Doctor of Philosophy / Biochemistry Interdepartmental Program / Qize Wei / This dissertation describes the role of myosin-interacting guanine nucleotide exchange factor (MyoGEF) and centrosome/spindle pole associated protein (CSPP) in mitotic progression and cytokinesis. We have identified three mouse isoforms of CSPP, all of which interact and colocalize with MyoGEF to the central spindle in anaphase cells. The N-terminus of MyoGEF interacts with myosin whereas the C terminus interacts with the N-terminus of CSPP, forming a complex. The N-terminus of CSPP appears to be important for both localization and interaction with MyoGEF. CSPP plays a role in mitotic progression since its depletion by RNAi resulted in metaphase arrest. MyoGEF is required for completion of cytokinesis. Both MyoGEF and CSPP are phosphorylated by mitotic kinases including Plk1 and Aurora. Importantly, MyoGEF is phosphorylated at Thr-574 in mitosis by Polo-like kinase 1, and this phosphorylation is required for activation of RhoA. Thr-543 of MyoGEF is required for Plk1 binding in mitosis and phosphorylation of MyoGEF by Cdk1/cyclinB, possibly at Thr-543 may generate a Plk1 docking site, i.e., Cdk1 can phosphorylate MyoGEF at Thr-543, thereby allowing Plk1 to bind and phosphorylate MyoGEF at Thr-574. Finally, MyoGEF and CSPP are also phosphorylated by Aurora-B kinase in vitro. Taken together, we propose that Aurora-B may phosphorylate and recruit MyoGEF and CSPP to the central spindle, where phosphorylation of MyoGEF at Thr-543 promotes Polo kinase binding and additional phosphorylation of MyoGEF, leading to the activation of RhoA at the cleavage furrow.

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.

Cell polarity is a key concept in plant biology. The subcellular localization of Pin- formed (PIN) auxin efflux carriers in the root of "#$%&'()*&* is remarkably asymmetrical, making PINs prominent markers to study cell polarity. In spite of its developmental importance and two decades of research, the molecular basis of PIN polarity remains largely unknown. In this thesis, I employed advanced transgenic and fluorescence microscopy approaches to gain insight into several aspects of PIN polarity regulation. I participated in establishing a novel genetically encoded inhibitor of endocytosis, an invaluable tool for the study of the importance of endocytosis for various cellular processes, including PIN polarity. I demonstrated that apical polarity of PIN2 needs to be re-established after cell division and that this process depends on endocytosis, '+!,(-( protein secretion and the action of WAG1 and related protein kinases, but not transcytosis, cell-cell signaling or intact cytoskeleton. Finally, I identified the previously unknown role of MAB4/MEL proteins in PIN polarity, which lies in the ability of MAB4/MELs to reduce PIN lateral diffusion and thus contribute to PIN polarity maintenance. My results, besides broadening current understanding of PIN polarity regulation, identify mechanisms that...

Cytokinesis involves constriction of the cell at the equator. Without decreasing in volume, a spherical cell requires a net increase in the surface area during this constriction. The constriction is driven by formation of an actomyosin contractile ring, and the surface increase by addition of membrane during the formation of the cleavage furrow. Both events depend on the central spindle microtubules at the midzone of the spindle and, in particular, on the centralspindlin protein complex. The communication between the central spindle microtubules and the actomyosin ring involves binding of a GAP and a GEF for RhoA to the centralspindlin kinesin Pavarotti/MKLP1. However, it is still unclear which molecular machinery connects the mitotic spindle to membrane trafficking during cleavage furrow ingression. ARF6 is a member of the ARF family of small GTPases, and previous studies suggest that it is an important regulator of membrane trafficking through the endocytic pathway, and cortical Actin remodelling. I generated an arf6 null mutant in Drosophila. arf6 null mutants survive to adulthood without obvious morphological defects, indicating that ARF6 is not required for Drosophila somatic development. However, ARF6 is required for cytokinesis in Drosophila spermatocytes. The centralspindlin kinesin Pavarotti, identified as an ARF6 interactor in a Yeast-2-Hybrid assay, binds ARF6 in GST pulldowns, and interacts genetically with the arf6 mutant. ARF6 localizes to the plasma membrane and a population of early and recycling endosomes. During cytokinesis, ARF6 is enriched on recycling endosomes at the central spindle. arf6 mutants form a cleavage furrow during cytokinesis, which later regresses. Cytokinesis in arf6 mutant spermatocytes lacks the rapid plasma membrane expansion observed during normal divisions. The results of this study suggest that ARF6 might promote rapid recycling of endosomal membrane stores at the central spindle to the plasma membrane during cytokinesis. ARF6 might be recruited to the central spindle via its interaction with Pavarotti, and act as part of the molecular link between the central spindle cytoskeleton and the rapid plasma membrane addition necessary for cytokinesis. Für die Ansicht der quick-time-Movies mit der Endung "avi" ist die Installation des "Apple QuickTime-Players" erforderlich.

Melanoma, the most lethal form of skin cancer, arises from altered cells in the melanocyte lineage, but the mechanisms by which these cells progress to melanoma are unknown. To understand the early cellular events that contribute to melanoma formation, we examined melanocytes in melanoma-prone zebrafish strains expressing BRAFV600E, the most common oncogenic form of the BRAF kinase that is mutated in nearly 50% of human melanomas. We found that, unlike wild-type melanocytes, melanocytes in transgenic BRAFV600Eanimals were binucleate and tetraploid. Furthermore, melanocytes in p53-deficient transgenic BRAFV600Eanimals exhibited 8N and greater DNA content, suggesting bypass of a p53-dependent arrest that stops cell cycle progression of tetraploid melanocytes. These data implicate tetraploids generated by increased BRAF pathway activity as contributors to melanoma initiation. Previous studies have used artificial means of generating tetraploids, raising the question of how these cells arise during actual tumor development. To gain insight into the mechanism by which BRAFV600E generates binucleate, tetraploid cells, we established an in vitro model by which such cells are generated following BRAFV600E expression. We demonstrate thatBRAFV600E-generated tetraploids arise via cytokinesis failure during mitosis due to reduced activity of the small GTPase RhoA. We also establish that oncogene-induced centrosome amplification in the G1/S phase of the cell cycle and subsequent increase in the activity of the small GTPase Rac1, partially contribute to this phenotype. These data are of significance as recent studies have shown that aneuploid progeny of tetraploid cells can be intermediates in tumor development, and deep sequencing data suggest that at least one third of melanomas and other solid tumors have undergone a whole genome doubling event during their progression. Taken together, our melanoma-prone zebrafish model and in vitro data suggest a role for BRAFV600E-inducedtetraploidy in the genesis of melanomas. To our knowledge, this is the first in vivo model showing spontaneous rise of tetraploid cells that can give rise to tumors. This novel role of the BRAF oncogene may contribute to tumorigenesis in a broader context.

The fission yeast Schizosaccharomyces pombe has become a powerful model system for studying cytokinesis, a process of cytoplasmic division by which one cell divides into two identical daughter cells. Like mammalian cells, S. pombe divides through the use of an actomyosin contractile ring, which is composed of a set of highly conserved cytoskeletal proteins. Cytokinesis in S. pombe is primarily regulated by the SIN pathway, which is activated in late mitosis and is required for actomyosin contractile ring and septum assembly, and also plays a role in spindle checkpoint inactivation, and telophase nuclear positioning. The various functions of the SIN are carried out by the terminal kinase in the pathway called Sid2. The lack of information in the downstream targets of Sid2 has limited our understanding of the different functions of the SIN. We recently showed that, in addition to its other functions, the SIN promotes cytokinesis through inhibition the MOR signaling pathway, which normally drives cell separation and initiation of polarized growth following completion of cytokinesis (Ray et al, 2010). The molecular details of this inhibition and the physiological significance of inhibiting MOR during cytokinesis was unclear. The results presented in Chapter II describe our approach to identify Sid2 substrates, particularly focusing on Nak1 and Sog2 that function in the MOR signaling cascade. We identified and characterized Sid2 phosphorylation sites on the Nak1 and Sog2 proteins. Chapter III explores how post translational modification of MOR proteins by Sid2 regulates polarized growth during cytokinesis. This includes delineating the effect of Sid2 mediated phosphorylation of Nak1 and Sog2 on protein-protein interactions in the MOR pathway as well as on the regulation of their localization during late mitosis. Finally, results in Chapter IV demonstrate that failure to inhibit MOR signaling is lethal because cells initiate septum degradation/cell separation before completing cytokinesis thereby emphasizing the importance of cross-regulation between the two pathways to prevent initiation of the interphase polarity program during cytokinesis.

Cell polarity is a key concept in plant biology. The subcellular localization of Pin- formed (PIN) auxin efflux carriers in the root of "#$%&'()*&* is remarkably asymmetrical, making PINs prominent markers to study cell polarity. In spite of its developmental importance and two decades of research, the molecular basis of PIN polarity remains largely unknown. In this thesis, I employed advanced transgenic and fluorescence microscopy approaches to gain insight into several aspects of PIN polarity regulation. I participated in establishing a novel genetically encoded inhibitor of endocytosis, an invaluable tool for the study of the importance of endocytosis for various cellular processes, including PIN polarity. I demonstrated that apical polarity of PIN2 needs to be re-established after cell division and that this process depends on endocytosis, '+!,(-( protein secretion and the action of WAG1 and related protein kinases, but not transcytosis, cell-cell signaling or intact cytoskeleton. Finally, I identified the previously unknown role of MAB4/MEL proteins in PIN polarity, which lies in the ability of MAB4/MELs to reduce PIN lateral diffusion and thus contribute to PIN polarity maintenance. My results, besides broadening current understanding of PIN polarity regulation, identify mechanisms that...

La cytokinèse est un processus minutieusement régulé par une structure corticale appelée l'anneau contractile d'actomyosine, sous le contrôle de la petite GTPase RhoA. La protéine d'échafaudage Anilline est un effecteur de RhoA et organise les différents éléments de l'anneau permettant sa fermeture. Via son N-terminus, l'Anilline interagit avec la Citron kinase Sticky, la myosine II et l'actine-F ce qui permet la stabilisation de l'anneau contractile et sa maturation en anneau du corps central ou «midbody». Via son domaine C-terminal, l'Anilline interagit avec la RhoA-GTP et les septines pour ancrer l'anneau à la membrane. De précédentes études du laboratoire ont montré que Sticky et les septines ont des actions opposées sur l'Anilline. Nous avons donc défini plusieurs ensembles d'interactions entre l'Anilline et les cytosquelettes d'actomyosine et de septines, qui possèdent des fonctions différentes voire opposées dans la cytokinèse: ce sont des réseaux cytosquelettiques. L'Anilline est ainsi impliquée dans la coordination de ces réseaux opposés et RhoA-dépendants. Nous devons encore déterminer si ces interactions de l'Anilline en N-ter et C-ter peuvent se produire en même temps ou si elles sont mutuellement exclusives pour coordonner les différents éléments cytosquelettiques pendant la fermeture de l'anneau contractile. Les cellules S2 de drosophile ont été utilisées pour déterminer comment l'Anilline coordonnait les réseaux cytosqulettiques de l'anneau contractile. Deux modèles ont été proposés : l'un où une seule molécule d'Anilline se lie simultanément aux réseaux en N-ter et C-ter; l'autre modèle suggère qu'une ou plusieurs populations d'Anilline coordonnent les réseaux de façon mutuellement exclusive. Pour distinguer entre ces deux modèles, des allèles de séparation de fonction de l'Anilline ont été testés : l'AnillinΔ1-5 qui n'interagit plus avec Sticky, ainsi que l'AnillinRBD* qui n'interagit plus avec Rho1-GTP et qui ne recrute plus les septines à l'anneau contractile. Des expériences de sauvetage suite à la déplétion de l'Anilline endogène ont été réalisées et les tentatives de division ont été captées par microscopie en temps réel. L'expression de chaque mutant individuellement menait à une fermeture de l’anneau décalée, ralentie et souvent incomplète. En revanche, la co-expression de l'AnillinΔ1-5 et AnillinRBD* en trans dans les mêmes cellules a restauré la cinétique normale de la fermeture de l'anneau. Ce résultat supporte le modèle des populations multiples d'Anilline. Cette étude avance significativement nos connaissances de l'organisation de l'anneau contractile qui gère la division de toutes cellules animales. / Cytokinesis is a process thoroughly regulated by a cortical structure called the actomyosine contractile ring, under the control of the RhoA GTPase. The scaffolding protein Anillin is a RhoA effector organizing the several elements of the ring, thus permitting its closure. The AnillinN-terminus interacts with the Citron kinase Sticky, Myosin II and F-actin to stabilize the contractile ring and drive its maturation to the midbody ring. The AnillinC-terminus interacts with the RhoA GTPase and the septins to anchor the ring to the membrane. Previous works revealed that Sticky retains Anillin while the septins shed Anillin from the ring. Therefore, Anillin is involved in opposed RhoA-dependent cytoskeletal sub-networks to generate or reduce the tension at the membrane, and their balance is necessary to improve the ring closure. This study aims to decipher the coordination between these opposed sub-networks. We proposed two models : either sub-networks on AnillinN-ter and AnillinC-ter are simultaneously organized by the same molecule of Anillin, or several pools of Anillin coordinate separately the opposed subnetworks. We generated and expressed several inducible Anillin mutants in drosophila S2 cells : AnillinΔ1-5 prevents the interaction with Sticky; AnillinRBD* does not interact with RhoA and perturbs the Anillo-septin assembly. The expression of each mutant individually delayed, slowed down and failed the ring closure. However, co-expression of single mutants in trans rescued the ring closure. Moreover, Sticky over-expression improved AnillinRBD* recruitment in the ring. These results support the multiple pools of Anillin model. This study improves our knowledge on the contractile ring organization, necessary to succeed cytokinesis in animal cells.

No description available.

Almost all animal cells maintain a thin layer of actin filaments and associated proteins underneath the cell membrane. The actomyosin cortex is subject to internal stress patterns which result from the spatiotemporally regulated activity of non-muscle myosin II motors in the actin network. We study how these active stresses drive changes in cell shape and flows within the cortical layer, and how these cytoskeletal deformations and flows govern processes such as cell migration, cell division and organelle transport. Following a continuum mechanics approach, we develop theoretical descriptions for three different cellular processes, to obtain - in collaboration with experimental groups - a detailed and quantitative understanding of the underlying cytoskeletal mechanics. We investigate the forces and cortex flows involved in adhesion-independent cell migration in confinement. Many types of cell migration rely on the extension of protrusions at the leading edge, where the cells attach to the substrate with specific focal adhesions, and pull themselves forward, exerting stresses in the kPa range. In confined environments however, cells exhibit migration modes which are independent of specific adhesions. Combining hydrodynamic theory, microfluidics and quantitative imaging of motile, non-adherent carcinosarcoma cells, we analyze the mechanical behavior of cells during adhesion-independent migration. We find that the accumulation of active myosin motors in the rear part of these cells results in a retrograde cortical flow as well as the contraction of the cell body in the rear and expansion in the front, and we describe how both processes contribute to the translocation of the cells, depending on the geometric and mechanical parameters of the system. Importantly, we find that the involved propulsive forces are several orders of magnitude lower than during adhesive motility while the achieved migration velocities are similar. Moreover, the distribution of forces on the substrate during non-adhesive migration is fundamentally different, giving rise to a positive force dipole. In contrast to adhesive migration modes, non-adhesive cells move by exerting pushing forces at the rear, acting to expand rather than contract their substrate as they move. These differences may strongly affect hydrodynamic and/or deformational interactions between collectively migrating cells. In addition to the work outlined above, we study contractile ring formation in the actin cytoskeleton before and during cell division. While in disordered actin networks, myosin motor activity gives rise to isotropic stresses, the alignment of actin filaments in the cortex during cell division introduces a preferred direction for motor-filament interactions, resulting in anisotropies in the cortical stress. Actin filaments align in myosin-dependent shear flows, resulting in possible feedback between motor activity, cortical flows and actin organization. We investigate how the mechanical interplay of these different cortical properties gives rise to the formation of a cleavage furrow during cell division, describing the level of actin filament alignment at different points on the cortex with a nematic order parameter, in analogy to liquid crystal physics. We show that cortical anisotropies arising from shear-flow induced alignment patterns are sufficient to drive the ingression of cellular furrows, even in the absence of localized biochemical myosin up-regulation. This mechanism explains the characteristic appearance of pseudocleavage furrows in polarizing cells. Finally, we study the characteristic nuclear movements in pseudostratified epithelia during development. These tissues consist of highly proliferative, tightly packed and elongated cells, with nuclei actively travelling to the apical side of the epithelium before each cell division. We explore how cytoskeletal properties act together with the mechanics of the surrounding tissue to control the shape of single cells embedded in the epithelium, and investigate potential mechanisms underlying the observed nuclear movements. These findings form a theoretical basis for a more detailed characterization of processes in pseudostratified epithelia. Taken together, we present a continuum mechanics description of the actomyosin cell cortex, and successfully apply it to several different cell biological processes. Combining our theory with experimental work from collaborating groups, we provide new insights into different aspects of cell mechanics.