Cell migration is required for a number of physiological processes and is implicated in pathologies such as tumor metastasis. Cell motility is dependent upon dynamic actin reorganization, and is regulated by the Rho family of small GTPases. Rho GTPases are molecular switches that cycle between their active and inactive conformations. The best-studied members of this family are Rac1, RhoA, and Cdc42. Each is responsible for the formation of specific actin structures. Diacylglycerol kinases (DGKs) act at the membrane to convert diacylglycerol (DAG) and phosphatidic acid (PA), maintaining the balance of these two lipid second messengers. Previous studies from our lab have demonstrated that the ζ isoform of DGK facilitates the release of Rac1 and RhoA from their inhibitor, RhoGDI. Here we studied a closely related isoform, DGKι, using mouse embryonic fibroblasts (MEFS) in which the gene for DGKι had been deleted. Aberrations in cell morphology, spreading, and migration were identified in DGKι-null MEFs. We showed that the activity of Rac1 and RhoC, but not RhoA, was impaired in the absence of DGKι, yet only RhoC protein levels were affected. Reduced activation of these Rho GTPases was accompanied by defects in Rac1- and RhoC- related actin structures. These data demonstrate that DGKι, in addition to DGKζ, contributes to the regulation of GTPase activation and remodeling of the actin cytoskeleton.

8 ABSTRACT Rab GTPases (Rabs) are the most populous branch of eukaryotic Ras GTPase superfamily. In active GTP-binding conformation, they serve as key instruments in defining transient membrane identity and through various effectors regulate formation, transport, conversion, and fusion of membrane vesicles. This is important for upkeep of compartmentalized structure of eukaryotic cells and for facilitating both endo- and exocytic processes. Rabs are converted into GDP-binding conformation by interactions with Rab GTPase activating proteins (Rab GAPs) that possess ability to significantly speed up weak intrinsic GTP hydrolytic activity of Rabs. Through this process, Rab GAPs can limit scope of the Rabs' activity and lay out spatiotemporal boundaries for varying Rab populations. In this thesis, I tried to characterize a Rab GAP, GAP2, seemingly necessary for standard development of thale cress plants. Besides TBC catalytic domain, GAP2 (product of At2g39280 gene) possesses a C-terminal coiled-coil motif, which was previously found to interact with Rab GTPases. Experiments aiming to complement T-DNA insertion mutant in GAP2, elucidate GAP2 intracellular localization, novel interacting partners, and character of interaction with the Rabs discovered in the pilot study were undertaken. The results suggest that...

Rab GTPases are small signaling molecules that play an important role in vesicle trafficking in eukaryotic cells. Correct signaling through small GTPases allows orchestration of vesicle transport among cellular organelles and also to the cell wall providing cell wall material for cell growth and elongation. Engagement of Rab GTPases in the regulation of endomembrane trafficking is one of the evolutionary conserved aspects of secretion regulation. The network of Rab GTPases interaction includes also various downstream effectors. One of them is the exocyst complex involved in vesicle docking at the plasma membrane. It is a complex composed of eight different subunits (Sec3, Sec5, Sec6, Sec8, Sec10, Sec15, Exo70 and Exo84). Exocyst was discovered as Sec4p Rab GTPase effector in yeast and also data from animal models describe the Sec15 exocyst subunit as the Rab-interacting partner, but data from plants are missing. On the other hand, numerous studies identified exocyst role in tip growth of pollen tube and root hairs, seed coat formation, cell plate and cell wall formation, hypocotyl elongation, and importantly also PIN auxin efflux carriers recycling and polar auxin transport. There are two paralogues of SEC15 in the Arabidopsis genome, SEC15a and SEC15b, the previous one already shown to be...

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Engulfment of dead cells by epithelial cells is crucial for the health of an organism. Defective engulfment can lead to serious conditions such as retinitis pigmentosa and asthma. In the Drosophila melanogaster ovary, protein starvation induces apoptotic cell death of germline cells, which are then engulfed by adjacent epithelial follicle cells. The follicle cells synchronously enlarge approximately four-to-five fold as they engulf the dying germline, suggesting that significant changes are required. However, the molecular changes needed to drive enlargement and engulfment by epithelial cells are still poorly understood. In this dissertation, I determined the role of integrins in engulfment, the interactions between the core engulfment machinery and the corpse processing pathway, and the roles of GTPases in engulfment by epithelial cells. First, I found that the integrin heterodimer, αPS3/βPS, becomes apically enriched and is required in the epithelial follicle cells for engulfment. αPS3/βPS is trafficked in a polarized fashion using much of the same machinery as migrating cells, suggesting similarities between engulfing and migrating cells. The canonical corpse processing pathway has been well-characterized, however, little is known about how the core engulfment machinery interacts with the corpse processing pathway. I found that the phagocytic receptor Draper is present on the phagocytic cup and early phagosomes, whereas integrins are maintained on the cell surface. Engulfment mutants fell into three distinct categories based on their specific effects on internalization and phagosome maturation, suggesting that components of the core engulfment machinery are required for distinct steps of corpse processing. Last, I investigated the roles of the Rho family GTPases during engulfment. Strikingly, Rac2 becomes induced during engulfment whereas Rac1 does not change its expression pattern. Both Rac1 and Rac2 are required for engulfment, however, Rac1 has defects in both enlargement and vesicle uptake whereas Rac2 only has defects in enlargement. Furthermore, I found that Rac1 and Rac2 may have differential effects on the Jun kinase signaling pathway, which suggests complexity in the regulatory network controlling engulfment in epithelial cells. Together, this work has provided a greater understanding of the molecular changes required within epithelial cells for proper engulfment. / 2016-12-01T00:00:00Z

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c-Cbl functions as a multifunctional adaptor and an E3 ubiquitin protein ligase. Several studies have shown that c-Cbl is involved in cytoskeleton-mediated events, but the molecular mechanisms linking c-Cbl to cytoskeletal rearrangements remain to be elucidated. Our previous results indicated that c-Cbl facilitates spreading and migration of v-Abl-transformed NIH 3T3 fibroblasts and suggested that small GTPases play important roles in the cytoskeletal effects of c-Cbl in this system. To elucidate the individual contributions of small GTPases to these effects, we assessed the roles of endogenous Rac1, RhoA and Rap1 in the c-Cbl-dependent spreading and migration of v-Abl-transformed fibroblasts overexpressing c-Cbl, using RNAi. Furthermore, since it has been shown that Rap1 can act as an upstream regulator of Rac1 in inducing cell spreading, we analyzed the interplay between Rap1 and Rac1 in the signaling pathways connecting c-Cbl to the cytoskeletal events. Our results indicate that Rac1 is essential for cell migration and spreading, whereas activation of RhoA exerts a negative effect. We have also shown that Rap1 is essential for cell spreading, although not for migration in our experimental system. Furthermore, we provide evidence that Rap1 is located upstream of Rac1 in one of the signaling pathways that regulate c-Cbl-facilitated cell spreading. Overall, our findings are consistent with the model describing the connection of c-Cbl to the cytoskeletal rearrangements via two pathways, one of which is mediated by PI3K and Rac1, and the other, by CrkL/C3G, Rap1 and Rac1. A major biological feature of glioma is the ability to invade normal brain tissue. The molecular mechanisms of glioma invasion are involved in multiple biological processes which are primarily associated with cytoskeleton-mediated events including adhesion, migration, degradation of extra cellular matrix (ECM). Biological functions of c-Cbl in glioma have not been elucidated. In this study, we examined biological roles of c-Cbl using RNAi-mediated depletion of endogenous c-Cbl and stably c-Cbl expressing glioma cells generated by lentiviral transduction and showed that c-Cbl increases invasion through degradation of ECM by upregulation of MMP2 but not through migration, adhesion, or growth of SNB19, a grade IV glioblastoma cell line. / Microbiology and Immunology / Accompanied by two .avi videos

Tableau d’honneur de la Faculté des études supérieures et postdoctorales, 2007-2008. / La mort cellulaire programmée assure l'élimination des cellules représentant une menace pour l'organisme. Des lésions génétiques permettant de résister à la mort par apoptose contribuent à la progression tumorale et à la résistance aux thérapies anticancéreuses classiques. Les travaux présentés dans cette thèse visent une meilleure compréhension des programmes alternatifs de mort cellulaire agissant efficacement dans les cellules tumorales. La protéine E4orf4 de l'adénovirus active un tel programme indépendant de p53 et de l'activité des caspases. Des données suggèrent que E4orf4 redirige l'activité oncogénique de Src vers la réorganisation de l'actine au détriment des voies de survie cellulaire. L'objectif de cette thèse est d'étudier la contribution de la dynamique de l'actine dans la mort cellulaire induite par E4orf4. Les résultats indiquent que E4orf4 induit deux programmes alternatifs de mort cellulaire selon sa localisation dans les cellules transformées. E4orf4 induit un programme indépendant de Src à partir du noyau et un programme dépendant de Src et de l'activité des calpaïnes à partir d'un compartiment membranaire. Une étude approfondie du programme dépendant de Src a révélé que la translocation de E4orf4 dans le cytoplasme et les membranes active la myosine II et stimule une nouvelle polymérisation de l'actine, laquelle est associée avec le recrutement et l'ancrage des endosomes de recyclage dans une région juxtanucléaire. Ce remodelage de l'actine requiert l'association de E4orf4 avec Src et une activation locale des voies de signalisation des petites GTPases RhoA/Rho kinase, Racl et Cdc42/N-Wasp. Certains résultats suggèrent que la présence de E4orf4 dans les radeaux lipidiques pourrait favoriser le recrutement de la machinerie de régulation de l'actine par la production de PIP2. E4orf4 détournerait le trafic endosomal pour permettre l'assemblage d'un anneau contractile juxtanucléaire ancré à la périphérie de la cellule par des fibres de stress. Des résultats solides indiquent que l'activité cytotoxique de E4orf4 relève de son effet sur la dynamique de l'actine. Cette thèse propose qu'un dérèglement de la dynamique de l'actine qui interfère avec le trafic membranaire normal peut déclencher un programme de mort cellulaire alternatif à partir des voies de sécrétion.