A maioria dos tumores sólidos apresentam características aneuplóides. Porém a relação entre aneuploidia e transformação maligna, ainda não está definida. Nos últimos anos diversas proteínas têm sido descritas como reguladoras de eventos durante a divisão celular, principalmente as relacionadas com a formação do fuso bipolar e segregação equacional dos cromossomos. Neste estudo propomo-nos a analisar os efeitos da interferência em dois pontos críticos da mitose, a segregação cromossômica e a citocinese, em relação à aneuploidia e à instabilidade genética tumoral. Nossos dados mostraram que o tratamento sequencial de Monastrol e Blebistatina determinou o surgimento de fusos mitóticos anormais, amplificação centrossômica, localização ectópica de Aurora A e aumento de micronúcleos. Esta interferência pode levar a um quadro de instabilidade genética e, consequentemente a progressão tumoral, abrindo novas possibilidades para o estudo dos mecanismos moleculares envolvidos na regulação do ponto de checagem mitótico e resistência a quimioterápicos. / Most solid tumors have aneuploid feature. Therefore the relationship between aneuploidy and malignant transformation is not yet understood. In the last years it has been described many proteins involved in regulation of mitosis, mainly those related to bipolar spindle and chromosome segregation. In this work we propose to study the effects of the interference on two mitotic critical points, the chromosome segregation and cytokinesis, in relation to aneuploidy and genetic tumor instability. Our data showed that sequential treatment with Monastrol and Blebbistatin led to abnormal mitotic spindle, centrosome amplification, Aurora A ectopic and micronucleus increased. This interference can lead to genetic instability and may be involved in a tumor progression, opening news possibilities to study the molecular mechanisms involved in regulation the checkpoint mitotic and resistance to chemotherapy found in genetically unstable cells.

La thèse porte sur la calibration d'un modèle biomathématique multi-échelle expliquant le phénomène de sélection des follicules ovariens à partir du niveau cellulaire. Le modèle EDP consiste en un système hyperbolique quasi linéaire de grande taille gouvernant l'évolution des fonctions de densité cellulaire pour une cohorte de follicules (en pratique, une vingtaine).Les équations sont couplées de manière non locale par l'intermédiaire de termes de contrôle faisant intervenir les moments de la solution, intégrée à l'échelle mésoscopique et macroscopique. Trois chapitres de la thèse présentent, sous forme d'articles publiés, la méthode développée pour simuler numériquement ce modèle. Elle est conçue pour être implémentée sur une architecture parallèle. Les EDP sont discrétisées avec un schéma Volumes Finis sur un maillage adaptatif piloté par une analyse multirésolution. Le modèle présente des discontinuités de flux aux interfaces entre les différents états cellulaires, qui nécessitent la mise en ½uvre d'un couplage spécifique, compatible avec le schéma d'ordre élevé et le raffinement de maillage.Un chapitre de la thèse est dévolu à la méthode de calibration, qui consiste à traduire les connaissances biologiques en contraintes sur les paramètres et sur les sorties du modèle. Le caractère multi-échelle est là encore crucial. Les paramètres interviennent au niveau microscopique dans les équations gouvernant l'évolution des densités de cellules au sein de chaque follicule, alors que les données biologiques quantitatives sont disponibles aux niveaux mésoscopique et macroscopique. / The thesis focuses on the numerical simulation of a biomathematical, multiscale model explaining the phenomenon of selection within the population of ovarian follicles, and grounded on a cellular basis. The PDE model consists of a large dimension hyperbolic quasilinear system governing the evolution of cell density functions for a cohort of follicles (around twenty in practice).The equations are coupled in a nonlocal way by control terms involving moments of the solution, defined on either the mesoscopic or macroscopic scale.Three chapters of the thesis, presented in the form of articles, develop the method used to simulate the model numerically. The numerical code is implemented on a parallel architecture. PDEs are discretized with a Finite Volume scheme on an adaptive mesh driven by a multiresolution analysis. Flux discontinuities, at the interfaces between different cellular states, require a specific treatment to be compatible with the high order numerical scheme and mesh refinement.A chapter of the thesis is devoted to the calibration method, which translates the biological knowledge into constraints on the parameters and model outputs. The multiscale character is crucial, since parameters are used at the microscopic level in the equations governing the evolution of the density of cells within each follicle, whereas quantitative biological data are rather available at the mesoscopic and macroscopic levels.The last chapter of the thesis focuses on the analysis of computational performances of the parallel code, based on statistical methods inspired from the field of uncertainty quantification.

Pendant le bris de l'enveloppe nucléaire, la kinase dépendante des cyclines liée à la cycline B (CDK1-cycline B) et d'autres kinases phosphorylent des protéines nucléaires conduisant au désassemblage des complexes de protéines de l'enveloppe nucléaire. Les protéines nucléaires phosphorylées sont ensuite déphosphorylées par un groupe de phosphatases en sortie mitotique. La protéine phosphatase 2A en complexe avec la sous-unité régulatrice B55 (PP2A-B55) est connue pour être la principale phosphatase à déphosphoryler les protéines critiques à la fin de la mitose. Cependant, les substrats nucléaires déphosphorylés par PP2A-B55 à la sortie mitotique sont peu connus. En utilisant des cellules de drosophile en culture, nous avons démontré que PP2A-B55 est nécessaire pour le recrutement de protéines de l'envelope nucléaire telles que BAF, la protéine de lamina nucléaire Lamin B et la nucléoporine Nup107. De plus, nous avons trouvé que les œufs de femelles des drosophiles hétérozygotes pour une mutation dans les gènes codant pour la Lamine B et Tws (B55 chez la drosophile) n’éclosent pas. Ces œufs présentent divers défauts au stade de la méiose et des divisions nucléaires de l’embryon syncytial. De plus, des tests in vitro et d'autres analyses biochimiques indiquent que PP2A-Tws se lie et déphosphoryle BAF. J'ai d'autres résultats qui suggèrent un rôle de la protéine Ankle2 dans la régulation du recrutement de BAF pour réassembler les noyaux à la sortie mitotique. Mes résultats suggèrent également que Ankle2 en complexe avec PP2A est responsable de la bonne progression mitotique. Mes résultats mettent en évidence l'utilité de la drosophile comme système modèle dans l'étude de différents aspects du cycle cellulaire. Ils démontrent également / During nuclear envelope breakdown, the cyclin-dependent kinase 1 bound to Cyclin B (CDK1-Cyclin B) and other kinases phosphorylate a number of nuclear proteins leading to the disassembly of nuclear envelope protein complexes. Phosphorylated nuclear proteins are then dephosphorylated by a group of phosphatases at mitotic exit. The protein phosphatase 2A in complex with the regulatory subunit B55 (PP2A-B55) is known to be the major phosphatase to dephosphorylate critical proteins at the end of mitosis. However, little was known about the nuclear substrates dephosphorylated by PP2A-B55 at mitotic exit. Using Drosophila cells in culture, we demonstrated that PP2A-B55 is required for the recruitment of nuclear envelope proteins such as BAF, the nuclear lamina protein Lamin B, and the nucleoporin Nup107. Also, eggs from Drosophila females heterozygous for a mutation in genes coding for Lamin B and Tws (B55 in Drosophila), didn’t hatch. These eggs showed various defects during the nuclear division stage and meiosis. Moreover, in vitro assays and other biochemical analyses indicate that PP2A-B55 binds and dephosphorylates BAF. I have other results that suggest a role of the protein Ankle2 in regulating BAF recruitment to reassembling nuclei at mitotic exit. My results also suggest that Ankle2 in complex with PP2A is responsible for the proper mitotic progression. Our results highlight the importance of Drosophila in investigating different aspects of the cell cycle. It also demonstrates a role of PP2A in the nuclear envelope reformation at the end of mitosis.

Eukaryotic cells have to divide constantly in order to promote the growth of certain organs, to replace dying or damaged cells, or to set up an entire organism. These essential processes are called mitosis in the case of somatic cell division. Mitotic cell division is the process during which chromosomes, centrosomes, and microtubules (MTs) are involved to set up a bipolar structure called the “mitotic spindle”. This bipolar spindle is formed by MTs, which are presumably mainly organized from the centrosomes. However, more data are being published that suggest MTs nucleation can occur from other MTs or even a chromosome surface. These biopolymers are built from α/β-tubulin heterodimers and can dynamically grow and shrink to exert forces necessary for chromosome segregation. Previous studies of spindles during mitosis have allowed the identification of different MT classes based on their plus-ends interaction with different cellular target sites. One of the MT classes is the kinetochore microtubules (KMTs), which physically connect chromosomes and centrosomes (i.e. spindle pole) via a specialized protein structure termed the “kinetochore”. This kinetochore-to-spindle pole connection has been studied in many organisms. In budding yeast, this connection is established by only a single KMT. In contrast, multiple KMTs bind to each mammalian kinetochore and form an MT bundle also called “k-fiber”. The ultrastructural architecture of the mammalian k-fiber connection is not well documented. Currently, different models concerning the nature of the kinetochore-to-spindle pole connection via k-fibers are discussed in the literature, i.e. a direct, semi-direct or indirect connection. The widely accepted ‘direct’ model proposes that all k-fibers of the mammalian spindle are formed through tight bundles of up to 20 KMTs, with all MT minus ends associated with the centrosome. However, it is necessary to understand the k-fibers structure in order to interpret its role during chromosome segregation. Here the architecture of the k-fiber was studied in human HeLa, U2OS and RPE-1 cell lines, as these different types of cells have been widely used in studies of mitosis. This thesis aimed to systematically investigate the characteristics of mammalian k-fibers and their attachment to the kinetochore within mammalian metaphase spindles. For that, the ultrastructure of mitotic spindles and k-fibers were analyzed using serial-section electron tomography primarily in HeLa cells. Furthermore, the spindle ultrastructure was compared by electron tomography to metaphase spindles in both U2OS and RPE-1 cells. Electron tomographic analysis of the mitotic spindle in HeLa cells revealed that the kinetochore-to-spindle pole connection is formed by k-fibers consisting of ~9 KMTs. Moreover, the data revealed that not all KMTs in k-fibers are directly associated with one of the spindle poles. Instead, KMT ends were located along the length of k-fibers indicating strongly for a semi-direct connection between the kinetochores and the spindle poles. Unexpectedly, by correlating the k-fiber ultrastructure with its position in the mitotic spindle, it can be demonstrated that the k-fiber structure varied depending on the position on the metaphase plate. It can also be shown that k-fibers located in the center of the metaphase plate had a tendency to form straighter and more bundled k-fibers. In contrast, k-fibers associated with the periphery of the metaphase plate had a more loose and disorganized structure resembling a fusiform shape. Furthermore, additional analysis of U2OS and RPE-1 cells indicated ultrastructural differences between the different cell lines. Mainly, differences between HeLa and RPE-1 cells were observed. K-fibers observed in RPE-1 cells showed a lower curvature and overall a more bundled ultrastructure compared to HeLa or U2OS cells. However, due to the small sample size for U2OS and RPE-1 cells, the results have to be confirmed in future experiments to conclude that there are indeed functional and structural differences in the k-fiber organization in different mammalian cell lines. Taken together, this work presents the first detailed quantitative ultrastructural analysis of KMTs in whole spindles in three different human cell lines. The data revealed that the currently favored direct model of k-fiber ultrastructure is oversimplified and needs to be corrected in terms of the k-fibers interaction with the spindle pole and the surrounding MT network within the mitotic spindle. The data here will serve as a structural basis for further analyses of mutant situations and contribute to our understanding of the overall organization and function of MTs in mitotic spindles.:Summary 6 Zusammenfassung 8 List of figures 10 List of tables 13 List of abbreviations and symbols 14 1 Introduction 19 1.1 The morphology of the mitotic spindle 21 1.1.1 Centrosomes 22 1.1.2 Microtubules 23 1.2 Kinetochores, KMTs and k-fibers 28 1.2.1 A brief history of k-fiber formation in mammalian cells 30 1.2.2 Models of the k-fiber ultrastructure in mammalian cells 32 2 Aims of this thesis 35 3 Materials and methods 36 3.1 Materials 37 3.1.1 Mammalian cell lines 37 3.1.2 Chemicals 38 3.1.3 Instrumentation and materials 40 3.1.4 Solutions and buffers 44 3.1.5 Software 46 3.2 Methods 47 3.2.1 Handling of cell cultures 47 3.2.2 Custom-designed incubation chambers 49 3.2.3 Specimen preparation for electron microscopy 51 3.2.4 Quality assessment of samples, acquisition of the tomographic data, and the 3D reconstruction 59 3.2.5 Ultrastructural analysis of MTs in mitotic spindles 62 3.2.6 Ultrastructural analysis of the k-fiber organization 70 4 Results 76 4.1 Initial characterization of mammalian mitotic spindles 77 4.2 Ultrastructure of KMTs 84 4.3 Curvature and tortuosity of KMTs 91 4.4 Ultrastructure of k-fibers 98 4.5 Effect of metaphase position on the k-fiber ultrastructure 102 5 Discussion 110 5.1. Comparison of data sets from different cell lines 111 5.2. Establishing a data analysis pipeline for the analysis of KMTs 113 5.3 Ultrastructural characterization of KMTs and k-fibers in HeLa cells 114 5.3.1 K-fibers have an unexpectedly low number of KMTs 115 5.3.2 Semi-direct kinetochores-to-spindle pole connection 117 5.3.3 Shape of k-fibers 121 5.4 Positional effect on the k-fiber shape 124 5.5 Comparison of k-fiber ultrastructure in different mammalian cells 127 5.6 Outlook 130 References 133 Appendix 1 149 Appendix 2 150 Appendix 3 151 Appendix 4 152 Acknowledgments 153

Dans le but de déterminer le rôle des Rho GTPases et de leurs régulateurs dans les cellules hématopoïétiques, une analyse des niveaux d’expressions de 300 gènes codant pour des protéines impliquées dans les voies de signalisation dépendantes de Rho a été faite à partir d’échantillons de patients atteints de leucémies de type T-ALL. Il a ainsi pu être mis en évidence qu’un groupe de gènes incluant notamment RacGAP1, Ect2, Citron et ARHGAP19 variaient parallèlement. A l’exception de ARHGAP19, ces gènes avaient une fonction connue au cours de la mitose. Il a donc été entrepris de caractériser ARHGAP19 qui, d’après les banques de données, est spécifique du système hématopoïétique, et pour laquelle aucune fonction n’avait encore été déterminée.Afin de déterminer la fonction biologique de GAP19, un anticorps a été généré. Cet outil nous a permis de montrer que l’expression de la protéine est régulée au cours du cycle cellulaire et que sa localisation varie au cours de la mitose. Par ailleurs, nous avons montré que GAP19, joue un rôle essentiel dans le changement de forme des lymphocytes en mitose, la ségrégation des chromatides sœurs et le recrutement membranaire des effecteurs de RhoA au cours de la mitose. Nous avons aussi mis en évidence le mécanisme par lequel GAP19 permet le changement de forme dans les lymphocytes.Nous avons aussi montré que GAP19 est phosphorylée par CDK1 sur deux résidus présents dans la partie C-Terminale. Afin de mettre en évidence le rôle de ces phosphorylations, nous avons généré des cellules Kit225 transfectées avec des plasmides pour les formes non-phosphorylables de la protéine. Ceci nous a permis de mettre en évidence que la phosphorylation des résidus T404 et T476 permet la localisation cytoplasmique de GAP19 en début de mitose. Nous avons aussi pu observer lors de l’anaphase la formation de ponts de chromatines, ainsi qu’une augmentation significative de cellules multinucléées. Par ailleurs, nous avons procédé à des expériences de cytogénétique et d’immunofluorescence afin de déterminer, si les ponts de chromatines avaient pour origine soit des défauts de condensation de la chromatine, soit un stress réplicatif.Enfin, un possible modèle de la protéine ARHGAP19 a été généré et des simulations de dynamiques moléculaires réalisées afin de comprendre le rôle des phosphorylations par CDK1 a un niveau structurel. / In an attempt to understand the role of Rho GTPases and their regulators in hematopoietic cell lines, expression levels of 300 genes were analyzed for proteins involved in Rho dependent signaling pathways from patients with T-ALL leukemia.It was shown that a group of genes consisting of RacGAP1, Ect2 and Citron varied concomitantly. With the exception of ARHGAP19, all already had a known function during mitosis. Consequently, it was decided to characterize ARHGAP19, which according to databases is specific of hematopoietic cell lines, and whose function was unknown. In order to determine the biological function of ARHGAP19, a specific antibody has been generated. This allowed us to demonstrate that the level of expression of the protein vary during the cell cycle and its localization varies during mitosis. In addition, we have shown that ARHGAP19 plays a central role in regulating cell shapes changes, sister chromatids segregation and RhoA effectors membrane recruitment during mitosis. We have also shown that this occurs by a previously undescribed pathway involving RhoA-ROCK-Vimentin.Finally, we have demonstrated that ARHGAP19 is a substrate of CDK1. It is phosphorylated on two residues located in the C-Terminal region of the protein. For investigating the role of these phosphorylations, we have generated Kit225 cell lines transfected with plasmids coding for the non-phosphorylable forms of the protein. This allowed us to show that phosphorylation of residue T404 and T476 are involved preventing GAP19 recruitment at the equatorial cell cortex during mitosis.In addition, we have observed the formation of chromatin bridges, as well as an increase in multinucleated cells. Thus, we have performed cytogenetic experiments for determining if chromatin bridges are due to chromosome condensation defects, or replicative stress. Finally, a possible tertiary structure of ARHGAP19 has been created de novo, and molecular dynamics simulations were generated in order to understand the role of these phosphorylations by CDK1 at a structural level.

L’équipe a identifié une nouvelle GAP de RhoA, ARHGAP19, majoritairement exprimée dans le système hématopoïétique. Le projet a consisté à étudier la régulation de cette protéine dans des lymphocytes T humains. Pour cela, les analyses se sont portées sur la phosphorylation d’ARHGAP19 et sur sa localisation au cours de la division des lymphocytes T. ARHGAP19 est phosphorylée par l’effecteur de RhoA, la protéine kinase ROCK, sur la Sérine 422 et par la protéine kinase mitotique CDK1 sur les Thréonines 404 et 476. La phosphorylation par ROCK permet à ARHGAP19 d’interagir avec la famille de protéines 14-3-3 qui la protège des déphosphorylations pouvant avoir lieu au cours de la division cellulaire. L'ensemble des phosphorylations est primordial pour la régulation de la localisation cellulaire d'ARHGAP19 et contribue à une division cellulaire correcte. En effet, en absence de phosphorylation, on observe des défauts lors de la cytodiérèse entrainant la formation de cellules multinucléées. De plus, des dérégulations de RhoGTPases comme l’absence de GAP, sont aujourd’hui mises en évidence dans les cancers. C’est pourquoi nous avons généré des souris arhgap19 KO pour étudier les conséquences de l’absence du gène codant pour ARHGAP19, dans le système hématopoïétique murin. L’ensemble des cellules progénitrices et matures intervenant dans l’hématopoïèse murine a été analysé. Par ce modèle d’invalidation conditionnelle d’arhgap19, aucun rôle majeur de la protéine n'a été mis en évidence mais les résultats suggèrent une implication aux différents stades de la différenciation hématopoïétique et un impact sur l'ensemble des populations de ce système. / The team identified a new GAP of RhoA, ARHGAP19, mostly expressed in the hematopoietic system. The project consisted in studying the regulation of this protein in human T lymphocytes. For this, the analyzes focused on the phosphorylation of ARHGAP19 and on its localization during the division of the T lymphocytes. ARHGAP19 is phosphorylated by the effector of RhoA, the protein kinase ROCK, on the Serine 422 and by the protein CDK1 mitotic kinase on Threonines 404 and 476. ROCK phosphorylation allows ARHGAP19 to interact with the 14-3-3 family of proteins that protects it from dephosphorylation that occur during cell division. All phosphorylations are essential for regulating the cellular localization of ARHGAP19 and contribute to correct cell division. Indeed, in the absence of phosphorylation, defects are observed during cytodiérèse resulting in the formation of multinucleate cells. In addition, deregulation of RhoGTPases, such as the absence of GAP, are now highlighted in cancers. This is why we generated arhgap19 KO mice to study the consequences of the absence of the gene coding for ARHGAP19, in the murine hematopoietic system. All progenitor and mature cells involved in murine hematopoiesis were analyzed. By this model of conditional invalidation of arhgap19, no major role of the protein has been demonstrated but the results suggest an involvement at different stages of hematopoietic differentiation and an impact on all populations of this system.

La mitose est précisément contrôlée par la signalisation via l'ubiquitine et est essentielle au maintien de l'intégrité du génome. Dans ce travail, j'ai étudié la fonction de l'enzyme de dé-ubiquitination, UCHL3 et de la ligase E3-ubiquitine, TRIM15. J'ai observé que TRIM15 régule l'adhésion et la mobilité des cellules. UCHL3 a été identifié par un criblage à haut contenu, en tant que facteur critique contrôlant l'alignement et la ségrégation des chromosomes. Fait intéressant, il a déjà été rapporté que les niveaux d’expression d’UCHL3 sont altérés dans divers types de cancer. En utilisant une approche protéomique, nous avons identifié la kinase Aurora B comme un médiateur potentiel de ces phénotypes. Comme l'aneuploïdie est la marque de nombreux cancers et que l'adhésion cellulaire joue un rôle important dans l'invasion des tumeurs et les métastases, mes résultats suggèrent que ces deux protéines pourraient jouer un rôle dans la carcinogenèse. / Mitosis is tightly controlled by ubiquitin signaling and is crucial to maintain genome integrity. In this work, I investigated the function of the deubiquitinating enzyme UCHL3 and the E3 ubiquitin ligase TRIM15. I observed that TRIM15 regulates cell adhesion and motility. UCHL3 was identified in a high-content screen, as a critical factor controlling the chromosome alignment and segregation. Interestingly, it has been previously reported that UCHL3 levels are altered in various cancer types. Using a proteomic approach, we identified Aurora B kinase as a potential mediator of these phenotypes. Since aneuploidy is a hallmark of many cancers, and cell adhesion plays an important role in tumor invasion and metastasis, my results suggest that both proteins could play a role in carcinogenesis.

Microtubules are tubular polymers that are involved in a variety of cellular processes such as cell movement, mitosis and intracellular transport. The dynamic behavior of microtubules makes this possible because all of these processes require quick responses. Embryonic stem (ES) cells were first isolated from mouse embryos and they have two unique characteristics; they can be kept undifferentiated for many passages with a stable karyotype and they can be differentiated into any type of cells under appropriate conditions. The pluripotency of ES cells, their ease of manipulation in culture, and their ability to contribute to the mouse germ-line provides us a model of differentiation both in vitro and in vivo. In my thesis I focused on the cell division and neuronal differentiation of ES cells and developed two methods to understand the effects of microtubule dynamics in spindle assembly and chromosome segregation and to reveal the roles of different Microtubule Associated Proteins (MAPs) in the neuronal morphology formation. In the first part, we developed a live-cell imaging method for ES cells to visualize, track and analyze the single cell behavior in a cell population over a time period. So far many techniques have been adapted and combined for imaging of cell lines, mainly for the cancer or immortalized ones. However, because ES cells are very prone to apoptosis, tend to form spheres and hard to stably label, it is quite tricky to image them in culture conditions. In our system, we combined the BAC-based gene expression with wide-field deconvolution microscopy for ES cells that are plated onto the laminin-511 coated surface and kept in CO2 independent culture conditions. This combined technique does not interfere with the growth of cells and keeps them healthy up to 24 hours on the microscope stage. In the second part, we analyzed the effects of MAPs chTOG, EB1, Kif18A and MCAK in the overall spindle morphology and mitotic progression in mES cells. For this purpose, we utilized our stable TUBB-GFP and H2A-GFP cell lines along with our live-cell imaging set-up to reveal the effects of the above-mentioned proteins and the interplay among each other. By using RNAi method we either single or co-depleted the genes by siRNAs and measured the spindle length and width in RNAi conditions. We further analyzed the mitotic progression in H2A-GFP cell line in terms of the metaphase timing and the percentage of chromosome segregation errors. Our results showed that, EB1 depletion did not cause any significant changes in the overall spindle morphology or in the metaphase timing. However, the co-depletion of EB1 with chTOG partially rescued the sichTOG specific mini-spindle phenotype. siKif18A produced longer spindles without any change in the spindle width. Surprisingly, the co-depletion of antagonistic chTOG and Kif18A proteins had additive effects on the spindle dynamics and on mitotic progression in a way that spindle assembly was severely disrupted by the absence of these two proteins and as a result of this, both metaphase timing and chromosome missegregation levels increased significantly. These results overall indicate that MAPs have important roles in the regulation of dynamic instability and these proteins have an interplay among each other to be able to control the morphology of the spindle as well as the correct segregation of chromosomes into daughter cells. In the last part, I will introduce you a new ES cell based differentiation and morphology model, which brings the advantages of high resolution imaging capacity, control over development and easy genetic manipulation and culturing. We have generated Tet-induced shRNA cell lines against chTOG, Kif18A and MCAK, which are also stably expressing TUBB-GFP. These labeled cells were mixed with unlabeled wild-type mES cells before differentiation at 1:1000 ratio and then they were differentiated into mouse cortical cells and spinal motor neurons. Our results showed that, all of the three genes could be successfully knocked-down by shRNA after 48 hours of Tet induction. After mixing the labeled and unlabeled cells, single neurons could be imaged at high resolution and their skeletons could be generated afterwards. The RNAi studies in shchTOG cell line showed that, the knock-down of this gene in early differentiation interferes with the neuronal differentiation.

Die mitotische Spindel ist ideal für die Erforschung der Selbstorganisation und Plastizität molekularer Kollektive im Zytoplasma. Die Geometrie der Spindel ist entscheidend für die korrekte Chromosomentrennung, muss sich aber an die Zellgröße anpassen. Es ist unbekannt, ob und wie Zellen während ihrer Differenzierung die Spindelarchitektur anpassen, was insbesondere während der Gehirnentwicklung relevant ist. Wir untersuchten dies mit Maus-Embryonalstammzellen, die in frühe neuronale Vorläuferzellen differenziert wurden. Wir entwickelten ein automatisiertes Mikroskopieprotokoll um einen umfassenden Datensatz von mitotischen Zellen zu generieren. Außerdem entwickelten wir Spindle3D, ein Werkzeug zur dreidimensionalen Analyse von Spindeln. Überraschenderweise waren die Spindelvolumina in differenzierenden Zellen bis zu 24% kleiner als in pluripotenten Zellen. Während die Wachstumsgeschwindigkeit der Mikrotubuli unverändert blieb, verschob sich in sich differenzierenden Zellen die Nukleation von Mikrotubuli zugunsten der astralen Population. Diese Veränderung der Spindelarchitektur basierte auf der differenzierungsbedingten Verdünnung des Zytoplasmas. Dies aktivierte CPAP, ein Protein, das die Zentrosomenreifung reguliert, was zur Superskalierung des perizentriolären Materials und verstärkte Rekrutierung von gamma-Tubulin an den Zentrosomen und somit zur Umlagerung von Mikrotubuli innerhalb der Spindel führte. Diese Veränderungen der mitotischen Architektur konnten durch externe osmotische Einwirkung in undifferenzierten Zellen nachgestellt werden. Insgesamt verbinden unsere Ergebnisse zelltypspezifische zytoplasmatische Materialeigenschaften mit der Spindelarchitektur. / The mitotic spindle provides an excellent system in which to study the plasticity of molecular collectives. To segregate chromosomes accurately, the spindle’s geometry must be adaptive to changes in cell size. It is unknown whether and how differentiating cells adjust spindle architecture, specifically during brain development when spindle defects have severe pathological consequences. Using murine embryonic stem cells, we recapitulated the transition from pluripotency to early neural cell identities in vitro. Aiming at a systematic exploration of spindle and cell morphology throughout this process, we developed an automated microscopy protocol and Spindle3D, a morphometric tool for the analysis of spindles in confocal images. Intriguingly, in cells with comparable cell volume, spindle volumes were up to 24% smaller in cells undergoing differentiation. While microtubule growth speed remained equal, we measured increased nucleation of astral microtubules at the expense of the spindle bulk in differentiating cells. The shift in spindle architecture was explained by the differentiation-driven cytoplasmic dilution. This activated the centrosomal regulator CPAP, causing the superscaling of the pericentriolar material and the concomitant increased recruitment of gamma-tubulin to the centrosomes, redistributing microtubule numbers within the spindle. Mimicking the dilution effect by osmotic challenge reproduced the same mitotic architecture in undifferentiated cells. Collectively, our results link cell state-specific cytoplasmic material properties to spindle architecture.

Le cycle cellulaire est hautement régulé par la phosphorylation réversible de plusieurs effecteurs. La kinase dépendante des cyclines Cdk1 déclenche la mitose en induisant le bris de l’enveloppe nucléaire, la condensation des chromosomes et la formation du fuseau mitotique. Chez les animaux métazoaires, ces évènements sont contrés par la protéine phosphatase PP2A-B55, qui déphosphoryle plusieurs substrats de Cdk1. La kinase Greatwall (Gwl) est activée par le complexe cycline B-Cdk1 en début de mitose et induit ensuite l’inhibition de PP2A-B55 via Endos/Arpp19. Toutefois, les mécanismes moléculaires qui régulent Gwl sont encore peu connus. Nous avons montré que Gwl a une activité s’opposant à PP2A-B55, qui collabore avec la kinase Polo pour assurer l’attachement du centrosome au noyau et la progression du cycle cellulaire dans le syncytium de l’embryon de la drosophile. Ensuite, nous avons trouvé dans des cellules de drosophile que Gwl est localisée au noyau pendant l’interphase, mais qu’elle se relocalise au cytoplasme dès la prophase, avant le bris de l’enveloppe nucléaire. Nous avons montré que cette translocation de Gwl est cruciale pour sa fonction et qu’elle dépend de la phosphorylation de plusieurs résidus de la région centrale de Gwl par les kinases Polo et Cdk1. Cette région centrale contient également deux séquences de localisation nucléaire (respectivement NLS1 et NLS2). De plus, nos résultats suggèrent que la phosphorylation de Gwl par la kinase Polo promeut sa liaison avec la protéine 14-3-3ε, ce qui favorise la rétention cytoplasmique de Gwl. Le rôle de Cdk1 dans cette translocation reste quant à lui inconnu. De plus, nous avons montré que le complexe cycline B-Cdk1 entre dans le noyau avant que Gwl ne soit transportée dans le cytoplasme. Cdk1 pourrait donc activer Gwl et phosphoryler ses substrats nucléaires, à l’abri de PP2A-B55 qui est largement cytoplasmique. Gwl est ensuite exclue du noyau et relocalisée dans le cytoplasme afin d’induire l’inhibition de PP2A-B55. Cela permet de synchroniser les événements de phosphorylation se produisant dans le noyau et dans le cytoplasme. Fait intéressant, un mécanisme de régulation de la localisation de Gwl similaire à cela a été découvert chez l’humain et chez la levure, suggérant que ce mécanisme est conservé entre différentes espèces. / Reversible phosphorylation of proteins, triggered by cyclically activated kinases and phosphatases, is a key mechanism to control cell cycle progression. CyclinB-Cdk1 is a crucial kinase phosphorylating a large number of substrates to trigger mitotic entry. However, in metazoans, it is counteracted mainly by a Protein Phosphatase 2A carrying the B55 regulatory subunit (PP2A-B55). On the other hand, the Greatwall (Gwl) kinase is activated by CyclinB-Cdk1 upon mitotic entry and subsequently induces the inhibition of PP2A-B55 by Endos/Arpp19, thus promoting mitotic entry and maintenance. Nonetheless, the regulatory mechanisms of Gwl are less clear. We demonstrated that in Drosophila syncytial embryos, PP2A-B55 is negatively regulated by Gwl, but collaborates with Polo kinase to ensure both nucleus attachment of centrosome and faithful cell cycle progression. Later, we discovered that in Drosophila, the subcellular localization of Gwl changes dramatically throughout the cell cycle. Gwl is nuclear in interphase but suddenly becomes mostly cytoplasmic in prophase before nuclear envelope breakdown. Such translocation is important for Gwl’s function and requires the phosphorylation of Gwl by both Polo kinase and Cdk1 in the region containing two Nuclear Localization Signals (NLSs). Phosphorylation of Gwl by Polo likely promotes its association with14-3-3ε thereby promoting Gwl cytoplasmic retention, whereas Cdk1’s role in this translocation remains elusive. Moreover, I found that most cyclin B is imported into the nucleus before Gwl translocates to the cytoplasm. Therefore, Cdk1 can activate Gwl and phosphorylate its nuclear substrates without the perturbation of PP2A-B55 which is largely cytoplasmic. Subsequently, Gwl translocates into cytoplasm to mediate the inhibition of PP2A-B55 so that the phosphorylation events can be synchronized between the nucleus and the cytoplasm. Interestingly, similar spatial regulation of Gwl was also uncovered in mammal cells and in yeast, implying a conserved regulatory mechanism across species.