algorithms and integration into the guide RNA selection tool CRISPOR[END_REF]. In addition, the DSB induction efficiency can be assessed by specific tests such as the T7 endonuclease assay (T7E1, used in this PhD). Importantly, the T7E1 assay is also inaccurate and does enable to discriminate very well between different efficiencies. Nonetheless, its results provide a visual representation of the absence or presence of DSB induction. The predicted efficiencies of the selected gRNA are presented in Table 4.

Cutting specificity

The second crucial parameter to be assessed when planning a CRISPR/Cas9 experiment is the specificity of the DSB. The specificity is defined by the probability that the Cas9 enzyme will create DSB at off-target site. Few parameters are important the establishment of the probability of cutting at non-target sites:

• The number of mismatches between the gRNA and the off-target site. The higher is the number of mismatches the lower is the probability of non-desired cuts.

• The localization of these mismatch on the gRNA. Mismatches located next the PAM decreases the probability of off-target cuts (12 adjacent to PAM, known as the seeding area).

• The localization of the off-target sites. Off-targets sites located on the same chromosome as the desired cutting site are more likely to be off-target.

A specificity score ranging from 0 to 100 is computed taking in to account the above parameters. Of note, because cutting efficiency of the enzymes for a specific location is computationally assessed but might vary in real conditions, it as a general advice to test different gRNA sequences before performing the genome editing experiment. As a consequence, the efficiencies of three different gRNA were tested for each gene with the T7E1 assay. The predicted efficiencies of the gRNA used are displayed in the Table 4.

Assessment of gRNA efficiencies

The cutting efficiencies of the gRNA designed above were assessed by T7E1 assay as described in the material and method section. In brief this test the T7E1 nucease cleaves the double stranded DNA sequences when detecting mismatches. Of note, this technique does not display strong sensitivity but enables to verify that gRNAs are inducing DSB. Several sets of experiment were required to obtain reliable results. Indeed, even though transfection rate of HEK cells is usually high, in the absence of enrichment in positively transfected cells before performing T7E1 cells, the DSB induction could not be detected (Figure 60A). Thus, enrichment due to puromycin selection following the transfection of the Cas9-2A-Puro plasmid was tested (Figure 60B), but T7E1 results were not satisfying probably because of the lower cell number resulting from antibiotic selection. Therefore, enrichment due to GFP selection by FACS following transfection of Cas9-A-GFP plasmid was performed before extracting cells gDNA and performing T7E1 test. This enrichment method allowed us to obtain acceptable T7E1 results (Figure 60C) and to select the ORAI1 gRNA3 and ORAI3 gRNA2 to conduct the recombination experiments. In order to confirm that our selected gRNAs were inducing DSB in our cells, and that these DSB could be efficiently repaired by the cells, we used a surrogate strategy [START_REF] Niccheri | An efficient method to enrich for knock-out and knockin cellular clones using the CRISPR/Cas9 system[END_REF]. In brief, this technique enables the visual validation of DSB induction through recording of green and red signal originating from eGFP and mCherry expression cassette (Please see material and method for full description of the assay). The result of this experiment confirmed that DSB were induced in our cells (Figure 61).

Recombination method

DSB are one of the most harmful type of DNA lesion. They can happen randomly during process such as mitosis or be induced due the exposure to deleterious element such as radiations or reactive oxygen species. Importantly, unrepaired DSB ultimately leads to cell death. As a consequence, cellular process evolved to specifically repair DSB. They can be divided in two mains pathways: the nonhomologous end joining (NHEJ) and the homology direct repair (HDR) pathway. While genome editing techniques aiming to induce gene KO mostly rely on NHEJ mechanisms, gene KI techniques are usually taking advantage of HDR mechanisms. Specifically, HDR pathway requires the presence of long stretches of sequences homologous to the region where the DSB took place to repair the lesion. Of note, the natural frequency of HDR occurrence in cells is considered to be significantly lower level than NHEJ. Especially, because NHEJ mechanisms are functionally throughout the cell cycle (excluding mitosis), while HDR mechanism is active only during G2/S phase. In addition, NHEJ process is faster than HDR and the NHEJ process induces the inhibition of HDR pathways [START_REF] Yang | Methods favoring homologydirected repair choice in response to crispr/cas9 induced-double strand breaks[END_REF]. Despite these limitations, the number of studies depicting strategies leading to successful integration of foreign DNA through HDR mechanism with impressive success rate, lead us consider that KI ORAI1 and ORAI3 genes with HDR were realistic goals to achieve. In addition, working with the easy-to-transfect cells HEK-293 and aiming to fluorescently tag protein led us to believe that even if only few cells would display fluorescence within millions of non-fluorescent cells, FACS would allow us to identify and select the desired cell clone. Thus, we tested different strategies to generate KI HEK-293 cells.

4.2.3.1. Considerations before conducting homologous direct repair (HDR) experiment

In almost any case, the induction of a recombination event aiming to insert a piece of DNA at a specific genomic location, requires the use of a donor template (donor plasmid) displaying sequence homology with the DSB induction site. In order to obtain the best efficiency during the KI procedure, the donor plasmid should respect few criteria. Ideally, the homologue region should perfectly match the DSB induction point. A distance superior 10 base pair between the beginning of the homologue region from the donor plasmid and the DSB induction drastically reduces the chances of homologous recombination events (HR) [START_REF] Boel | CRISPR/Cas9-mediated homology-directed repair by ssODNs in zebrafish induces complex mutational patterns resulting from genomic integration of repair-template fragments[END_REF]. In addition, the design of the donor plasmid should be carefully studied. Indeed, if the donor plasmid displays the PAM as well as the intact targeting gRNA sequence, the CRISPR-Cas might degrade the plasmid before it could be used for the HR process. In a similar way, if following a successful HR event, the edited gDNA presents the PAM and the gRNA sequences, it is not excluded that CRISPR-Cas9 might induce a new DSB and thus ruins the outcome of the experiment (Figure 62). Thus, it is common practice to introduce one (or several) silent mutation(s) in the donor plasmid sequence corresponding to the PAM or gRNA sequence of the target gene.

Donor plasmid

The most common and most described way to generate homologous recombination for generating KI is represented by the co-transfection of a donor plasmid together with the Cas9-gRNA plasmid complex. The specific designs of both donor plasmids used for inducing HR in ORAI1 and ORAI3 genes sequences are presented in Figure 63. Specifically, for ORAI1 gene, the donor plasmid was designed as follow: the mTurquoise2 sequence was surrounded by an 800 base pair sequence corresponding to the ORAI1 5'UTR region, and by an 800 base pair sequence corresponding the genomic region following the ORAI1 start codon (including the totality of the ORAI1 first exon and part of its first intron). A similar design was used for ORAI3 gene, with the sYFP2 sequence as fluorescent tag and with regions corresponding the ORAI3 start codon surroundings.

In our first attempts to generate KI in HEK-293 cells, the Cas9-gRNA plasmids (coding for ORAI1gRNA3 or ORAIgRNA2) were transfected together with the donor plasmids (ORAI1 HR mTurquoise2 or ORAI1 HR sYFP2) in HEK-293. Assessment of a successful insertion of fluorescent tags was performed by FACS one week after transfection. However, no fluorescent signal could be detected and thus HR recombination could not be identified. Due to the low expected number of HR event in the cells, we hypothesized that performing FACS without confirmation of the occurrence of HR event was not the best strategy. Therefore, in the following experiments we decided to assess the successful insertion of fluorescent sequences by PCR. Indeed, in case of successful integration of fluorescent tag, PCR performed with primers surrounding the region of integration should display an additional band of higher molecular weight compared to WT HEK-293. Nonetheless, using the strategy described above, we remained unable to identify positive HR event on gDNA level (Fig 64A). Some studies reported that HR rate was improved due to transfection of linearized plasmid [START_REF] Ran | Genome engineering using the CRISPR-Cas9 system[END_REF].

Therefore, we tested this approach, by performing restriction digest and purification of the donor plasmid before transfecting it together with the Cas9-gRNA plasmid. Interestingly, PCR performed on gDNA displayed bands of a high molecular weight corresponding to successful recombination event potential successful HR, we reasoned that the absence of fluorescent detection in the cell population could be attributed due a low level of HR events. Thus, we looked for way to improve it. Importantly, some publications reported that small molecules could increase the rate of HR event in the cells, by unknown mechanism [START_REF] Yu | Small Molecules Enhance CRISPR Genome Editing in Pluripotent Stem Cells[END_REF]. Therefore, we used one of the mentioned molecules (Brefeldin A) in our subsequent experiments. In addition, we decided to select only cells transfected by the CRISPR-Cas9 system. To achieve this selection, we used two strategies:

• The first strategy tested was to transfect CRISPR-Cas9 plasmid encoding for a puromycin resistance cassette. Unfortunately, addition of puromycin to the transfected cells resulted in a high death rate, and PCR results were negative. In addition, we hypothesized that using a selection agent might lead to the creation of cellular clone constantly expressing the Cas9 protein and thus abandoned this way of selection.

• The second strategy tested was to transfect CRISPR-Cas9 plasmid coding for GFP. Thus, following transfection, GFP-positive cells were sorted using FACS. They were further expanded, and their mRNA were extracted to perform PCR and assess for successful HR event (Fig 64D). The results of this experiment indicated on the existence of potential successful HR event at a substantially higher rate.

Nevertheless, detection of fluorescence in confocal microscopy or cell sorting due to FACS remained impossible.

Increasing the endogenous expression level of ORAI1 and ORAI3

PCRs results indicated that recombination events were occurring in HEK-293 cells, but fluorescence could not be detected neither by FACS nor by confocal imaging. We hypothesize that the fluorescence level might be too dim to be effectively detected and hypothesized that it could be attributed to a low expression level of ORAI protein. Thus, we tried to increase ORAI1 and ORAI3 endogenous expression level. Importantly, publications showed that inactive Cas9 protein (which do not present cleavage activity) fused to transcriptional activating factor such as VP64-p65-Rta (VPR) or to the core of the human acetyltransferase p300 (p300) could be used to enhance expression of genes targeted by gRNAs [START_REF] Hilton | Epigenome editing by a CRISPR-Cas9-based acetyltransferase activates genes from promoters and enhancers[END_REF][START_REF] Chavez | Highly efficient Cas9-mediated transcriptional programming[END_REF]. Thus, we set out to design new gRNA targeting 5'UTR regions of ORAI1 and ORAI3 with the aim of increasing expression level of these proteins. Unfortunately, we could not detect any increase in expression level of ORAI1 or ORAI3 following transfection with these plasmids.

Precise Integration into Target Chromosome (PITCh) technique

Because KI experiments using the HDR reparation mechanism were not successful, we thought of using a strategy relying on the microhomology-mediated end-joining (MMEJ) to perform gene KI.

Specifically, an experimental approach named precise integration into target chromosome (PITCh) was considered. This technique relies on the insertion of short sequences (5 to 25 bp) identical to the DSB induction region on each side of a template plasmid bearing the desired sequence to inset. In addition, plasmid linearization is induced through DSB induced by the Cas9-gRNA plasmid. Plasmids enabling this approach were constructed. Nonetheless, these plasmids were constructed in parallel to the experiments where we could detect HR event with the classic HR donor plasmid, and by lack of time, they were not use for trying to generate KI in HEK-293 cells.

Conclusion on the gene KI experiments with CRISPR-Cas9 system.

While quite straightforward in theory, the generation of gene KI remained in our hand a complex task. Obviously, the CRISPR/Cas9 revolution offered the possibility to virtually any scientist to attend gene KI, nonetheless several pitfalls are still present. Specifically, during our attempts to generate KI we faced three main difficulties:

• First, the design, induction, and verification of the successful induction of DSB at the desired gene location were already well described at the beginning of this PhD. Nonetheless, several protocol adaptations were required in order to succeed in these tasks.

• Second, following the DSB induction, one desire to specifically insert a sequence of interest at the specific genomic location. When starting this work, the main technique described to reach this goal was to take advantage of HDR process. However, the low rate of success described with the classic technique of providing a plasmid template for recombination led us to adapt our initial strategy. Thus, we specifically selected positively transfected cells, tried to use a linearized donor plasmid, and tested drugs supposedly able to enhance the rate of recombination. In addition, several other techniques were considered such as the PITCh technique. These adjustments allowed us to detect by PCR the presence of recombination event. Nonetheless positively edited cells could not be identified.

• The final difficulty we faced the identification and selection of positively edited cells. Of note, most of the publications describing successful KI did not highlight this point probably for two main reasons. First, while generating KI, several publications used a donor plasmid bearing a selection cassette that would ease the selection of positively edited cells. Second, works describing fluorescent labeling of endogenously proteins were targeting high expression level proteins. In our case, the low expression level of ORAI protein prevented us to easily identify KI cells. As a consequence, we attempted to use the newly developed CRISPR tools allowing for specific increase of protein expression., which unfortunately remained unsuccessful.

As a conclusion, the field of gene KI with CRISPR/Cas9 system has continuously developed through the year, with roughly a 3-fold expansion in the number of publications reporting gene KI with CRISPR since 2017. Thus, several strategies have been developed that should ease the process of generating KI and are worth to be tested when trying to generate KI. Of note, the most appealing of these strategies will be briefly described in the discussion section. Anyway, due to time pressure and to our inability to select and identify cells that underwent recombination event, we had to adapt our strategy for reaching our aim to study ORAI protein remodeling. Therefore, because during our attempts to generate KI we had successfully induced DSB, we thought that knocking out ORAI1 and ORAI3protein before reexpressing them due to transfection would be a valid strategy. Identification of a suitable screening method for KO identification Since KI generation turned out to be unsuccessful, we decided to modify our strategy and to generate KO cells for ORAI1 and ORAI3 in which fluorescently labelled protein will be later re-expressed.

Creation of gene KO due to CRISPR-Cas9 technique takes advantage of the NHEJ reparation mechanism. Specifically, NHEJ mechanism takes place following the induction of DSB in the cell and will ligate the DNA extremities together. This mechanism is useful when intending to create KO, because it is error prone. In fact, NHEJ leads to the creation of small insertions or deletion (Indels) in the region surrounding the DSB. The size of the Indels usually ranges from 1 to 10 bp, nonetheless they are extremely heterogenous. The overall proportion of out-of-frame reparation is about 2/3, thus it represents an efficient way to generate KO. The T7E1 assay performed while attempting to generate KI, demonstrated that NHEJ mechanism were taking place in our cells and thus one could hypothesize that KO cells for ORAI1 and ORAI3 were effectively created. Nevertheless, we had to think of a way to select our KO cells from the whole population of transfected cells. To do so, we considered screening single-cell clones by immunoblotting, or by Ca 2+ imaging techniques. However, we did not possess a reliable antibody allowing to screen for the absence of ORAI1 of ORAI3 protein at endogenous level.

In addition, screening hundreds of clones through Ca 2+ imaging technique would be expensive and time consuming. Therefore, we find another way to screen for knockout generation. Interestingly, Chen et al., proposed a dual gRNA method enabling to screen for edited cell by PCR performed on gDNA [START_REF] Chen | Dual sgRNA-directed gene knockout using CRISPR/Cas9 technology in Caenorhabditis elegans[END_REF]. The procedure involves the use to two gRNAs targeting the same gene but separated by hundreds of base pairs. The induction of two DSB would lead to the excision of the small portion of DNA located between the DSB induced by gRNA. Thus, a simple PCR amplifying the gDNA region targeted by the gRNA allows for identifying cellular clone that experienced genomic fragment excision.

Generation of ORAI1 KO HEK-293 cells

The most efficient ORAI1-KO gRNA were cloned into the Cas9-2A-Puro plasmid. This plasmid was used because the generation of KO cells required the transfection of two Cas9-gRNA plasmids. We thought of using two different backbones plasmids so we could select double transfected cells. Indeed, the plasmid coding for Cas9-2A-GFP allowed for sorting of transfected cells by FACS, while the plasmid coding for Cas9-2A-Puro allowed us to use puromycin for selecting only double transfected cells. To validate our double gRNA strategy, we performed PCR on gDNA extracted from the double transfected cells. Our results validated our strategy since we could visualize that HEK-293 cells underwent ORAI1 gene excision (Figure 69A). Therefore, in a subsequent experiment, the similar strategy was used, and double transfected cells were further isolated by limit dilution in 96 well-plates.

Once single-cell colonies became visible, their gDNA were extracted and PCR were performed to identify cell clone displaying excision of ORAI1 fragment (Figure 69B). The positive clones were stored for further analysis.

Immunoblotting

Several ORAI1 antibodies were available in the laboratory at the time of the study, but their ability to detect endogenous level of ORAI1 in HEK-293 cells was not demonstrated. Even though results of Ca 2+ imaging experiment demonstrated that ORAI1 protein was absent in our cells, we decided to test these antibodies with the aim of identifying the most suitable for later studies. Of note, the expression level of ORAI1 in HEK-293 is relatively low. Thus, PC3 cells, supposedly expressing higher level of ORAI1, wereu sed as a positive control in the experiment. Unfortunately, all the antibodies tested failed to provide clear evidence for the effective ORAI1 KO. Indeed, antibodies purchased from Abcam and ProteinTech, were clearly not functional as they exhibited an apparent random signal all over the membranes (Figure 71A). Other antibodies tested (purchased from Alomone, GeneTex and Sigma) displayed more convincing but not perfect signal. In fact, the three antibodies were effectively lacking bands compared to WT HEK-293 and PC3 cells. Nonetheless, the molecular weight of these bands was not identic between antibodies, with some antibodies displaying bands at 50 kilodaltons, corresponding to glycosylated for of ORAI1, or 35 kDa (Figure 71B), corresponding to the expected size of ORAI1.

In addition, some nonspecific bands were present at the expected size for ORAI with the GenTex antibody. Taken together the results of immunoblotting confirmed that ORAI1 protein was effectively deleted in our cells. Nonetheless, because of the lack of quality of the available antibodies we decided to avoid using them for the conduction of the study. The previous study from our laboratory suggested the existence of a dynamic remodeling between ORAI1 and ORAI3 proteins was performed using two different cancerous cell lines: LNCaP and PC3.

On the one hand, LNCaP cells present a modal number of 76 to 91 chromosomes. On the other hand, PC3 presents a modal number of 62 chromosomes Thus, we decided to use the PC3 cells to create double KO for ORAI1 and ORAI3 genes. We used the same strategy to induce KO in the PC3 cells as the one designed for HEK-293 cells. Specifically, we indented to create ORAI1 KO PC3 and to use these KO cells to generate a second KO for the ORAI3 gene. Indeed, we rationalized that the generation of ORAI1 KO could be quickly confirmed through Ca 2+ imaging experiment and would allow us to quickly select ORAI1 KO cell clones to generate the double KO. Conversely, using a strategy where ORAI3 KO would be generated first would force us to confirm ORAI3 KO through genomic sequencing before generating double KO for ORAI1, and thus would be more time consuming. entry pathway in non-excitable cells [START_REF] Prakriya | Store-operated calcium channels[END_REF]. Because Ca 2+ is a central intracellular second messenger controlling several cellular functions such as proliferation and migration [START_REF] Clapham | Calcium Signaling[END_REF][START_REF] Berridge | Calcium signalling: Dynamics, homeostasis and remodelling[END_REF], we hypothesize that ORAI1 deletion and SOCE impairment should affect HEK-293 physiological properties. Of note, it was already shown that ORAI1 and SOCE are altered in cancer [START_REF] Jardin | STIM and calcium channel complexes in cancer[END_REF]. Nonetheless, their role in the control of cancerous properties of the cells seems to be dependent on the type of cancer studied. For example, it was shown that SOCE does not control the proliferation of primary cultures of human metastatic renal cellular carcinoma [START_REF] Dragoni | Store-operated Ca2+ entry does not control proliferation in primary cultures of human metastatic renal cellular carcinoma[END_REF] while other studies reported that ORAI1 and STIM1 downregulation were inducing cell cycle arrest in multiple myeloma cells (Wang et al, 2018b). Similar contradictions were described for the role of ORAI1 and SOCE in migration, as breast cancer cells display migration impairment due to ORAI1 downregulation [START_REF] Yang | Orai1 and STIM1 Are Critical for Breast Tumor Cell Migration and Metastasis[END_REF] while colorectal carcinoma cells migrative properties were not affected by the downregulation of SOCE components [START_REF] Zuccolo | Stim and Orai mediate constitutive Ca2+ entry and control endoplasmic reticulum Ca2+ refilling in primary cultures of colorectal carcinoma cells[END_REF]. In a physiological context, ORAI1 function is also cell type dependent as it is involved in the proliferation of endothelial cells [START_REF] Abdullaev | Stim1 and orai1 mediate crac currents and store-operated calcium entry important for endothelial cell proliferation[END_REF], inhibits the migration of myeloblastic cells ((Diez-Bello et al, 2017), and controls the differentiation of myoblasts [START_REF] Darbellay | STIM1and Orai1-dependent store-operated calcium entry regulates human myoblast differentiation[END_REF]. In addition, studies from our laboratory suggested that ORAI1 was involved in HEK-293 cells proliferation, but that this role was SOCE independent [START_REF] El Boustany | Differential roles of STIM1, STIM2 and Orai1 in the control of cell proliferation and SOCE amplitude in HEK293 cells[END_REF][START_REF] Borowiec | Are Orai1 and Orai3 channels more important than calcium influx for cell proliferation?[END_REF]. Finally, patients with loss of function (LoF) or gain of function mutation (GoF) leading to absence or constitutive activation of SOCE present syndromes mostly affecting specific tissues or organs (Lacruz & Feske, 2015b). These data question the general assumption that ORAI1 and SOCE are controlling most of the cellular function. Overall, the different conclusion on the role of ORAI1 and SOCE in the control of cellular processes can be attributed to the techniques that were used to study their role. Indeed, downregulation as well as pharmacological inhibition of SOCE represent so far, the most widely tolls used to determine the roles of ORAI1 and SOCE. Although these techniques have proven to be powerful, pitfalls such as residual endogenous We continued investigating the role of ORAI1 in the migration of HEK-293 by performing the collective migration cell assay represented by the wound healing experiment. We analyzed several features of the collective migration. First, we determined that the collective speed migration was increased for ORAI1 KO cells only during the first 10 hours of the experiment. Further measurements of the collective migration speed from the 10 th hours until total wound closure did not show any differences (Figure 81A). Second, we compared the time of total wound closure between our cell line and observed that there was no difference (Figure 81B, left). Nonetheless, because we observed that migration speed was different in the beginning of our experiment, and because the initial size of the wound is susceptible to variation, we performed a ratio between the half-and full-wound closure time.

This analysis confirmed that the collective migration speed of HEK-293 was significantly slower than for ORAI1 KO in the beginning of the experiment (Figure 81B, right). Finally, we measured the leading-edge migration speed of our cell lines. Specifically, we measured the shortest distance between the two migration front every five hours period (Figure 82). This analysis demonstrated that ORAI1 KO cells are migrating faster than WT HEK-293 during the 1 st -5 th hours and 5 th -10 th hours periods.

Altogether, our data of wound healing experiment indicates that collective WT HEK-293 migration present a delay in the migration initiation compared to ORAI1 KO cells. Finally, we wanted to assess whether the effect observed was restricted to collective migration, or if ORAI1 KO was also affective single cell migration. Therefore, we performed Transwell ® assay on our cell line. Our results indicate that single-cell migration is not affected by ORAI1 KO (Figure 83). Thus, we concluded that ORAI1 is involved in the control of HEK-293 cell collective migration.

Conclusions

In this study we have confirmed that ORAI1 is the main SOCE effector in HEK-293. In addition, we revealed that a small proportion of SOCE is not attributed to ORAI1 channel. RT-qPCR data suggest that the remaining SOCE observed is not due to ORAI 2 and ORAI3 overexpression. was modified due to ORAI channel activation. This work originates from one of the hypotheses suggested by the publication of Dubois et al. (Dubois et al, 2014a). Specifically, the authors demonstrated that the disruption of the equilibrium between ORAI1 and ORAI3 expression represents an oncogenic switch. In particular, the authors showed that ORAI3 is overexpressed in prostatic primary tumors. Moreover, they overexpressed ORAI3 in prostate cancer cell line, to resemble the remodeling observed during cancer progression, and demonstrated that ORAI3 overexpression promotes its association with ORAI1 and leads to the formation of heteromeric ORAI1-ORAI3 channel. The association between these two proteins induces a modification in the cellular Ca 2+ answers provided by the cell thereby promoting cancerization processes. Finally, the authors proposed that ORAI channel composition as homo-or hetero-multimers depends on the expression level of the different ORAI proteins, but also on the specific stimulation received by the cells (Dubois et al, 2014b). The later proposition suggests that ORAI channel composition is susceptible to undergo a dynamic remodeling depending on the stimulation received by the cells. This suggestion led us to hypothesize that ORAI proteins could undergo an on-site assembly leading to the creation of specific ORAI channels. Of note, such a mechanism of an "on site" assembly of the ORAI channel, while highly hypothetical, was also proposed by Li and al (Li et al, 2016b). Alternatively, one can consider the existence of pre-assembled channel of different composition which would be specifically activated depending on the signal received by the cells. To address these possibilities, we took advantage on a collaboration with a team specialized in the use of FLIM-FRET technique to identify if ORAI protein interaction level was modified during activation of ORAI channels.

4.5.2. Impact of ORAI1 and ORAI3 overexpression modulation on SOCE level 4.5.2.1.

Effect of transient overexpression of ORAI1 and ORAI3 on SOCE

The previous publication from our laboratory demonstrated that ORAI3 overexpression was impairing SOCE in PC3 cells. Of note, this result was obtained in PC3 cells stably overexpressing ORAI3. However, the expression level of ORAI3 in the cells was 20 times higher than in WT PC3 cells.

Of note, this large overexpression of ORAI3 might have allowed detection of ORAI3 effect of SOCE which are marginal in physiological conditions. In addition, the creation of stable clones potentially induced compensatory mechanisms affecting the SOCE process. Therefore, we decided to confirm and extend their results by transiently overexpressing ORAI3 in WT PC3 cells. We hypothesized that transient overexpression would prohibit the apparition of compensatory mechanisms and would result in a cellular population overexpressing various level of ORAI3. Hence, the recording of SOCE from the whole cell population might depict a more accurate picture of ORAI3 impact on SOCE. In addition, we decided to assess if the overexpression of ORAI1 would also affect SOCE.

Accordingly, we transfected PC3 cells with ORAI3 or ORAI1 plasmids and performed Ca 2+ imaging experiments where store depletion was induced with TG. The results of the Ca 2+ imaging experiment indicated that the transient ORAI3 overexpression decreased the level of SOCE compared to PC3 WT, while ORAI1 seemed to enhance SOCE (Figure 84). The quantification of the amplitude of SOCE revealed that ORAI3 transient overexpression was significantly decreasing SOCE (Figure 85).

However, ORAI1 overexpression did not significantly affected the level of SOCE compared to WR cells (Figure 85). Thus, these results confirmed the previous data obtained by the laboratory that ORAI3 impairs SOCE. To extend further these results, we decided to use the ORAI1 and ORAI3 KO cells and to measure their SOCE level. In addition, we aimed to perform rescue experiment where the deleted protein (ORAI1 or ORAI3) would be re-expressed in order to verify that the effect observed in the KO cells could not be attributed compensatory mechanisms. Thus, these results suggest the existence of heteromeric ORAI channels composed of ORAI1 and ORAI3 proteins. Since we showed that ORAI3 was modulating SOCE entry we decided to study the interaction level between ORAI1 and ORAI3 following ORAI channel activation. Specifically, HEK-293 were transfected with different ratio of ORAI1-mTurquoise2 (ORAI3-mT2) and ORAI3-sYFP2 plasmids (i.e, ratio 1:1 and ratio 1:3 for ORAI1-mT2:ORAI3-sYFP2. Indeed, the publications from Dubois et al., identified that ORAI3 expression level was three time higher to ORAI1 in prostate cancer tissue. In their work they used these transfections ratio to mimic the cancerous phenotype. Therefore, we used the same strategy to compare the interaction level between the two proteins depending on their relative expression level. Of note, for all the FLIM-FRET experiments, ORAI1-mT2 was used as the donor molecule, while ORAI3-sYFP2 was used as the acceptor molecule.

In addition, only the donor lifetime (ORAI1-mT2 only transfection) was measured for each experiment to allow the calculation of the FRET efficiency. Finally, because lifetime is sensitive to external condition such as the temperature, and pH, the data obtained for each experiment were statistically treated as paired because they were performed in the exact same conditions (same solutions for example).

Basal interaction

In a first set of experiment, we evaluated the interaction level between ORAI1 and ORAI3 before activation of ORAI channels. Specifically, ORAI1-mT2 and ORAI3-sYFP2 were transfected in WT HEK-293 as the acceptor molecule with different ratios (1:1 and 1:3), and changes in the ORAI1mT2 lifetime were measured. Of note, in case of FRET, the lifetime of ORAI1-mT2 is expected to decrease because FRET represent an additional pathway for de-excitation of the excited molecule. First, we measured lifetime of ORAI1-mT2 alone and in the presence of ORAI3-sYFP2 in equimolar quantity (i.e, transfection, ratio of 1:1) and in a three-fold higher expression rate (i.e, transfection ratio of 1:3).

ORAI1-mT2 lifetime alone was measured at 3.97 ns, which is consistent with the lifetime of the "free" mTurquoise2 fluorescent protein (4 ns) (Figure 89A). When ORAI3-sYFP was transfected together with ORAI1-mT2, a significant lifetime decrease was observed, revealing the existence of FRET and thus the existence of basal interaction between ORAI1 and ORAI3 (Figure 89A). Increasing the ratio of ORAI3-sYFP2 expression (ratio 1:3), decreased even further the ORAI1 mTurquoise2 lifetime indicating that the level of interaction between ORAI1 and ORAI3 was increased (Figure 89A). Thus, we demonstrated that ORAI1 and ORAI3 are interacting in basal condition. In addition, we demonstrated that the level of interaction between these two proteins is dependent on ORAI3 level of expression. In the subsequent experiments we wanted to evaluate the ORAI1-ORAI3 interaction following ORAI channel activation. Because STIM1 can be limiting during ORAI channel activation and thus its overexpression might be required. We performed the same experiment as above with the additional overexpression of the STIM1-mCherry plasmid to determine if this overexpression was affecting the basal interaction between ORAI1 and ORAI3. The measured lifetime of ORAI1-mT2 in the presence of STIM1 not deeply modified in the presence or absence of STIM (Figure 89B).

Arachidonic acid activated interaction

Next, we assessed if the activation of ORAI channels was modifying the interaction level between ORAI1 and ORAI3 proteins. First, we used arachidonic acid (AA) to specifically activate the ARC channel. Of note ARC channel is hypothesized to be constituted by a pentameric assembly of three ORAI1 proteins and two ORAI3 proteins. Because it was shown that STIM1 is required for ARC activation, measurements were performed both with endogenous STIM1 expression, and overexpressed STIM1. Using the same ratio strategy as described above we observed that in the 1:1 expression ratio, the addition of AA on the cells did not influence the lifetime of ORAI1mT2, independently on the overexpression presence of STIM1 (Figure 90 A andB). At the opposite, using a 1:3 transfection ratio, we observed that addition of AA on the cells was increasing the interaction level between ORAI1 and ORAI3, only when STIM was not overexpressed (Figure 91 A and B). Because AA is a highly unstable molecule, we thought that the difference in the effect observed when using AA might be attributed to its degradation when experiments were performed using a non-freshly open vial. Thus, we inspected single experiments result to determine if experiment performed on the day vial-opening were displaying different result than other repeats, but it was not the case (Figure 90 and 91 low panels). Overall, our results indicate that the addition of AA on the cells modifies the interaction level between ORAI1 and ORAI3 only when ORAI3 is overexpressed and that this increased interaction is prevented when STIM1 overexpressed.

4.5.3.1.3.

Store dependent interaction

Further, we decided to measure the interaction between ORAI1 and ORAI3 following SOCE activation. Indeed, the Ca 2+ imaging experiments performed in overexpression system, and in KO models suggest that ORAI3 is involved in the SOCE. Because STIM1 is known to be a limiting factor for SOCE development when ORAI1 is overexpressed, all the experiment were carried out with STIM1 overexpressed together with ORAI1 and ORAI3. Our results indicate that SOCE activation leads to an increased interaction between ORAI1 and ORAI3 when these proteins were expressed at the same level (ratio 1:1) (Figure 92A). On the contrary, when ORAI3 was overexpressed (ratio 1:3), SOCE activation did not modify their interaction level (Figure 92B).

Sequential activation of ARC and CRAC

Overall, our results demonstrate that ORAI1-ORAI3 interaction is modified by 1-AA when ORAI3 is overexpressed compared to ORAI1 (ratio1:3) in the absence of STIM1 overexpression; 2-TG when ORAI1 and ORAI3 are expressed in the same quantity and with STIM1 overexpressed. In order to obtain a better understanding of these opposite effects, we performed experiments where AA was added on the cell to induce activation of ARC channels, followed by the addition of TG to induce SOCE. Because we planned to focus on the SOCE activated interaction between ORAI1 and ORAI3, we performed these experiments only when STIM1 was overexpressed. Interestingly, the sequential activation of ARC channel followed by SOCE channel activation indicates that when expressed in the same proportions (ratio 1:1), neither the activation of ARC, nor the activation of SOCE modified the interaction level between ORAI1 and ORAI3 (Figure 93A). Nonetheless, it is interesting to note that ARC activation prevented the increase of interaction between ORAI1 and ORAI3 that was observed following SOCE activation (Figure 92A). The same experiment carried-out when ORAI3 was overexpressed compared to ORAI1 (ratio 1:3) did not lead to any significant differences in the lifetime of ORAI1mT2 (Figure 93B). These data demonstrate that AA pre-treatment prevents modification of the ORAI1-ORAI3 interaction induced by SOCE activation.

especially because their endogenous expression is significantly higher in PC3 compared to HEK-293.

Thus, we pursued our experiments in the PC3 double knockout cell line. Following the same strategy as for HEK-293, we started by evaluating the level of interaction between ORAI1 and ORAI3 in basal condition. In addition, because ORAI1 and ORAI3 were deleted in our cells, we decided to verify if STIM overexpression was mandatory to observe any effect in the double KO cell line. First, we confirmed that ORAI1 and ORAI3 are interacting in basal condition and revealed that increasing the proportion of ORAI3 enhanced the interaction between ORAI1 and ORAI3 (Figure 95 A). This effect was reproducible independently on the overexpression of STIM1 (Figure 95 B).

Store dependent interaction

Subsequently, we induced SOCE with TG, and measured the interaction level between ORAI1 and ORAI3. We started by performing this experiment without overexpressing STIM1. Our results indicate that induction of SOCE leads to increased interaction between ORAI1 and ORAI3 when they are expressed in the same proportion (ratio 1:1) (Figure 96 A) but not when ORAI3 was overexpressed compared to ORAI1 (ratio 1:3) (Figure 96 B). Then, we repeated this experiment in cells overexpressing STIM1. In line with the former results, when ORAI1 and ORAI3 were expressed respecting the 1:1 ratio, their interaction level was reinforced upon SOCE induction (Figure 97 A). In addition, for the ratio 1:3 in the presence of STIM, the induction of SOCE led to an increase of interaction between ORAI1 and ORAI3 (Figure 97 B).

Carbachol induced interactions

Finally, because TG is an irreversible blocker of SERCA pumps, its addition in the cell medium ultimately leads to a complete Ca 2+ store depletion, which does not depict accurately physiological level of SOCE activation. Thus, we decided to use carbachol (Cch) to induce transient and reversible store depletion. Of note, carbachol is a ligand of muscarinic acetylcholine receptor, whose activation leads to activation of phospholipase C inducing the degradation of the phosphatidyl inositol phosphate into diacylglycerol and inositol triphosphate (IP3). The later activating the ER-residing IP3 receptor which leads to Ca 2+ store release. We repeated one more time our classic protocol by replacing TG by Cch. As we demonstrated that STIM1 overexpression was mandatory to observe effects on interaction between ORAI1 and ORAI3, the experiments were performed with STIM1 overexpressed. Our results indicate that in the ratio 1:1, SOCE induction with Cch leads to an increased interaction between ORAI1 and ORAI3 (Figure 98 A), while no differences in interaction were visible when using the ratio 1:3 (Figure

B).

Conclusion on the interactions between ORAI1 and ORAI3 in PC3 cells

Our data on PC3 cells demonstrate the importance of ORAI1 and ORAI3 deletion to detect fine variation in their interaction level. Indeed, no difference in the interaction level between ORAI1 and ORAI3 due to ORAI3 overexpression was identified in PC3 WT, but the deletion of these endogenous protein in our KO model allowed to identify that ORAI1 and ORAI3 interaction level was dependent on ORAI3 expression level. In line with these results, SOCE activation did not alter the interaction level between ORAI1 and ORAI3 in PC3 WT but increased their interaction in the double KO cells. In addition, in store-depleted condition, we showed that ORAI1 and ORAI3 were displaying an increased interaction when they were expressed at the same level (1:1 ratio) or when ORAI3 was overexpressed

(1:3 ratio). Moreover, we showed that this effect was dependent on the STIM1 expression level. Finally, using a more physiological SOCE activator, we showed that the increased interaction between ORAI1 and ORAI3 was effective only when they were expressed in the same ratio.

5. Discussion and perspectives 5.1. Challenges in generating gene KI with the CRISPR/Cas9 system

In the first part of this PhD, we aimed to create gene KI to fluorescently label the ORAI1 and ORAI3

proteins. Through our different tentative, we were able to successfully induce a DSB on the desired genomic location, and we could identify HR recombination event by PCR screening. Nonetheless, we were unable to identify and select the cells that positively underwent recombination. The reasons of our inability to create gene KI are not clearly defined and are probably the consequences of multiple factors, that will be exposed thereafter.

HDR-based method of KI

First, the method we used to generate KI might not have been optimal. Indeed, HR techniques relying on the cellular reparation mechanisms HDR was considered being the standard technique to generate KI in the beginning of this PhD work. Nonetheless, diverse techniques using modified donor sequence were developed and reported better efficiencies than the classic HR method. For example, the easi-CRISPR method relying on single stranded donor (ssODN) bearing homology arms displayed efficiencies up to 100% depending on the loci considered (Figure 99A) [START_REF] Miura | Easi-CRISPR for creating knock-in and conditional knockout mouse models using long ssDNA donors[END_REF]. However, ssODN production is complicated and subject to mistake (i.e, single nucleotide polymorphism) during production (kit for in vitro production of ssODN exists but are relying on reverse transcriptase which are not proof-reading). In addition, modification of the standard protocol for inducing HR with the use of a donor plasmids have been developed such as cell synchronization (HDR events are more frequent during G/S phase of the cycle), or inhibition of NHEJ mechanisms. Nonetheless, it appears that success rates in generation of KI are cell type / loci / size and type of insertion dependent, and thus several techniques need to be tested for a specific desired application [START_REF] Yamamoto | Making ends meet: targeted integration of DNA fragments by genome editing[END_REF].

Non-HDR based method for KI

Other techniques non relying on HDR mechanisms were proposed to generate KI. Of note, the PITCh method developed in 2014 represented an interesting alternative to the HDR-induced KI methods (Figure 99B) [START_REF] Nakade | Microhomology-mediated end-joining-dependent integration of donor DNA in cells and animals using TALENs and CRISPR/Cas9[END_REF][START_REF] Sakuma | MMEJ-Assisted gene knock-in using TALENs and CRISPR-Cas9 with the PITCh systems[END_REF]. Especially, this strategy involves donor plasmid linearization performed in vivo due to specifically designed gRNA sequences as well as antibiotic selection of successful recombination event. Nonetheless, the original plasmid construct available were intended for KI of GFP tag in the C-terminal extremity of protein. Since our aim was to tag our protein of interest in the N-terminal extremity with the mTurquoise2 of sYFP2 protein, several rounds of cloning were required to obtain a construct allowing the tagging of N-terminal extremities with desired fluorescent tags. This laborious and time-consuming procedure were performed nonetheless, due to time pressure and modification of our global strategy (use of KO models instead of KI) the experiments for generating KI could not be performed (see material and method for detailed procedure). Alternative methods including the incorporation of the target gRNA sequences on each side of the homologue sequences leading to creation of a donor template bearing the same DSB-induced extremities as the targeted genome were proven successful for generating homology-independent targeted integration (HITI) through NHEJ (Figure 99C) (Suzuki et al, 2016b) with success rate up to 30-40 %. Alternatively the use of Cas9 variants leading to the creation of cohesive ends following DSB induction were proposed to induced incorporation of DNA fragment in the DSB site by designing donor plasmid bearing the same DSB induction site in its sequence (Figure 99D) [START_REF] Yamamoto | Making ends meet: targeted integration of DNA fragments by genome editing[END_REF]. Of note, several techniques homology-independent gene insertion technique were developed in the recent years such as the homology-independent universal genome engineering (HiUGE) using adenoviral vector to generate insertion at specific sites, or the CRISPR/Cas9-mediated Homology-independent PCR-product integration (CHoP-In) using PCR products as donor template, or the Open Resource for the Application of Neuronal Genome Editing (ORANGE), a modified HITI method developed to enable KI in neurons [START_REF] Gao | Plug-and-Play Protein Modification Using Homology-Independent Universal Genome Engineering[END_REF][START_REF] Manna | Fast and cloning-free CRISPR/Cas9-mediated genomic editing in mammalian cells[END_REF][START_REF] Willems | ORANGE: A CRISPR/Cas9-based genome editing toolbox for epitope tagging of endogenous proteins in neurons[END_REF]. The development of all these techniques indicates that improvement in efficiencies rate is required to generalize the use of the CRISPR-Cas9 method to generate KI.

Hypothesis on the non-detection of recombination events

The fact that PCR amplification of the targeted region for KI indicated a successful fluorescent tag insertion, suggest that the KI were achieved in our cells. Nonetheless, cells displaying such a positive recombination event could not be selected. Several reasons could explain this result. First, the HEK-293 being hypotriploid cells one can consider that HR events were occurring on a single allele and thus leading to a fluorescence intensity that would be below detection level of FACS. In addition, as discussed earlier, the endogenous level of expression of ORAI1 and ORAI3 protein might be too low to enable their detection with fluorescent tags. The CRISPR strategies allowing enhancement of protein expression, have been refined in the last years, and might present an interesting solution to increase expression level of ORAI proteins. Finally, while the HDR technique is reputed error-free it is not obligatory the case, and insertion of small insertion or deletion (InDels) at the junctions sites occurs due to the existence of NHEJ reparation mechanisms concomitant to HDR mechanisms [START_REF] Paquet | Efficient introduction of specific homozygous and heterozygous mutations using CRISPR/Cas9[END_REF]. The generation of ORAI KO in HEK-293 and PC3 cell lines was achieved relatively easily. Indeed, the experimental design of the experiment, especially the use of dual gRNA strategy allowing for an easy screening procedure, and the experience acquired with the use of CRISPR/Cas9 system during the KI experiments allowed us to create KO cell line for ORAI proteins in HEK-293, but also in cancerous PC3 cells. While our strategy was successful, there are still rooms for improvements for every step involved in the KO generation with CRISPR/Cas9 genome editing technique.

Design of gRNA

The French-based CRISPOR website (http://crispor.tefor.net/) was used to design gRNAs in this work. Nonetheless, several online tools are allowing the design of gRNA. Some of these tools are developed and maintained by public laboratory (For example: https://chopchop.cbu.uib.no/, and https://portals.broadinstitute.org/gppx/crispick/public), or by private companies specialized in CRISPR related products (such as Synthego: https://design.synthego.com/). Within all these tools, the computed efficiency and specificity scores for the proposed gRNA are somewhat similar. Nonetheless, each website displays different features (indication of the GC content of the gRNA, or self-complementarity of gRNA sequence), or enable to screen for gRNA to be used for specific usages (gene interference, gene enhancement of expression, nickase experiment). Thus, crossing their results might be useful to define the more suitable sequences required for a specific experiment.

Assessment of gRNA efficiency

Efficiency assessment of the gRNA represents another important procedure in the development of KO cell lines. In our case, the identification of suitable primers pairs allowing to perform robust PCR on gDNA was complex. Of note, non-coding genomic sequences can display specific structures requiring longer denaturation times to allow polymerase to access the amplification site. In addition, these regions can display highly repetitive sequences or rich GC content where the polymerases are usually poorly performing. Finally, the design of primers amplifying specifically a defined region is sometimes complex due to the increased size of the genomic DNA compared the size of the transcriptome. In our case, several primer pairs were tested, and PCR protocol adaptations were required to obtain robust and reproductible PCR amplifications. While we cannot exclude that these difficulties were gene specific, we believe that a sufficient amount of time should be devoted to the primer design and PCR condition adjustments. On a similar note, the assessment of DSB induction efficiency with the T7E1 assay was also a time-consuming procedure. Indeed, the incubation time of the PCR products with the enzyme as well as the level of amplification of the target region, or the size of the amplicon to be digested required adjustments to allow the obtention of clear results. For this last point, we found that increasing the size of the PCR amplicon greatly helps to obtain more reliable results because of the stronger intensity displayed by the digested PCR products of high molecular weight. Thus, T7E1 assays performed with KO gRNAs, where the PCR product were in the range of a thousand kb, exhibited more reliable results than the T7E1 assays performed for the KI gRNAs, where the size of PCR product was around 500 pb. Overall, because the sensitivity of the assay is reputed quite low, this assay should mostly be seen as a tool to assess the induction of DSB by the designed gRNAs than a precise method allowing the clear identification the most efficient gRNA. Finally, other strategies such as the Tracking of Indels by Decomposition (http://shinyapps.datacurators.nl/tide/), might represent an interesting tool to assess the efficiency of Indels induction of a gRNA. 5.2.3. Screening method for selection of KO cell lines and validation of KO.

Due to the dual gRNA strategy, the screening of potential KO was greatly facilitated. In addition, the re-use of primers pairs designed for T7E1 assay allowed efficient PCR to be performed without additional difficulties. One of the limitations of this strategy is the multiplication of potential off-target sites, thus choosing highly specific gRNA is advised. In addition, one should keep in mind that the gene excision procedure might be inducing a higher rate of complex genomic recombination as several clones screened by PCRs were displaying additional bands of non-expected sizes.

Finally, KO validation through the use of antibodies was particularly complex as different antibodies were displaying bands at different sizes for ORAI1. These differences might be explained by the differences in the epitope binding of ORAI1 antibodies. Of note, ORAI1 exhibit glycosylation site on N223 (asparagine 223 on the 2nd extracellular loop or ORAI1). Importantly, Alomone antibody was raised with synthetic peptide sequence in close proximity with the glycosylation site. We believe that glycosylation prevents ORAI1 binding to its site and thus. On the contrary, Sigma and GeneTex antibodies were raised against N-terminal which are distant from the glycosylation site and thus recognizes specifically this form (50kDa). Nonetheless, it is clear that reliable antibody for ORAI protein was not available during this study and thus their use was avoided. methods allowing for the screening and sequencing of whole genomes or transcriptome. However, they represent expensive technique to conduct for simply ensuring the absence of off-target effect.

Nonetheless, the development of affordable sequencing techniques such as Nanopore sequencing technology might represent an interesting alternative. In addition, using NGS techniques might allow to identify compensatory mechanisms and off-target effects in a single experiment. Finally, because a list of off-target sites is usually provided when identifying suitable gRNA, a simple amplification of these sites for off-target represents an easy method for assessing the induction of DSB at unwanted genomic sites but would allow to identify events involving relatively big insertion or deletion events (+/-50 bp).

5.3. Dispensability of ORAI1 in the control of the HEK-293 physiology.

Following the establishment of the ORAI1 KO HEK-293 cell line, we set-out to study the effect of this deletion on the physiology of HEK-293 cells. Specifically, we identified that ORAI1 deletion was greatly impairing SOCE but was not totally abolishing it. In addition, we evaluated if this remaining SOCE could be attributed to compensatory mechanism involving modulation of expression of other ORAI homologues and concluded that it was not the case. Further, we evaluated the proliferative properties of the ORAI1 KO cells and compared them to WT HEK-293. We observed that the deletion of ORAI1 protein did not modify the proliferation of HEK293 cells. Finally, we studied the migrative properties of the ORAI1 KO cells in comparison the WT HEK-293. We noted that adhesive properties were not modify following ORAI1 deletion. In addition, we demonstrated that ORAI1 deletion was increasing the speed at which HEK-293 cells are initiating their collective migration but was not affecting single cell migration. Others have used a single gRNA strategy leading the induction of an ORF shift toward the middle of the ORAI1 protein sequence. While the possibility that "half-ORAI1" protein could possess ill-function is hypothetical, it represents a potential explanation for the differences observed in SOCE level.

Alternatively, we proposed that the remaining SOCE could be attributed to ORAI2 or ORAI3.

Nevertheless, our RT-qPCR data indicate that the expression level of both genes is decreases in ORAI1 KO cells. Another hypothesis is that the observed SOCE entry is potentiated in our experiment due to the use of a supra physiological Ca 2+ concentration (4mM) while Cai et al., and Yoast et al., used a physiological Ca 2+ concentration [START_REF] Cai | The Orai1 store-operated calcium channel functions as a hexamer[END_REF]Yoast et al, 2020b). Finally, we cannot exclude that expression level of other channels might be altered to compensate for ORAI1 deletion such as TRCPs since they have been shown to interact with STIM and to participates in the SOCE entry.

ORAI1 deletion does not affect proliferative properties of HEK-293

In the second part of our work, we studied and demonstrated that ORAI1 did not control the proliferation of HEK-293 cells. Interestingly, in immune cells, it was demonstrated that SOCE control proliferation through Nuclear Factor of activated T-cells (NFAT) activation [START_REF] Cai | The Orai1 store-operated calcium channel functions as a hexamer[END_REF]Yoast et al, 2020b). Specifically, NFAT is known to regulate cell cycle proteins such as cyclins [START_REF] Mognol | Cell cycle and apoptosis regulation by NFAT transcription factors: new roles for an old player[END_REF]. Moreover, work from our laboratory demonstrated that ORAI1 knockdown was decreasing the expression level of cyclin D1 in a prostate cancer cell line. In addition, several studies established a role for ORAI1 and SOCE in the control of cancer cell proliferation [START_REF] Dragoni | Store-operated Ca2+ entry does not control proliferation in primary cultures of human metastatic renal cellular carcinoma[END_REF]. Nonetheless, these results were obtained in cancerous model where ORAI1 and SOCE are dysregulated. On a physiological level, our laboratory demonstrated a role for ORAI1 and ORAI3 proteins in HEK-293 proliferation [START_REF] El Boustany | Differential roles of STIM1, STIM2 and Orai1 in the control of cell proliferation and SOCE amplitude in HEK293 cells[END_REF][START_REF] Borowiec | Are Orai1 and Orai3 channels more important than calcium influx for cell proliferation?[END_REF]. However, these results were obtained due to transient downregulation and acute treatment, while we induced a stable deletion of the ORAI1 protein. We hypothesize that the total deletion of ORAI1 protein probably led to the apparition of compensatory mechanisms allowing HEK-293 cells to assure their proliferation. Of note, other groups have demonstrated an impairment of NFAT translocation following ORAI KO in HEK-293 cells. Nonetheless the physiological consequences of these impairment were not studied. One can hypothesize that if ORAI1 deletion was inducing strong modifications in the behavior of their KO HEK-293 cells, these studies would have studied the physiological consequence of the NFAT translocation impairment. As a conclusion, our results demonstrate that ORAI1 is not a crucial regulator in the control of HEK-293 proliferation, even though a limited role for this protein cannot be excluded. Indeed, the creation of complete KO might have induced the apparition of compensatory mechanisms. In addition, since HEK-293 are cultivated in FBS-containing media, is it conceivable that the FBS-specific composition contributes to the variations observed in other studies about a role of ORAI1 in proliferation.

5.3.3. ORAI1 deletion is not fundamental for the control of HEK-293 migration

In the last part of our work, we demonstrate that ORAI1 affects the collective migration properties of HEK-293. Of note, Ca 2+ has been shown to be involved in several migration processes through the control of proteases, phosphatases, and Ca 2+ -dependent kinases [START_REF] Wei | Calcium gradients underlying cell migration[END_REF][START_REF] Sun | Cholesterol-induced activation of TRPM7 regulates cell proliferation, migration, and viability of human prostate cells[END_REF].

Therefore, it is not surprising that SOCE has also been shown to control the migration process. For example, it was demonstrated that SOCE and ORAI1 were controlling migration properties of melanoma and breast cancer cells [START_REF] Umemura | Store-operated Ca2+ entry (SOCE) regulates melanoma proliferation and cell migration[END_REF][START_REF] Yang | Orai1 and STIM1 Are Critical for Breast Tumor Cell Migration and Metastasis[END_REF]. Nonetheless, in a physiological context suppression of SOCE did not impairs the migration of CD8+ cells [START_REF] Weidinger | STIM1 and STIM2-mediated Ca2+ influx regulates antitumour immunity by CD8+ T cells[END_REF]. Our results suggest that ORAI1 is involved specifically in the collective migration but does not take part in the single-cell migration. More specifically, our data demonstrates that the initiation of the collective migration is accelerated in the absence of ORAI1. Interestingly, studies performed in Xenopus embryos demonstrated that during collective migration, the cells leading-edge migration was due to Ca 2+

transients [START_REF] Hayashi | Intracellular calcium signal at the leading edge regulates mesodermal sheet migration during Xenopus gastrulation[END_REF]. Of note, cellular migration has been increasingly linked to Ca 2+ transient [START_REF] Li | Calcium oscillations coordinate feather mesenchymal cell movement by SHH dependent modulation of gap junction networks[END_REF][START_REF] Catacuzzeno | Role of KCa3.1 channels in modulating ca2+ oscillations during glioblastoma cell migration and invasion[END_REF][START_REF] Tang | Mertk deficiency affects macrophage directional migration via disruption of cytoskeletal organization[END_REF], and a recent study using HEK-293 triple KO (for all ORAI homologues) demonstrated that ORAI1 did not prevent apparition of Ca 2+ oscillations while ORAI2 and ORAI3 were crucial actors in the maintenance of such oscillations (Yoast et al, 2020b). Thus, the observed increase in the migration initiation speed due to ORAI1 KO could be attributed to a deregulation of Ca 2+ oscillations induced by the ORAI1 deletion, and the ORAI2 and ORAI3 down regulation. Taken together, these data are in line with our results indicating that ORAI1 is only playing a minimal role in the overall cell migration process. 5.4. In addition to the data presented and discussed above, we noted that the Ca 2+ content of the ER was surprisingly higher in the ORAI1-KO cells compared to the WT HEK-293 (Annex 1).

These data indicates that the whole HEK-293 Ca 2+ homeostasis is probably affected by ORAI1-KO. Thus, we also performed preliminary experiments using a physiological inducer of Ca 2+ store depletion, carbachol. We realized during Ca 2+ imaging experiment that the use of carbachol to induce ER store release was leading to the generation of regenerative oscillations.

Of note, these oscillations were not abolished in ORAI1 KO, but they were clearly displaying a different pattern than the one from WT HEK-293 (Annex 2). These data led us to consider that the other minor Ca 2+ stores compartments (such as mitochondria and nuclear envelop) might be affected due to ORAI1 KO. We considered studying the level of these stores due to targeted GECI. Because oscillations are important for NFAT translocation to the cell nuclei, we also assessed its translocation level to the nuclei following carbachol activation and our data indicated that its translocation was impaired in ORAI1 KO cells (Annex 3). Of note, part of the results discussed above were already published by other groups who showed in HEK-293 KO for ORAI proteins, that their Ca 2+ oscillations pattern was modified upon by ORAI proteins deletion, and that these deregulations were impairing the translocation of NFAT to the cells nuclei (Yoast et al, 2020b;[START_REF] Kar | The N terminus of Orai1 couples to the AKAP79 signaling complex to drive NFAT1 activation by local Ca2+entry[END_REF]. Nonetheless, the consequences of ORIA1 KO on the overall Ca 2+ dialog between compartment has not been studied. Evidence of an ORAI1-ORAI3 dynamic remodeling

In the last part of the PhD work, we studied the dynamic interactions between ORAI1 and ORAI3.

First, we performed a set of Ca 2+ imaging experiments using PC3 WT and PC3 KO for ORAI1 and ORAI3. These experiments allowed us to confirm that ORAI3 overexpression was impairing SOCE. In addition, we demonstrated that endogenous ORAI3 was impairing the native SOCE in PC3. These data suggesting that ORAI1 and ORAI3 are component of a heteromeric ORAI channels. We further performed FLIM-FRET experiments to evaluate how the ORAI1-ORAI3 interaction was evolving in basal conditions and following activation of the ORAI channels. This study conducted in HEK-293 and in PC3 cells revealed that ORAI1 and ORAI3 are interacting in basal conditions and that the strength of this interaction was dependent on the expression level of ORAI3. Using HEK-293 cells, we tried to decipher the mode of association between ORAI proteins during the formation of ORAI channels. We proposed and tested few models, but the complexity of and diversity of potential association prevented us to clearly identify whether ORAI proteins were able to associate on site to form functional channel or if pre-assembled ORAI channels were already present at the PM. Nonetheless, these results allowed us to determine that upon induction of SOCE, the interaction level between ORAI1 and ORAI3 is increased. We deepened these results by repeating the experiment in the cancer cell line PC3 where ORAI1 and ORAI3 were deleted. In this model, where background due to endogenous ORAI1 and ORAI3 expression was abolished, we confirmed that the ORAI1-ORAI3 interaction was reinforced by SOCE activation and that the level of SOCE activation was affecting the strength of their interaction. WT. Nonetheless, while we believe that transient overexpression of ORAI3 represent a refine approach compared to stable overexpression of ORAI3, we could not control the exact level of ORAI3 overexpression in our cells, and thus could not identify in which proportion ORAI3 overexpression impact SOCE. An interesting approach to accurately evaluate the involvement of ORAI3 in SOCE would be to use CRISPR transcription activating plasmid, which can be activated through the addition of small molecules in a dose-dependent manner, such a system would allow for a tighter control of ORAI3 overexpression level and allow the study of the consequences of modifications in ORAI3 expression level [START_REF] Chiarella | Dose-dependent activation of gene expression is achieved using CRISPR and small molecules that recruit endogenous chromatin machinery[END_REF]. Further, we showed that ORAI1 overexpression did not enhance SOCE amplitude. Although this result might seem surprising, other publications reported that STIM1 expression level could be limiting when trying to increase SOCE level [START_REF] Liao | Functional interactions among Orai1, TRPCs, and STIM1 suggest a STIM-regulated heteromeric Orai/TRPC model for SOCE/Icrac channels[END_REF]. Thus, the lack of effect of ORAI1 overexpression on the measured SOCE level might be attributed to a low expression level of the STIM1 protein preventing the formation and the activation of the totality of the ORAI1 channel. Overall, it is interesting to note that on the one hand, STIM1 might be a limiting factor for increasing SOCE level when overexpressing ORAI1, while, on the other hand STIM expression level was not a limiting for the SOCE impairment observed with the ORAI3 overexpression. This is explained by the fact that ORAI3 overexpression leads to the creation of heteromeric ORAI channels, which possess different characteristic compared to purely ORAI1-channel, such as lower Ca2+ selectivity for example, which explains the impairment of SOCE observed (Yoast et al, 2020a).

Effect of ORAI proteins deletion on SOCE in PC3 cells

In the second part of our Ca 2+ imaging experiments we used KO cell lines to determine the role of endogenous ORAI proteins in SOCE. The abolition of SOCE due to ORAI1 deletion was expected.

Nonetheless, it is interesting to compare the results obtained in PC3 to the ones obtained in HEK-293 cells. Indeed, while ORAI1 KO HEK-293 cells were still displaying a limited SOCE, such a reminiscent Ca 2+ entry is barely detectable in PC3. It seems evident that the channel expression pattern of these two highlight that the crystal structure of the hexameric Drosophila ORAI channel is consistent with this hypothesis as it displays two adjacent ORAI1 protein interacting together through hydrophobic interaction between their C-termini (Hou et al, 2012b). Thus, in a first model we hypothesized that ORAI proteins were existing as dimers at rest and that depending on the cell stimulation they were assembled on site in distinct channels.

5.4.2.1.2. Putative model N°2: reorganization of pre-formed ORAI channels

A second putative model that might explain our result is the existence of pre-assembled channels that will be specifically activated depending on the type of stimulation received by the cell. In fact, while the structural data obtained from the Drosophila CRAC channel display an architecture constituted by trimer of dimers, nothing indicates the channels are not formed and assembled in the ER and exported as "channel units" to the PM. Similarly, the biochemical studies indicating on the existence of low oligomeric states of ORAI proteins did not deciphers the localization of this oligomers in different cellular compartments. In addition, the microscopic experiments such as the counting of TIRFm photobleaching steps were performed in Xenopus oocytes, that might not possess all the required human proteins allowing the classic assembly of ORAI channel. Moreover, some of these experiments were performed in overexpression system where endogenous protein might have affected the counting. Also, contradictory microscopic studies identified that ORAI are diffusing ad homotetramers in the PM questioning the possibility of "on site" assembly of dimers (Madl et al, 2010b). Finally, the original architecture of the ARC channel consisting of a pentamer assembly of three ORAI1 and two ORAI3 proteins does not fit with a model of an "on site "assembly of ORAI dimers [START_REF] Mignen | The molecular architecture of the arachidonateregulated Ca2+ -selective ARC channel is a pentameric assembly of Orai1 and Orai3 subunits[END_REF]. Thus, in the second model, we hypothesized that ORAI protein are pre-assembled in ARC and CRAC channels. In addition, we considered that ARC channels were pentameric association of three ORAI1

and two ORAI3 channels (in a 31113 or 31311 stoichiometry) as proposed by Mignen et al., and that CRAC channels were heteromeric assemblies of ORAI dimers [START_REF] Mignen | The molecular architecture of the arachidonateregulated Ca2+ -selective ARC channel is a pentameric assembly of Orai1 and Orai3 subunits[END_REF].

FLIM-FRET results in regard with the interaction models

We tried to explain the results obtained in HEK-293 cells by confronting the FLIM-FRET data obtained in HEK-293 to the two putative models presented above.

5.4.2.1.3.1. Basal interaction suggests a pre-assembled channel model

Our results clearly demonstrates that ORAI1 and ORAI3 are interacting in basal condition and that this interaction is dependent on the expression level of ORAI3. The data indicating that ORAI1 and ORAI3 are interacting in basal condition when expressed in the same quantity (1:1 ratio) do not allow to differentiate between our two hypothetical models. However, the data indicating that the interaction between ORAI1 and ORAI3 is increased when ORAI3 is overexpressed points toward a pre-assembled model of the ORAI channels. Indeed, considering the existence of ORAI dimers, and that the probability 5.4.2.1.

Conclusion on the hypothetical models of interaction

To conclude, most of the data obtained in our experiments can be explained by our simplified models. On the one hand, the ORAI1-ORAI3 basal interaction and its dependency on the ORAI3 expression level find an explanation with the pre-assembled channel model. In addition, the increased interaction between ORAI1 and ORAI3 following store depletion can also be explained by the preassembled channel scenario. On the other hand, the results of the consecutive treatment by AA and TG, are finding an explanation with the "on-site assembly" model. Finally, the data demonstrating that AA treatment increased ORAI1 -ORAI3 interaction only when ORAI3 is overexpressed cannot be explained by any of our model. Thus, we believe that 1-both models are not mutually exclusive, 2-our models are a simplification of complex processes occurring within cells which explains why we could not interpret the totality of the results obtained. In any case, further experiments are required to confirm the results obtained with the FLIM-FRET measurement, especially, Ca 2+ imaging experiments demonstrating the successful activation of ARC channel due to acid arachidonic. Overall, because WT HEK-293, expressing endogenous ORAI1 and ORAI3 proteins, were used to perform the experiments, the interpretation of results is limited by the existence of "invisible" ORAI molecules that might affect the measurable interaction level between ORAI1 and ORAI3.

5.4.2.2.

Study of ORAI1-ORAI3 interaction in PC3 5.4.2.2.1. ORAI1-ORAI3 interaction in PC3-WT

For the specific study of the ORAI1-ORAI3 interaction level following SOCE activation, we started by evaluating the basal interaction and store-depleted interaction levels between ORAI1 and ORAI3 in PC3 WT and verify whether we could reach the same conclusions as in HEK-293. On the contrary to what was observed in HEK-293, overexpressing ORAI3 compared to ORAI1 did not allow to increase their interaction level. In addition, SOCE activation did not allow to observe modification of FRET level between ORAI1 and ORAI3. These differences might be explained by the different expression level of endogenous ORAI1 and ORAI3 protein in the two cell lines. Indeed, both ORAI1 and ORAI3 protein display a higher expression level in PC3 than HEK-293. As a consequence, we hypothesize that the higher proportion of non-tagged ORAI proteins in PC3 cells prevented us from detecting modification in the interaction level between ORAI1 and ORAI3.

ORAI1-ORAI3 interactions in double KO (ORAI1+ORAI3) PC3

Using the double KO (ORAI1+ORAI3) PC3 cells we demonstrated that the interaction level between ORAI1 and ORAI3 was dependent on the expression level of ORAI3. In addition, we demonstrated that the interaction level between both molecules was increased by the maximal activation of SOCE induced by TG for ORAI1:ORAI3 transfection ratio of 1:1, and 1:3. Finally, we demonstrated, using the more physiological activator carbachol, that the increased interaction observed following SOCE activation when ORAI3 was overexpressed (i.e, ratio 1:3) was limited to the maximal SOCE activation only. Taken together our data indicates that the modification of interaction observed cannot be attributed to the ORAI channel opening. Indeed, FLIM-FRET is an extremely sensitive method allowing to evaluate distance between proteins. As it was demonstrated that store depletion leads to STIM1 binding to ORAI1 inducing a conformational change in ORAI1 allowing CRAC channel opening, one could hypothesize that the change in lifetime observed following SOCE activation is the consequence of this conformational change. If this would be the case, changes in lifetimes would occurs for any expression ratio and with any type of agent leading to store depletion, which is not the case in our experiments. Our results rather suggest that ORAI1-ORAI3 interactions are dynamic as they evolve depending on, 1-their relative expression level, and 2-the type of activation of the CRAC channel. Of note, other FRET experiments were performed to study the interaction level between ORAI proteins during SOCE activation. On the one hand, a study evaluating the interaction level between ORAI1 protein during SOCE activation concluded that CRAC activation was decreasing FRET [START_REF] Navarro-Borelly | STIM1-Orai1 interactions and Orai1 conformational changes revealed by live-cell FRET microscopy[END_REF]. Nonetheless, the author indicated that changing the location of the fluorescent tag from the C-terminal extremity to the N-terminal extremity of ORAI1 was almost abolishing the decrease of FRET associated with SOCE activation. On the other hand, another study conducted in triple KO (for ORAI1, ORAI2, and ORAI3) HEK-293 have concluded on the absence of changes in interaction level between ORAI and ORAI3 following SOCE activation (Yoast et al, 2020b). Of note, in these works the FRET measurement were performed based on signal intensity due to sensitized emission, which is sensible to the donor and acceptor concentration and less accurate than FLIM-FRET measurements in resolving level of FRET interaction. In addition, the latter publication used ORAI proteins tagged in their C-terminal extremity while we used N-terminally tagged ORAI proteins which might also explain the difference observed between our two studies.

5.4.2.3.

Perspectives on the study of ORAI1-ORAI3 interactions 5.4.2.3.1.

Additional analysis of the results

Our results have highlighted that, while we could measure and detect variations in the interaction level between ORAI1 and ORAI3 in various condition, the interpretation of the data turned out to be complex. Indeed, during the FLIM-FRET experiment we are measuring the lifetime of the donor molecule (O1-mT2), which is directly related the FRET efficiency between the donor molecule and the acceptor molecule (equation 7). As described in the introduction, FLIM-FRET measurements allow the determination of the proportion of interacting molecules 𝛼 𝑖 (equation 6), which in our case would help getting a better understanding of the measured lifetime changes. Nonetheless, the use of phasor representation which eases the calculation of the mean lifetime for each condition, makes difficult the determination of the proportion of interacting molecules in each pixel of the image. Thus, additional analysis of our data might ease the interpretation of our data. For example, an updated version of software used in our study to determine the mean lifetime of images allows the determination of the MFD (minimal fraction of interacting donor) and represent an additional analysis to be performed, that would allow us to map the interaction level pixel by pixel for each cell. In addition, performing new experiments for which images of O1-mT2 and O3-sYFP2 intensities would be performed in addition to lifetime measurement would allow to correlate the signal intensity of each fluorophore to the measured lifetime and might allow detection a better understanding of the proportion of ORAI molecule interacting depending on the transfection ratio used or of the treatment applied on the cells. Finally, new analysis developments specifically accounting for specific FRET phenomenon such as the FRET antenna effect would be of great interest to get better understanding of our results [START_REF] Bunt | FRET from single to multiplexed signaling events[END_REF].

Additional experimental procedures

Finally, several additional experiments considered in order to obtain a better understanding of our results. For example, some publications hypothesized that ORAI channels could be located in premembranaire vesicles and be exported to the PM following store-depletion. Such a mechanism could provide better understanding of the model indicating on the existence of pre-assembled ORAI channels that would undergo rearrangement following SOCE activation. Due to the acquisition in the laboratory of a microscope equipped to perform TIRF, preliminary experiments were performed using ORAI1 and ORAI3 tagged protein transfected in PC3 double KO cells (Annex 4). While only preliminary (a single repeat, allowing the visualization of two cells), we could observe in these experiment that ORAI1 and ORAI3 signals were not totally overlapping, both before and after TG treatment (Annex 5). In addition, visual inspection of the acquired images did not allow us to detect any "apparition" of signal that would indicate fusion of ORAI-containing vesicles to the membrane. Unfortunately, we could not proceed further with experimental acquisitions. Nonetheless, we believe that adjusting the experimental TIRF conditions (level of transfections, laser power for instance) in order to obtain single molecule signal would allow us to decipher if the non-localized signals were corresponding to ORAI1-ORAI3 molecules or to ORAI1 or ORAI3 oligomers (photobleaching steps). In addition, single particle tracking of ORAI1 and ORAI3 proteins was also considered, and few preliminary experiments were performed through a collaboration. Unfortunately, we faced several problems in the determination of optimal conditions allowing to perform the spt experiments. Especially, we used the SNAP-/ CLIP-technology allowing the tag proteins with a chemical dye, unfortunately addition of this chemical dye to our cells resulted in a strong impairment of the adhesion of the PC3 cells and thus preventing us to obtain reliable data.

Finally, to obtain a better understanding on the association between ORAI1 and ORAI3 protein we also used BifC strategy. Specifically, we used two different constructs, the dimerization-dependent fluorescent proteins (dd-FP) and the split Venus proteins. The dd-FP, allow the reconstitution of fluorescent signal between the two parts a split FP if they are in close proximity [START_REF] Alford | A fluorogenic red fluorescent protein heterodimer[END_REF].

We cloned the dd-FP in frame to the ORAI1 and ORAI3 sequences and transfected them in our PC3 double KO cells but could not identify signal. Thus, we used the split Venus system with the aim to study how CRAC activation was modifying, ORAI1-STIM1, ORAI3-STIM1, and ORAI1-ORAI3 interactions, nonetheless, only preliminary data on ORAI1-STIM1 and ORAI3-STIM1 interactions could be obtained (Annex 6). Finally, we also thought of using GCaMP and GECO constructs fused to ORAI1 and ORAI3 to study the Ca 2+ signal generated by the CRAC activation in our double KO PC3 cells transfected with ORAI1 / ORAI3 or both homologues, while the constructs were generated only preliminary experiments were performed on WT PC3(Annex 7).

The general idea with these constructs was to confirm and deepen our understanding of the modification of the ORAI1-ORAI3 interaction due to CRAC channel activation:

-The TIRF experiments in which eGFP-ORAI1 and mCherry-ORAI3 would be transfect in PC3

double KO cells would allow us to perform photobleaching steps experiments before and after CRAC channel activation to determine the oligomeric state of these proteins before and after channel activation.

-The spt experiments would allow us to to follow single ORAI1 and ORAI3 molecules and to verify if their movement following CRAC activation were synchronous or not. Indeed, a synchronous movement would suggest that the proteins are already interacting before the channel activation -The split FP experiments would allow us to visualize if ORAI1-ORAI3 interaction were effectively increased following CRAC channel activation.

-Finally, we planned to transfect GECI fused to ORAI1 together with a second plasmid coding for mCherry-ORAI3 (and vice versa) in order to observe the Ca 2+ signal intensity resulting from homomeric versus heteromeric CRAC channel activation.

General conclusion

The main objective of this PhD was to study the interaction between ORAI1 and ORAI3 proteins and to determine the existence of a dynamic remodeling between these proteins. Throughout this work, several milestones and auxiliary aims were defined:

6.1. Generation of KI cell lines with CRISPR/Cas9

Our specific aim was to generate HEK-293 knocked-in in such a way that the mTurquoise2 and sYPF2 fluorescent sequences would be inserted in frame with the ORAI1 and ORAI3, respectively.

Despite the use different strategies, the generation of KI cells remained unsuccessful. Of note, we used a strategy relying on the HDR cellular reparation mechanism, and thus intended to induce HR between a donor plasmid and the genomic sequence cleaved by the CRISPR/Cas9 system. While we could successfully detect HR event by PCR, we were unable to identify and specifically select edited cells, highlighting the overall difficulty of generating KI cells.

Generation of KO cell lines with CRISPR/Cas9

Using the CRISPR/Cas9 system, we were able to successfully generate the following KO cell lines:

HEK-293 KO for ORAI1; PC3 KO for ORAI1; PC KO for ORAI3; PC3 double KO for ORAI1 and ORAI3. The generation of these cell lines was facilitated by the implementation of the double gRNA strategy leading to the creation of genomic deletion facilitating the screening procedures for KO. Of note, the relative efficiency of KO generation could be attributed to the dominance of NHEJ reparation mechanisms compared to HR in our cell lines.

Study of ORAI1 role in fundamental physiology of HEK-293

Using the ORAI1 HEK-293 cells, we could evaluate the role of ORAI1 in the physiology of this cell line. We demonstrated that ORAI1 was the main effector of SOCE in HEK-293, but not the only one as a slight remaining SOCE could be detected following ORAI1 deletion. In addition, we showed that its deletion was leading to the downregulation of ORAI2 and ORAI3 expression, and thus we suggest that the remaining SOCE observed is probably not due to these homologues. Further, we demonstrated that the role of ORAI1 in the maintenance of HEK-293 physiology was marginal, as its deletion did not modify the proliferative not the adhesive properties of HEK-293. In addition, we showed that ORAI1 KO only slightly influences the HEK-293 migrative properties by increases the speed of the initiation of collective cell migration. This study put in perspective the assumed fundamental role of ORAI1 in the maintenance of cellular physiology and highlight the probable existence of alternative pathway in providing Ca 2+ entry to the cells following store depletion.

Study of ORAI1-ORAI3 interaction with FLIM-FRET

In order to investigate the interaction between ORAI1 and ORAI3, different cellular models were used. First, experiments performed in HEK-293 led us to imagine two potential models describing the mode of formation of the ORAI channels. Confronting our results to our hypothetical models, we concluded that ORAI proteins might exist in a pre-assembled channel state as well as a in a lower oligomeric state allowing on site assembly into functional channels. Second, we used the double KO PC3 cells to demonstrate that the CRAC channel in these cells is a heteromeric assembly of ORAI1 and ORAI3 proteins. In addition, we studied the interaction level of ORAI1 and ORAI3 in this KO model and established that maximal SOCE activation was increasing their interaction, strengthening our conclusion that ORAI1 and ORAI3 form a heteromeric channel in PC3.

8.

  Second, the observation of tissue auto-fluorescence by August Kohler in 1904, as well as the successful labelling of pneumococcal anti-serum (1941, Coons) and secondary antibodies (Weller and Coons 1954) with fluorescent compounds (fluoresceine), paved the way for a general use of fluorescence in biological

  photons hitting the light sensitive surface are transformed into photoelectrons. These photoelectrons are accumulated in the specific picture elements of the array usually described as a bucket. This accumulation of photoelectrons creates an electric charge of a

  Therefore, any part of the sample being under the light beam is emitting fluorescence. This often results in a noisy image. Especially, because the light coming from the unfocused background is reaching the detector. Confocal microscopy allows the removal of the light coming from unfocused parts of the sample, resulting in a lower background. The original idea of confocal microscopy is attributed to Marvin Minsky who had the idea to, 1-focus coincidentally the excitation light and the detected light to decrease the amount out-of-focus light reaching the detector, and 2-scan the sample point by point before reconstructing the image (Nikolaus Naredi-Rainer, Jens Prescher, Achim Hartschuh, 2013). Specifically, the focusing of the light was obtained by the addition of two pinholes: an illumination pinhole located right after the light source, allowing to focus the incident light on a diffraction limited spot, and a detection pinhole, preventing the out-of-focus light to reach the detector. The scanning of the sample was executed by shifting sample under the light beam. Minsky presented a prototype of his confocal microscope in 1958, and patented it 1961 (Figure 31) (Nikolaus Naredi-Rainer, Jens Prescher, Achim Hartschuh, 2013). Nonetheless, the general endorsement on the technique occurred in 1987 due to two papers showing that confocal microscopy was greatly improving contrast and resolution compared to classic widefield microscopy.

Finally

  , their quantum efficiency is about 30% (Nikolaus Naredi-Rainer, Jens Prescher, Achim Hartschuh, 2013). Of note, confocal microscopes are usually equipped with multiple PMT to allow acquisition of different light wavelength (i.e colors).o On the other hand, APD, which are also based on the photoelectric effect, are silicon-based photodiode. Specifically, photodiode includes a "pn" junction consisting of two semi-conductor regions positively (p-) and negatively (n-) doped, and a neutral area (depletion region). When a voltage is applied to the semiconductors the photons hitting the "pn" junction are generating electron-holes pairs which transit to the depletion zone where they generate a flow of electrons and electron holes (avalanche) proportional to the number of incident photons (Figure33B)(Dobrucki, 2013). Of note, single-photon APD (SPAD) are able to detect single photon and are usually equipped on microscope used to perform low-light experiment such as fluorescence lifetime imaging (FLIM) or fluorescence correlation spectroscopy (FCS).o Finally, hybrid combination of both technologies allows to overcome their specific limitations (low gain for PMT, and restriction to lows signals for APD).Overall, confocal microscopy produces high signal to noise ratio (SNR) and provides good resolution only limited by the light diffraction barrier. However, because the image in constructed pointby-point through the sample's scanning, the speed of image acquisition is limited (around 10 frames by second), thus limiting its application for live cellular process of fast kinetic.1.3.3.3.3. Total internal reflection fluorescence (TIRF) microcopyAs mentioned above, widefield fluorescence microscopy suffers from an important noise due to outof-focus light reaching detectors. TIRF microscopy allows to drastically decrease out-of-focus light by taking advantage of a specific light property indicating that light rays can be totally reflected at the interface between two media displaying different refractive indexes when shined at a specific angle.

  1.3.4.1.2.Ca 2+ imaging with genetically encoded Ca 2+ indicators (GECI) GECI were developed to address the main limitations of chemical Ca 2+ dyes namely, the difficulty to target specific intracellular region and to observe calcium signal within living animals. GECIs are engineered fluorescent or luminescent proteins with the ability of binding Ca 2+ . Due to the fusion of GECI to specific tags or addressing signal they can be targeted to specific organelles. In addition, since they are genetic tools, they allow the creation of transgenic animals allowing the monitoring of Ca 2+ fluctuation within their tissues(Suzuki et al, 2016a)[START_REF] Redolfi | Lighting up ca2+ dynamics in animal models[END_REF]. One can distinguish three classes of GECI: bioluminescent probes derived from aequorin, single-fluorescent protein probes such as GCaMPs, and FRET-based probed. Importantly, members of the GCaMP family were fused to ORAI proteins allowing the observation of Ca 2+ signals induced by their specific opening. GCaMP proteins are based on chimeric construct including the GFP sequence, a partial calmodulin sequence and a M13peptide sequence. The development of GCaMP proteins was allowed due to the discovery made by Tsien's team that rearrangements within GFP structure were changing its ability to fluoresce. Specifically, they demonstrates that circularly permuted GFP (cpGFP), a construct where the C-terminal and N-terminal of the GFP are inverted (the protein sequence starts with the C-terminal sequence and finishes by its N-terminal7), can reconstitute the fluorescent GFP chromophore following a conformational change (Baird et al, 1999). Fusion of the M13 peptide (i.e., the binding domain of Ca 2+calmodulin, a 13 amino acid sequence issued from the myosin light chain kinase), and N-terminal segment of Calmodulin (a ubiquitous Ca 2+ binding protein) on each extremity of cpGFP enables, following Ca 2+ detection, the induction of a conformational change reconstituting the original structure of the GFP protein. The reconstituted structure of GFP allows the formation of the classic β-barrel

  to FRAP, fluorescence correlation microscopy (FCS) techniques have also been used to study the behavior of ORAI and STIM. FCS technique allows the specific study of the diffusive properties of single molecules within the confocal volume (i.e, the cellular volume being illuminated by the laser beam of the confocal microscope). Especially, this technique is based on the level of fluorescence intensity fluctuations observed in the restricted area of a confocal volume. The correlation of the fluorescence intensity signal over time allow to establish the diffusion properties of the observed molecules. This technique was used in TIRF modality to show that cholesterol level within PM was affecting the ORAI1 diffusive behavior. Specifically, this study demonstrated that reduction of plasmalemmal cholesterol induces internalization of ORAI prior to SOCE activation, thus decreasing SOCE level. In addition, the authors also demonstrated that cholesterol depletion from PM alters the diffusion of ORAI1 protein. Indeed, in normal condition (i.e., non-cholesterol depleted) ORAI1 was mostly diffusive within confined compartments, while following cholesterol depletion from PM, the proportion of ORAI residing in confined structure was drastically reduced (Bohórquez-Hernández et al, 2017). These findings were confirmed by Qin et al, through the use of single particle tracking (spt-) techniques and simulations (Figure40)[START_REF] Qin | Increased Confinement and Polydispersity of STIM1 and Orai1 after Ca2+ Store Depletion[END_REF]. Another study using combination of super resolution techniques, FRAP, TIRFm and electron microscopy claimed that STIM and ORAI are interacting through the unimolecular model where one STIM dimer interacts with ORAI units of distinct SOCE channels leading to cross linking of these channels. In addition, they confirmed that crosslinked CRAC channels were displaying different mobilities compared to the non-activated channels. Of note, they also suggested that addition of fluorescent tag in C-terminal extremity of ORAI1 might create a steric hindrance preventing the detection of unimolecular model but not affecting the ORAI1 function(Zhou et al, 2018a). 1.3.4.2.3. Usage of super resolution microscopy techniques 1.3.4.2.3.1. Principle of single particle tracking technique.

  again, investigation of protein-protein interactions in living cells can be investigated with microscopy techniques. Especially, FRET is considered as the main microscopy method allowing the study of protein-protein interactions, and as such has been used to study and demonstrate the existence of interactions within ORAI proteins and between STIM and ORAI. Nonetheless, other microscopy techniques also demonstrated their interest in the study of protein-protein interactions such as the bimolecular fluorescence complementation (BiFC) that has been used to study the interaction between SOCE components. 1.3.4.3.1. Usage of Bimolecular fluorescence complementation (BiFC) BiFC is based on ability of circularly permuted FP to reconstitute their intact structure and thus produce fluorescence. Specifically, BiFC refers to the fact that a given FP split in the middle of its sequence in two individual fragments can assemble when brought in close proximity and reconstitute the chromophore of the FP. Therefore, by fusing the circularly permuted fragments to different protein of interest, one can assess their interaction level depending on the fluorescence displayed by the split FP

  Finally, a BiFC-derived super resolution technique named reconstituted fluorescence-based stochastic optical fluctuation imaging (refSOFI), has been developed, and tested on the specific interaction between ORAI and STIM during SOCE interaction. Specifically, fragments of the Venus FP were fused to N-terminal and C-terminal extremities of ORAI1 and STIM1 respectively.The authors compared the results of TIRFm and refSOFI technique to evaluate the size of ORAI-STIM interaction sites. Due to refSOFI technique they improved measurements obtained with TIRFm, and demonstrated that the activation of SOCE leads to an increase in the interaction site number between ORAI1 and STIM1 and not in a size expansion of pre-existing interaction puncta[START_REF] Hertel | RefSOFI for Mapping Nanoscale Organization of Protein-Protein Interactions in Living Cells[END_REF].1.3.4.3.2.Usage of Förster resonance energy transfer (FRET)1.3.4.3.2.1. FRET principleFRET is a non-radiative energy transfer process that occurs between two fluorescent molecules in close proximity. Specifically, considering two fluorescent molecules (i.e, donor and acceptor molecules), if the emission spectrum of the donor overlaps the excitation spectrum of the acceptor molecule, and if the two molecules are in close proximity (i.e., <10 nm), then an excited donor molecule can transmit its energy to the acceptor fluorophore through non-radiative process (i.e., without the emission of a photon from the excited molecule). This phenomenon ultimately leads to the emission of fluorescence by the acceptor molecule (Figure45). While simple in principle, mastering the use of FRET technique requires to understand the physical principles allowing its existence.

(

  Figure 20). The FRET represents an additional non radiative de-excitation pathway for the excited molecule. Since FRET ultimately lead to emission of photon by the donor molecule and to the return to the ground state by the acceptor molecule without photon emission, the visual consequence of FRET is a decrease in donor fluorescence intensity accompanied by an increased fluorescence of the acceptor molecule.

  1.3.4.3.2.2. Intensity based FRET measurements FRET principle is based on the transfer of energy from a donor fluorophore to an acceptor fluorophore. This transfer leads to a decreased in the fluorescence intensity of the donor associated to an increase in the fluorescence intensity of the acceptor. Thus, the simplest way to measure FRET between to molecules is represented by the ratiometric imaging, where the ratio between the acceptor (FA) and donor (FD) fluorescence intensities is calculated within each pixel of the image. Nonetheless, because of the spectral properties of the FPs, it is practically impossible to excite only the donor fluorophore, and to detect only the acceptor fluorescence (Figure48). In addition, the concentrations of donor and acceptor molecules affects the intensity-based FRET measurements. Thus, its use is restricted to experiments where the stoichiometry between acceptor and donor is fixed (i.e attached on the same polypeptidic chain)[START_REF] Yasuda | Imaging spatiotemporal dynamics of neuronal signaling using fluorescence resonance energy transfer and fluorescence lifetime imaging microscopy[END_REF]. A good example of such use is represented by sensors such as the FRET-based sensor Cameleon where the change in FRET intensity can be directly related to conformational modification. Despite these limits, the application of corrections to account for 1-the direct excitation of the acceptor during donor excitation, and 2-the bleedthrough from donor emission into the acceptor emission channel allow to measure EFRET through intensity-based techniques. The corrected ratiometric imaging of FRET is called sensitized emission FRET. It enables to access semiquantitative information on the level of interaction between molecules. In brief, in sensitized emission FRET, one need to account for: 1-the excitation of the acceptor molecule when exciting the donor molecule, and 2-the amount of donor emission in spilling over the emission spectrum of the acceptor.

  6)With, 𝛽 • 𝐷 𝑒𝑥 𝐷 𝑒𝑚 corresponding to the correction for donor bleedthrough, and 𝛿 • 𝐴 𝑒𝑥 𝐴 𝑒𝑚 corresponding to the correction for direct acceptor excitation. Thus, sensitized emission requires the recording of three images: a donor excitation/acceptor emission image (DexAem); a donor excitation/donor emission image (DexDem); and an acceptor excitation/acceptor emission (image AexAem). In addition, images of donor and acceptor alone in separate experiments to obtain the correction factors (β and δ). One the one hand, this type of measurement is easy to implement on a widefield or a confocal microscope, and it allows fast acquisitions to study dynamic interactions in live cells, explaining its wide usage in biological studies. On the other hand, sensitized emission is only semiquantitative, and requires to perform several calibration measurements leading to an increased risk of error as well as an increase in the background noise(Ishikawa-Ankerhold et al, 2012a). Of note, other intensity-based FRET measurements exist such as acceptor photobleaching but are limited to fixed sample, thus it they will not be developed here.1.3.4.3.2.3. Study of SOCE with intensity-based FRET measurements.

  1.3.4.3.2.4. Study of ORAI-ORAI intensity-based FRET measurements FRET studies were also conducted to study level of interaction within ORAI homologues. For examples, Navarro-Borelly et al., studied the interaction level between ORAI1 proteins during SOCE activation. They demonstrated that ORAI1 proteins are interacting in basal condition. In addition, they observed a reduction in EFRET when SOCE was activated and attributed this decrease to molecular rearrangement[START_REF] Navarro-Borelly | STIM1-Orai1 interactions and Orai1 conformational changes revealed by live-cell FRET microscopy[END_REF]. Of note, the EFRET decrease was strictly dependent on the overexpression of the STIM protein in the model. In addition,Schindl et al., used ORAI1 and ORAI3

  limitation of the intensity-based method in evaluating EFRET, alternative methods have been developed such as measuring the fluorescence lifetime of the donor molecule to calculate EFRET.

  and cosine Fourier transform to each experimentally constructed histogram. In this new two-dimensional histogram, called phasor or polar, each pixel from the original image corresponds to a point in the phasor define by its coordinates [u; v] and vice versa (Figure51)[START_REF] Leray | Generalization of the polar representation in time domain fluorescence lifetime imaging microscopy for biological applications: Practical implementation[END_REF]. In addition, the phasor representation allows the deduction of phase and modulation lifetimes (𝜏 𝜑 , and 𝜏 𝑚 , respectively) which are well known parameters of frequency domain FLIM measurements. Of note, for a fluorescent emitter presenting a mono-exponential decay, the values of the phase, modulation, and the true lifetime (𝜏) are all equal (10), while in the case a multi-exponential decay, which can be represented by FRET, the phase lifetime is shorter than the modulation lifetime (11): Mono exponential decay: 𝜏 = 𝜏 𝜑 = 𝜏 𝑚 (10) Multi exponential decay: 𝜏 𝜑 < 𝜏 𝑚 (11) Moreover, on the phasor diagram, mono-exponential decay lifetimes will be plotted along a semicircle centered a [0.5, 0] with a radius of 0.5, with longer lifetimes located close to the origin of the diagram (coordinates [0, 0]), and short lifetimes located closer to the coordinates [1, 0]. Of note, multiple lifetime decays are located inside of the semi-circle (Figure 51). Thus, on the one hand, the phasor diagram presents the advantage of providing a visual representation of the fluorescence lifetimes measured in an image. In addition, one can calculate the mean lifetime value for the original image averaging the values of phase and modulation lifetimes (12) (Leray et al, 2011):

  on the use of microscopy techniques for the study of ORAI proteins As developed above, several microscopy techniques were used to study ORAI proteins and their associated channels. In the year following the ORAI proteins discovery and until the determination of the Drosophila CRAC channel structure by crystallization, several microscopy techniques such as counting of fluorescence photobleaching steps in TIRF modality, FRAP, and FRET techniques were used to assess the stoichiometry of ORAI channels. Even though a considerable amount of data were generated by these techniques, no consensus on the exact ORAI channel composition could be reached. This might be explained by one of the biggest limitations of imaging techniques: the rely on the overexpression of fluorescently labelled protein or on antibodies whose specificity, and capability of identifying all protein with a cell are limited. This limitation leads to the presence of an "invisible" population of protein of interest during the imaging experiment that complexify the interpretation of the results obtained. Of note, the recent revolution caused by the genome engineering technique CRISPR/Cas9 open the possibility of get rid of the aforementioned limitation by tagging virtually all the protein of interest within a cell through the insertion a genomic sequence coding for a fluorescent protein in frame with the sequence of the protein of interest. Thus, in addition to our initial aim of studying the ORAI1-ORAI3 protein interaction with FLIM-FRET technique, we also aimed to use the CRISPR/Cas9 technique to generate cell line displaying endogenous ORAI1 and ORAI3 protein fused to different FP. Therefore, a short introduction of the CRISPR/Cas9 system and its application for genome editing will be described thereafter. 123 1.4. The CRISPR-Cas9 system 1.4.1. Discovery of the CRISR-Cas system The identification of Clustered regularly interspaced palindromic repeats (CRISPR) is dated 1987

  the overhangs for cloning. Bold nucleotides represent the addition of guanin for increasing efficiency of U6 transcription initiation 3.3.2. gRNA cloning gRNA oligonucleotides sequences were cloned either in pX330 derived plasmids (addgene

  Four different plasmids were constructed to perform homologous recombination experiments (HR): HR-ORAI1-sYFP2, HR-ORAI1-mT2, HR-ORAI3-mT2, HR-ORAI3-sYFP2. The structure of the HR plasmids included 4 elements (Figure 56):

  protein (mTurquoise2/sYFP2). gDNA : genomic DNA mRNA : messenger RNA KI: TE71 experiment to assess gRNA efficiency for knockin experiment KO: TE71 experiment to assess gRNA efficiency for knockout experiment

  used in this study are shown in the table below: cloning experiments were performed using NEBuilder system (described thereafter) which involves the use of primers annealing to the target sequence and overlapping to the sequences to be joined. In the following tables, the sequences indicated in uppercase correspond to sequences annealing to the target, while lowercase sequences correspond to the overlapping part.

  was performed on cell suspension by using counting chamber Malassez (Marienfeld, Germany) according to the manufacturer's instructions. The cell doubling was calculated as according to the formula: 𝐷𝑜𝑢𝑏𝑙𝑖𝑛𝑔 𝑡𝑖𝑚𝑒 (ℎ𝑜𝑢𝑟𝑠) = 𝑇𝑖𝑚𝑒 𝑏𝑒𝑡𝑤𝑒𝑒𝑛 𝑚𝑒𝑎𝑠𝑢𝑟𝑒𝑚𝑒𝑛𝑡𝑠 (ℎ𝑜𝑢𝑟𝑠) × log 2 log(𝐶𝑒𝑙𝑙 𝑛𝑢𝑚𝑏𝑒𝑟 [𝐷𝑎𝑦 𝑥]) -log (𝐶𝑒𝑙𝑙 𝑛𝑢𝑚𝑏𝑒𝑟 [𝐷𝑎𝑦 𝑥 -1])

Condition

  the cell medium was discarded, and cells were washed twice with 2 mM Ca 2+ HBBS pre-warmed at 37°C. During the recordings, cells were incubated in 2 mM Ca 2+ HBSS, treatment with TG or AA were performed 5 minutes before acquisition. Before fluorescence lifetime recording, expression of the different plasmid was confirmed by visual inspection of the fluorescent signal on the confocal microscope. During imaging, cells were maintained at 37°C using stage incubator. FLIM experiments were performed with a Nikon A1 Eclipse Ti confocal, operated with NIS software. A pulsed diode laser, PDL 800-B (PicoQuant), delivered 20-MHz repetitive rate pulses at 405 nm. Single photons were collected through a 63x objective and detected through a 483/32 singlebandpass filter (Semrock) on single photon avalanche photodiodes (PDM Series; PicoQuant). Arrival time of single photons was measured with a time correlated single photon counting (TCSPC) counting card (HydraHarp 400; PicoQuant). The data were acquired with the SymPhoTime software (PicoQuant).

(

  Fig 64B). Nevertheless, because PCR were performed on gDNA extracted from cells transfected with the donor plasmid, we could not exclude that PCR bands correspond to plasmid amplification. Thus, we performed PCR experiment on mRNA extracted from the transfected cells and detected extremely faint bands corresponding to successful HR (Fig 64C). Nonetheless, no fluorescence signal could be detected, and no cells could be isolated by FACS. Because of the low intensity of the PCR bands indicating a

4. 3 .

 3 Generating knockout (KO) with CRISPR-Cas9 system. 4.3.1. General consideration for creating KO with CRISPR-Cas9 4.3.1.1.

  4.3.1.2.Design of gRNA for ORAI1-and ORAI3-KO Since our strategy involved the use of two gRNAs per target gene, and because we had already established that the gRNA created for the KI generation were effective, we created a second set of gRNAs located in the end of ORAI1 and ORAI3 first exons. As a reminder, the gRNA selected for our KI experiment were inducing DSB 11bp prior to the start codon of ORAI1, and on the start codon of ORAI3. Therefore, successful fragment excision would delete the start codon eliminating the possibility of creating an ORAI1 or ORAI3 protein presenting an "in-frame" deletion. The CRISPOR website was used to generate a set 3gRNAs to test per gene. The selected gRNAs with their efficiency and specificity characteristics are displayed in Table5. The strategy and localization of gRNAs used for this study are displayed in Figures 66 and 67.4.3.1.3. Assessment of gRNA efficienciesThe gRNAs were cloned in plasmids coding for Cas9 protein fused to GFP via 2A peptide. As a consequence, transfected cells were selected by FACS. Therefore, the PCR performed on gDNA and subjected to T7E1 assays were enriched in positively transfected cells. The T7E1 assay results indicated that the ORAI1-KO gRNA2 and ORAI3-KO gRNA3 were the most efficient and thus they were selected for the KO generation (Fig68).

  of ORAI1 KO in HEK-293 cells To validate the effective ORAI1 KO in the HEK-293 clones selected, we performed Ca 2+ imaging experiments as well as immunoblotting and gene sequencing. 4.3.2.1.1. Ca 2+ imaging experiments First, we performed Ca 2+ imaging experiment on the three clones displaying gene excision. As ORAI1 is known to be the main effector of SOCE, we used a protocol mimicking ER store depletion and measured the subsequent Ca 2+ entry in the cell. Specifically, we used Thapsigargin (TG), an irreversible inhibitor of sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) pump, to induce ER store depletion. TG was added on HEK-293 cells in a Ca2 +-free medium leading store depletion (Figure 70, grey inserts). Subsequent addition of Ca 2+ in the medium led to the generation of SOCE in the WT cells, while in ORAI1 KO cells, Ca 2+ entry was strongly decreased (Figure 70, yellow inserts).

  verification of ORAI1 effective KO, the area surrounding the deleted portion of ORAI1 was sequenced. The region of interest was amplified by PCR with proof-reading polymerase, and PCR products were cloned into pMiniT2.0 plasmid (NEB) and send for sequencing. Thus, the genomic sequencing results indicated that the ORAI1 gene presented a deletion including both ORAI1 start codons (i.e., ATG corresponding to the classic full length ORAI1 and to the ORAI1-β isoform). The exact coordinates of the induced deletion are displayed in the Figure 72. 4.3.2.2. Conclusion on the generation of HEK ORAI1 KO The generation of ORAI1 KO was a surprisingly straightforward procedure. In addition, the double gRNA strategy greatly facilitated the screening procedure for the detection of potential KO and thus accelerated the obtention of KO cell clones. Of note, the main aim of this PhD was to study dynamic remodeling of ORAI1 and ORAI3 proteins. The hypothesis of the existence of such mechanism was developed due to results obtained in cancer model cell line PC3 and LNCaP. Because of the relative efficiency of KO generation in HEK, we thought that applying the same strategy in the PC3 cancer cell line would be successful and provide a more relevant model for our study. As a consequence, we did not develop ORAI1-ORAI3 double KO HEK-293 cells as initially planned, but we rather decided to generate this double KO in PC3 cells. In addition, we decided to take advantage of the ORAI1 KO HEK-293 cells to study the physiological effect of ORAI1 KO in this cell line. 4.3.3. Generation of double KO for ORAI1 and ORAI3 in PC3 cells.

  of ORAI1 KO in PC3 cells The same strategy as previously described as used to generate ORIA1 KO in PC3 cells. The results of the PCR screening procedure are shown in Figure 73A. Validation of effective ORAI1 KO was performed through Ca 2+ imaging experiments Figure 74. Clones displaying gene deletion as well as SOCE impairment were selected for ORAI3 KO generation or stored for further experiment. Final validation of effective ORAI1 KO was established by ORAI1 gene sequencing Figure 75 A. Four ORAI1 KO clones were used to induce ORAI3 KO and thus generate double KO for ORAI1 and ORAI3. The procedure for generating ORAI3 KO similar to the one used for ORAI1. PCR screening for ORAI3 gene deletion allowed us to select three clones double KO for ORAI1 and ORAI3 (Figure 73B). Final verification of effective ORAI3 deletion was established by ORAI3 gene sequencing (Figure 75 B). 4.3.3.2. Screening and validation of ORAI3 KO in PC3 cells In addition to ORAI1 KO and ORAI1-ORAI3 double KO cell line, we thought of generating ORAI3 KO cell line in order to later compared the physiological functions of these protein in PC3 cells. The procedure for generating ORAI3 KO was reiterated and allowed us to generate four clones displaying ORAI3 gene partial excision (Figure 76). The confirmation of effective ORAI3 deletion was established by ORAI3 gene sequencing (Figure 75 C). 4.3.4. General conclusions on the generation of KO cells KO generation in both HEK-293 and PC3 cells was a relatively straightforward task. Contrarily to generation of KI, the process of KO generation is well established and clearly defined, and several protocols are readily available to ensure success with the KO generation. At the start of this PhD, the most problematic task was the screening for KO step. Indeed, without the double gRNA strategy, this step is time consuming and would rely on expensive technique such as Ca 2+ imaging experiment, or on the existence of reliable antibody. Overall, the crucial points of KO generation involved: the design of specific gRNAs, the assessment of their efficiency, the screening procedure for identifying KO cells. To conclude, the generation of KO did not present strong difficulties. During this PhD, several other teams generated HEK cells knocked out for ORAI proteins demonstrating the global usage of CRISPR-Cas9 for generating KO. However, generation of ORAI KO in cancer cell lines was not described by other teams, probably because of a more complex genotype than the classic HEK-293 cell line. 4.4. Impact of ORAI1 deletion on the HEK-293 physiology 4.4.1. Different roles for ORAI1 in HEK-293 physiology ORAI1 is identified as the main effector of the SOCE. In addition, SOCE represent the main Ca 2+

  ORAI1 protein after knockdown, lack of specificity of pharmacological agents inhibiting SOCE, and potential compensation due to the expression of other ORAI isoforms might prevent the identification of clear roles for ORAI1 and SOCE. Therefore, our ORAI1 KO HEK-293 cells present a new model to study the fundamental role of ORAI1 in the HEK physiology. 4.4.2. Ca 2+ signaling First, we assessed the effect of the ORAI1 KO on the SOCE level displayed by HEK-293 cells. We used TG to induce store depletion and measured and compared the level of SOCE in our cell lines. As 4.4.6. Migration

  Further we demonstrate that ORAI1 does not control the proliferation of HEK-293. Similarly, ORAI1 does not impact the adhesion level of HEK-293. Finally, we establish that ORAI1 is partially involved in the control of HEK-293 migration. Specifically, we demonstrated that the collective cell migration properties are affected by ORAI1 KO while single-cell migration is unaffected by ORAI1-KO. As a conclusion, we demonstrated that ORAI1 and SOCE are dispensable in the maintenance of HEK-293 physiology. Whether the absence of an effect of ORAI1 deletion is the result of compensatory mechanisms or whether the impact of SOCE in HEK-293 physiology is indeed limited remain to be investigated. 4.5. Study of ORAI1 -ORAI3 interactions with FLIM-FRET 4.5.1. Hypotheses on the existence of a dynamic re-arrangement of ORAI protein The last part of the PhD work was devoted to the study of ORAI1-ORAI3 interactions. Specifically, we aimed to use the FLIM-FRET technique to determine if the interaction between ORAI1 and ORAI3

  4.5.2.2. Effect ORAI1 deletion on SOCE We started by measuring the level of SOCE in ORAI1 KO PC3 cells. As expected, ORAI1 deletion almost totally abolished SOCE, confirming that ORAI1 is the major effector of SOCE (Figure 86 A, C). Further we performed rescue experiments, where a plasmid coding for ORAI1 was transfected in ORAI KO cells. Unexpectedly, the rescue of ORAI1 did not allow a restoration of SOCE level comparable to the one observed in PC3 WT (Figure 86 A, D). Nonetheless, inspection of single cell Ca 2+ traces allowed us to identify that some cells were displaying SOCE of a similar intensity to the one observed in PC3 WT (Figure 86 B, D). Therefore, we assumed that the difference observed in the mean intensity of SOCE level between PC3 WT and rescued ORAI1 KO cells could be attributed to a low transfection level. 4.5.2.3. Effect of ORAI3 deletion on SOCE Next, we measured the level of SOCE in ORAI3 KO cells. With this experiment, we intended to determine if endogenous ORAI3 was affecting the amplitude of the SOCE in the PC3 cells. Ca 2+ imaging experiment revealed that SOCE was enhanced following ORAI3 deletion, suggesting that endogenous ORAI3 impairs SOCE in PC3 WT (Figure 87 A). Quantification of the SOCE amplitude allowed us to determine that the improvement of SOCE following ORAI3 deletion was statistically significant (Figure 87 B). Further, we re-expressed ORAI3 in the cells where it was deleted. The Ca 2+ traces as well as quantification of SOCE level demonstrated that ORAI3 rescue in ORAI3 KO cells significantly decreased the SOCE amplitude (Figure 88). 4.5.2.4. Conclusion on the roles of ORAI1 and ORAI3 in endogenous SOCE Our results confirm and extend the finding of Dubois et al. indicating that ORAI3 impairs SOCE. Indeed, we used a transient overexpression model to show that ORAI3 impairs SOCE in PC3-WT. In addition, we demonstrate that the deletion of ORAI3 in PC3 cells leads to a SOCE enhancement indicating that endogenous ORAI3 expression possess an inhibitory role on SOCE level. In addition, we show that this effect is not the consequence of compensatory mechanisms since re-expression of ORAI3 in ORAI3 KO cells, leads to a decrease of the SOCE amplitude. We have also studied the effect of ORAI1 expression modulation on the SOCE level. The absence SOCE modulation following ORAI1 overexpression might be explained by a low expression level of STIM proteins compared to the overexpressed ORAI1, preventing the activation of all ORAI channel present in the cells. Finally, the experiments performed in ORAI1 KO cell line demonstrates that ORAI1 is the major effector of SOCE in PC3 cells. In addition, rescue experiments demonstrated that ORAI1 re-expression restored SOCE in PC3 cells. Overall, these data demonstrate the involvement of ORAI3 and ORAI1 in the SOCE in PC3.

  of this PhD work, we considered studying ORAI1-ORAI3 interaction in HEK-293 cells bearing endogenously tagged ORAI proteins with FLIM-FRET technique. Even though, we could not establish this cell line, we had started performing FLIM-FRET experiments in HEK-293.

  This might explain why PCR results indicated on successful KI while fluorescence ere not detectable, indeed, if InDels occurred and resulted in open reading frame (ORF) shift, fluorescent protein would not be expressed. Of note, a newly developed KI technique named, CRISPR-mediated insertion of exon (CRISPIE) includes the insertion of fluorescent tag in an intronic region leading to the splice out of InDels insertion, might present a good strategy to prevent the apparition of ORF shifted KI.5.1.4. Conclusions and perspectives for the generation of gene KIAs a conclusion, the current abundance of methods allowing creation of KI cells might ease the process of conducting such experiments. Nonetheless, success rates described in scientific papers should be taken with precaution as modifications, of the cell line used, or the gene to integrated might greatly affect the outcome of the experiment. In addition, careful design of the experiment should be performed in order to avoid pitfall such as the low expression of the targeted gene to KI. In our specific case, one could probably achieve the labelling ORAI1 and ORAI3 with fluorescent tag with gene KI. An adequate strategy would probably involve: 1-the use of a cell line bearing a high expression of ORAI protein and displaying a diploid genome. Alternatively, using HEK-293 cells would require to successfully increase the expression level of ORAI proteins; 2-the creation and the use of several methods for inducing gene recombination. Especially, conducting experiments relying on different reparation mechanisms such as NHEJ (like the HITI method), MMEJ (like the PITCh method), and HDR (like the easi-CRISPR method) should allow to successfully create knockin to fluorescently label the ORAI proteins. 5.2. Challenges in generating gene KO with the CRISPR/Cas9 system

5. 3

 3 .4. Conclusion on the role of ORAI1 in HEK-293 physiology.Overall, the results obtained in our ORAI1 KO cell line allows us to claim that ORAI1 is not an indispensable actor of HEK-293 cell physiology. Indeed, proliferative and migratory properties of HEK-293 cells were not fundamentally affected by ORAI1 deletion. While we cannot exclude those compensatory mechanisms allowed the maintenance of basic HEK-293 properties, it appears unlikely that ORAI1 KO cellular clones would arise if the function of the protein was of utmost importance for HEK-293 cells. Nonetheless, it remains under question how ORAI1 KO HEK-293 cells are able to restore their Ca 2+ store following depletion, and further experiments assessing for modulation of expression of channels would help elicits this mechanism. 5.3.5. Perspectives on the role of ORAI1 in HEK-293 physiology.

5. 4

 4 .1. Influence of ORAI1 and ORAI3 expression on SOCE level 5.4.1.1. Overexpression of ORAI in WT PC3 In our Ca 2+ imaging experiments, we started by overexpressing ORAI1 and ORAI3 in WT PC3. The results obtained allowed us to confirm that ORAI3 overexpression decreases the level of SOCE in PC3

  than the critical angle TIRFm objective should present a NA > 1.45. Finally, while lasers are preferentially used to perform TIRFm, arc lamp can be used but requires the addition of mask in incidental optical path. Overall, TIRFm possess the advantage of both, widefield microscopes (speed of while confocal microscopy, allow the detection of faint signal and better resolution, but is slower in acquiring images. In the meantime, TIRFm present a good SNR and as well as a fast image acquisition but is restricted to phenomenon occurring within the few hundred of nanometers following the PM. Thus, all these techniques have allowed the visualization and the description of different cellular processes. Importantly, a variety of theses microscopy techniques have been used to describe the properties of ORAI protein and will be discussed below.1.3.4. Study of properties of ORAI proteins with microscopy techniquesThe specific features of ORAI channels, made them good subjects to be studied with microscopy techniques. For example, because CRAC channels is Ca 2+ selective, the SOCE activation was studied with Ca 2+ imaging techniques using chemical dyes or genetically encoded Ca 2+ indicators (GECI).

	image acquisition,
	acquisition), and confocal microscopes (good SNR and optical sectioning). Nonetheless, it is limited to
	region illuminated by the evanescent wave, and thus its use is usually restricted to imaging and studying
	of membrane proteins.
	1.3.3.4.	Summary of the advantage and limitations of fluorescence microscopy
	techniques

  .1. Stocks solution 3.1.1. Buffers and standard solutions Recipes of the buffers and standard solutions used in this study are indicated in the table below.When needed buffers and standard solutions were autoclaved at 120°C or sterilized with 0.22 µm filters.

	Name		Used for	Final working concentration
	Carbenicilin	Bacterial selection	30 µg/mL
	Kanamycin	Bacterial selection	50 µg/mL
	3.2. Molecular biology
	Name	Composition 3.2.1. NEBuilder Cloning
	0 Ca 2+ solution	150 mM NaCl, 5 mM KCl, 3 mM MgCl2, 10 mM HEPES, 10 mM Glucose, pH=7.4
	4 mM Ca 2+ solution	150 mM NaCl, 5 mM KCl, 4 mM CaCl2, 1 mM MgCl2, 10 mM HEPES, 10 mM Glucose, pH=7.4
	8 mM Ca 2+ solution	150 mM NaCl, 5 mM KCl, 8 mM CaCl2, 10 mM HEPES, 5.6 mM Glucose, pH=7.4
	Hank's balanced salt	150 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 10 mM
	solution (HBSS)	HEPES, 10 mM Glucose, pH=7.4
	Lysogeny Broth (LB)	1% tryptone, 0.5% Yeast extract, 1% NaCl, pH=7.0
	Phosphate buffered saline	137 mM NaCl, 2.7 mM KCl, 0.2 mM KH2PO4, 1.42 mM
	(PBS)		Na2HPO4•2H20, pH=7.4
	Radioimmunoprecipitation assay buffer (RIPA)	15 mM KH2PO4, 100 mM Na2HPO4, 100 mM NaH2PO4, pH=7.2
	Tris buffered saline-Tween (TBS-T)	1.5 mM Tris, 14 mM NaCl, 0,05% Tween20, pH=7.4
		3.1.2. Pharmacological agents
	Pharmacological agents were used as indicated in the table below.
	Name		Mode of action	Final working concentration
	Acetylcholine (ACh)	Agonist of cholinergic receptors	100 µM
	Arachidonic acid	Agonist of ARC channel	8 µM
	Carbachol (CCh)	Agonist of cholinergic receptors	100 µM
	Thapsigargin (TG)	Irreversible inhibitor of SERCA	1µM
	Brefeldin A	Unknown
		3.1.3. Antibiotics
	The antibiotics used in this study are indicated in the table below.

  Femto Maximum Sensitivity Substrate and the SuperSignal™ West Dura Maximum Sensitivity Substrate (Thermo Fischer Scientific). The antibodies used in the study are shown in the table below:

	ORAI1	3.23 µg/mL (1/200)	Rabbit	35kDa	Alomone (ACC-006)
	ORAI1	1µg/mL (1/500)	Rabbit	35kDa	Abcam (ab115558)
	ORAI1	0.4 µg/mL (1/250)	Rabbit	35kDa	Sigma (HPA 016583)
	ORAI1	1µg/mL (1/500)	Rabbit	35kDa	GeneTex (85057)
	Β-ACTINE	1/1000	Mouse	42 kDa	Sigma (A5441)
	3.9. Calcium imaging
	For calcium experiments, cells were plated on glass-bottom dishes pre-coated with Poly-L-Lysine
	(Ibidi) 3 days before the experiment. The ratiometric dye Fura-2-acetoxymethyl ester (AM) (Interchim)
	Target	Working concentration	Secondary antibody	Expected size	Source (reference)
	ORAI1	0.5µg/mL (1/500)	Rabbit	35kDa	ProteinTech (13130-1-AP)

  5.2.4. Conclusion on the generation of gene KO with the CRISPR/Cas9 systemAs a conclusion, due to the advent of CRISPR/Cas9 system, generation of KO cell line does not represent a big challenge anymore. However, one should bear in mind that a good transfection efficiency and a proliferative cell line are mandatory to generate KO. Overall, KO generation with CRISPR/Cas9 is low-priced, as several Cas9 plasmids are available through addgene, and the generation steps are involving classic techniques used in biological laboratory (cloning, and PCR techniques mostly).Nonetheless, it remains a time-consuming procedure as many steps require careful assessment of the results before proceeding to the following step. Of note, several companies are now proposing to generate KO for specific cell line and might represent an interesting alternative for obtaining KO cells.Finally, it is important to bear in mind, that the use of CRISPR/Cas9 system might generate off-target DSB, which might result in unwanted genomic modifications. While it can be considered of relatively low importance if conducting mechanistic studies, it might represent a burden if one wants to assess the physiological relevance of a specific KO. In our study we used two different clones for assessing the physiological role of ORAI1 KO in HEK-293 cells. However deeper studies might require a specific assessment of DSB induction at the potential off-target sites. The most evident technique for evaluating the generation of unwanted genomic modification is represented by next generation sequencing (NGS)

  5.3.1. ORAI1 deletion abolishes SOCEWe identified the existence of a residual SOCE following ORAI1 deletion. Of note, other studies have reported various level of SOCE in ORAI KO cells. For example,Cai et al., as well as Yoast et al., observed a total SOCE abolition in ORAI1 KO cells(Yoast et al, 2020b;[START_REF] Cai | The Orai1 store-operated calcium channel functions as a hexamer[END_REF], while Alansary et al., observed a residual SOCE entry following ORAI1 deletion in HEK-293, the latter attributed this remaining SOCE to the presence of ORAI3 protein[START_REF] Alansary | Detecting single ORAI1 proteins within the plasma membrane reveals higher-order channel complexes[END_REF]. The differences in the SOCE level observed within these studies and ours might be explained by the strategies used to generate ORAI1 KO. Indeed, we are the only ones to use the double gRNA strategy to generate ORAI1 KO. This strategy allowed us to generate cell line displaying a total absence of the ORAI1 protein (due to ATG deletion).

Annexes

Remerciements

We have seen above that each microscopy technique presents its own advantages and limitations.

For example, widefield microscopy allows the study of fast and dynamic processes due to its rapidity in

anticipated, ORAI1 KO strongly impaired SOCE level in our cells. Nonetheless, it was not totally abolished. Quantification of SOCE entry by integration of the curve area following Ca 2+ addition in the extracellular medium revealed that SOCE amplitude was decreased about 80% in ORAI1 KO cells (Figure 77).

ORAI homologues expression

It was demonstrated that all ORAI homologues are able to provide SOCE when overexpressed.

Since we observed a small but non-null SOCE in the ORAI1 KO cells, we hypothesized that this Ca 2+ entry could be attributed to ORAI2 and ORAI3. Thus, we verified their expression level in our cell line by RT-qPCR. Surprisingly, our results indicated that both genes are downregulated in our ORAI1 KO HEK-293 (Figure 78). Other studies using HEK-293 cells where ORAI protein were knocked out reported that only overexpression of ORAI2 and/or ORAI3 was able to restore SOCE in small proportion [START_REF] Kar | The N terminus of Orai1 couples to the AKAP79 signaling complex to drive NFAT1 activation by local Ca2+entry[END_REF]Yoast et al, 2020b). In addition, patch-clamp recordings performed in ORAI1 KO HEK-293 cells demonstrated the absence of ORAI current in these cells. Taken together these data suggest that the remaining SOCE observed in our cells in not due to ORAI2 and ORAI3.

Proliferation

We hypothesized that the strong impairment of SOCE observed in ORAI1 KO cells should have physiological consequences on the HEK-293 properties. Thus, we first compared the level of proliferation between WT HEK-293 and ORAI1 KO cells. First, the doubling time of both cell lines was established by manual and automated counting. The result of this experiment indicated that the doubling time of HEK-293 was not modified by ORAI1 KO (25.7 hours for WT HEK-293, versus 26.8 hours for ORAI1 KO cells) (Figure 79A). To confirm this result, we measured the proliferative rate of these cell lines with SRB assay. In agreement with the result of doubling time we observed that the proliferative rate of ORAI1 KO cell line was not affected (Figure 79B). Finally, we measured the DNA content of our cells due to propidium iodide (PI) staining and cell-cycle analysis. This experiment proved that ORAI1 did not control proliferation of HEK-293 cells as no differences were observed in the proportion of cells in each phase of the cycle between WT HEK-293 and ORAI1 KO HEK (Figure 79C).

Adhesion

Following the assessment of the role of ORAI1 and SOCE in the control of HEK-293 proliferation we considered studying the impact of ORAI1 deletion on their migrative properties. Cell migration is a multi-step process involving release of cell adhesion spots from the substrate, creation of protrusion, renewal of adhesion spots, and cytoskeletal contraction [START_REF] Ridley | Cell Migration: Integrating Signals from Front to Back[END_REF]. Thus, we first set out to compare if our two cell lines were displaying differences in their adhesion level. Adhesion experiment revealed that adhesion properties were not affected by ORAI1 KO (Figure 80).

Conclusion on the ORAI1-ORAI3 interaction in HEK-293

Overall, the results obtained on the measurement of interaction level between ORAI1 and ORAI3 obtained in HEK-293 demonstrate a complex level of regulation between ORAI1 and ORAI3 as suggested by the publication of Dubois et al. (Dubois et al, 2014a) .While it is clear that ORAI1-ORAI3 are interacting in basal condition and that their interaction level is dependent on the expression level of ORAI3, the data obtained in ORAI channel activated condition are more complex to interpret. On the one hand, ARC activation due to AA addition modified the interaction between ORAI1 and ORAI3 only for the 1:3 transfection ratio. In addition, overexpression of STIM1 prevented this phenomenon. On the other hand, induction of SOCE due to TG addition led to an increase in the ORAI1-ORAI3 interaction only in the 1:1 ratio when STIM1 as overexpressed. A potential interpretation of these puzzling data will be described in the discussion. We started by evaluating the interaction level of ORAI1 and ORAI3 in basal conditions in the PC3 WT. In line with the results obtained in HEK-293 WT, the measured lifetime of ORAI1-mT2 was within the range of the lifetime corresponding to the free mTurquoise protein (i.e, 3.96 ns, Figure 94A). The transfection of the of ORAI1-mT2 and ORAI3-sYFP2 in the same proportion decreased the lifetime of ORAI1-mT2 indicating that ORAI1 and ORAI3 were interacting in basal condition (Figure 94A).

However, increasing the proportion of ORAI3-sYFP due to the 1:3 transfection ratio did lead to a significant modification of the ORAI1-mT2 lifetime (Figure 94A).

Store dependent interaction in PC3 WT

Subsequently, we assessed if the interaction level between ORAI1 and ORAI3 was affected by SOCE activation. Surprisingly, no significant differences were observed following SOCE activation independently on the ORAI3 expression level in the cells (Figure 94 B,C). While surprising, we hypothesize that this result might be explained by the presence of endogenous ORAI1 and ORAI3, cell lines is different and might explain the difference observed between these two KO models. Yet, exploring the differences in channel expression between these two cell lines could allow the identification of specific actors of SOCE. The results obtained in the ORAI1 rescue experiment showed For evaluating the interaction between ORAI1 and ORAI3, we performed a first set of experiment using WT HEK-293 cells. The results obtained following ORAI channel activation were puzzling. While this complexity might be attributed to the existence of an endogenous background due to the presence of endogenously expressed ORAI1 and ORAI3 proteins. We have tried to imagine models that might explain the results observed. Thus, we considered two putative models based on our data and on suggestions from previous publications.

5.4.2.1.1.

Putative model N°1: on site assembly of ORAI dimers.

First, in one model we consider that ORAI protein can assemble as channel "on site". Of note, few publications suggested that ORAI channel could be assembled on site (Li et al, 2016b;[START_REF] Penna | The CRAC channel consists of a tetramer formed by Stim-induced dimerization of Orai dimers[END_REF]. In addition, early publications on ORAI channel structure using biochemical analysis, as well as microscopy techniques have demonstrated that ORAI protein could be found in different oligomeric states such as monomers, dimers, trimers, and tetramers [START_REF] Maruyama | Tetrameric Orai1 is a teardrop-shaped molecule with a long, tapered cytoplasmic domain[END_REF]Gwack et al, 2007a;[START_REF] Zhou | Pore architecture of the ORAI1 store-operated calcium channel[END_REF]Ji et al, 2008a). Nonetheless, from the different oligomeric states identified, dimeric state is believed to be the most represented, and in addition a dimeric association of ORAI proteins would explain the formation of higher oligomeric states of ORAI proteins (tetramers, hexamers) upon store depletion [START_REF] Penna | The CRAC channel consists of a tetramer formed by Stim-induced dimerization of Orai dimers[END_REF]Gwack et al, 2007a). Finally, Cai et al., proposed that the SOCE activated ORAI channel is a 'trimer of a ORAI dimer' [START_REF] Cai | The Orai1 store-operated calcium channel functions as a hexamer[END_REF], in this publication the authors of forming homo or heterodimer is equivalent, in a 1:1 transfection ratio one expects to observe 50% of heterodimers (ORAI1-ORAI3) displaying FRET, 25% of ORAI1-ORAI1 homodimers, and 25% of homodimers ORAI3-ORAI3, the two later not displaying FRET. For a 1:3 transfection ratio the proportion of ORAI3-ORAI3 homodimers would increase at the expense of both ORAI1-ORAI1

homodimers, but the amount of interacting donor and acceptor molecule should not be strongly affected as a consequence, one would expect that FRET level would not be modified (Figure 100 A). On the contrary, considering a mixture of pre-assembled ARC and CRAC channels, the increase of ORAI3 expression induced by the 1:3 transfection ratio would favor the creation of heteromeric CRAC channel leading to an increased number of FRET acceptor molecules (ORAI3-sYFP2) for a single donor molecule (ORAI1-mT2), and thus induce a general increase of FRET level observed (Figure 100 B).

AA-activated interaction does not agree with the proposed models

The data obtained following activation of ARC channel due to AA treatment indicates that in the 1:1 ratio, the interaction level between ORAI1 and ORAI3 is not modified while in the 1:3 ratio their interaction is reinforced. In addition, this effect is observed only when STIM1 is expressed at its endogenous level.

Considering the 1:1 transfection condition for our two models, both can explain the absence of effect observed following AA treatment. If considering the "on site" channel assembly model, AA treatment would lead to the creation of ARC channel. The differences in FRET that would occur following ARC channel formation is not clear. Indeed, in the basal interaction, one can observe that half of the ORAI dimers are displaying FRET (ORAI1-ORAI3 heterodimers) and half of the dimer are not displaying FRET (25% of ORAI1-ORAI1 homodimer, and 25% of ORAI3-ORAI3 homodimers). Thus, the measured FRET would relate to a situation in which one donor molecule (O1-mT2) interacts with one acceptor molecule (O3sYFP2). On the other hand, the assembled channels would bring together homodimers that were previously not able to display FRET, and thus would increase FRET. However, because of the specific architecture of ARC channel, the ratio of donor to acceptor molecule would be of three donor molecules (O1-mT2) for two acceptor molecules (O3-sYFP2), and so lower than in the condition basal condition (Figure 101 A). Thus, the observation that AA treatment does not modify the interaction between ORAI1 and ORAI3 might agree with this model. If considering the pre-assembled model, activation of ARC channel would not modify the proportion of ORAI molecules interacting together, and thus does not impact the measured lifetime of O1-mT2 (Figure 101 B). Of note, in such a scenario, our data would suggest that a conformational change due to ARC channel opening, would not be transmitted to the FP, as their interaction level is not modified. This is plausible due to the length and flexibility of the linker used in our experiment. Of note, in our experiment the presence of STIM1 protein at endogenous or overexpressed level did not modify the results and thus does not seem of utmost importance.

Considering the 1:3 transfection condition, both models fail to explain the results observed. Indeed, both of our models would lead to the same conclusion as for the 1:1 transfection ratio (Figure 102). The conduction of similar experiments in ORAI KO cells as well as the ability to define the exact proportion of ORAI1 and ORAI3 with the use of concatemers might help deepen this result and obtain a better understanding of the observed results.

5.4.2.1.3.3. CRAC-activated interaction indicates a pre-assembled channel model

Because our Ca 2+ imaging data suggested that ORAI3 was involved in native SOCE, we have studied the interaction level of ORAI1 and ORAI3 following TG treatment. Our data indicate that the interaction between ORAI1 and ORAI3 was reinforced following SOCE activation only when the two proteins were expressed at the same level. These data can be explained exclusively by the pre-assembled channel models. Indeed, if for the 1:1 ratio, both models could explain an increase of FRET following store depletion; the absence of modification in the interaction level between ORAI1 and ORAI3 for a 1:3 expression ratio can be explained only with the pre-assembled channel model.

Specifically, for the 1:1 expression ratio; considering the "on site" assembly model, association of ORAI dimers into a channel might not increase the FRET level, however the clustering of CRAC channels induced by store depletion would increase the proportion of interacting donor (O1-mT2) and acceptor (O3-sYFP) molecules. If considering the pre-assembled channels model, the same phenomenon would occur (Figure 103). As for the 1:3 transfection ratio, in the "on site" assembly model, the FRET level of the basal condition would be relative to the number of ORAI-ORAI3 dimers. Due to SOCE activation, ORAI3-ORAI3 dimers would be included in the CRAC channel and thus increase the acceptor/donor ratio ultimately leading to a FRET increase (Figure 104A). On contrary, if considering the pre-assembled channel model, one could consider that the high ratio of acceptor to donor molecules in the pre-assembled channels do not allow a further increased in FRET when channels are brought in close proximity by STIM1 proteins (Figure 104 B).

5.4.2.1.3.4. Consecutive activation of ARC and CRAC channels suggest an on site assembly model

The final experiment conducted in HEK-293 was to consecutively induce ARC formation followed by SOCE activation. The results of these experiments showed that AA treatment was preventing the modification of interaction observed when SOCE was activated for the 1:1 ratio of expression between ORAI1 and ORAI3. Still considering our two models, this last result finds an explanation with the "on site" assembly model. Indeed, we explained earlier that this model would allow an increase of FRET