La sécrétion d'hormones et de neuropeptides par les cellules neuroendocrines est assurée par un processus d'exocytose, contrôlé notamment par les GTPases Rho. La compréhension des mécanismes moléculaires régulant la sécrétion neuroendocrine est primordiale. En effet, la majorité des cancers neuroendocrines tels que les phéochromocytomes, sont associés à une perturbation du processus de sécrétion. Actuellement, les mécanismes moléculaires qui induisent de telles perturbations de la sécrétion ainsi que l’impact de l’activité sécrétrice sur le développement des tumeurs neuroendocrines ne sont pas élucidés. Mes travaux de thèse proposent pour la première fois un lien fonctionnel direct entre l'activité sécrétrice des cellules et la vitesse de développement des phéochromocytomes ainsi qu’une altération des voies moléculaires impliquant certaines protéines Rho, en démontrant un lien entre la baisse de l’activité de Rac1 et Cdc42 observée dans les phéochromocytomes et la diminution de l’expression de leurs régulateurs ARHGEF1 et FARP1. / Neuroendocrine cells secrete hormones and neuropeptides through calcium-regulated exocytosis, controlled especially by Rho GTPases. Neuroendocrine tumours, such pheochromocytomas, are generally associated with a dysfunction of secretion. Although this aspect is well known by clinicians, it has never been explored at the molecular level. Moreover, the potential link between secretion and tumour development remains uninvestigated. Altogether, our results demonstrate for the first time the importance of secretion in tumor development of pheochromocytomas and an alteration of the Rho GTPase pathway, by demonstrating a link between the inhibition of Rac1 and Cdc42 activity observed in pheochromocytomas and the decrease of their activators ARHGEF1 and FARP1 expression.

O Corpo Ciliar (CC) é uma fonte de células tronco da retina de animais adultos, mas sua ativação permanece desconhecida. GTPases Rho são proteínas que reorganizam do citoesqueleto de actina, regulam vias de sinalização e transcrição gênica, sobrevivência celular e proliferação. Neste trabalho, investigamos a expressão das GTPases Rho nas células do CC e seu efeito na regulação do ciclo celular. As GTPases RhoA, RhoB e Rac1 foram expressas nas células do CC e sua ativação pelo ácido lisofosfatidico (LPA) aumentou a expressão dos genes progenitores retinianos Pax6 e Chx10. A inibição das proteínas por Toxina A de Clostridium difficile aumentou a proliferação no CC e potencializou o efeito proliferativo dos fatores de crescimento. A inibição especifica destas proteínas diminuiu a expressão dos transcritos de p21cip, p27kip, p16INK4a e p19INK4d e aumentou de Ki67, CiclinaA e D1. O estudo da via de Wnt indicou que Rac1 regulou os genes de componentes da degradação de -catenina e Lef1. Concluímos que a inativação das GTPases Rho induziu a proliferação das células progenitoras retinianas localizadas no CC e regulou a via de Wnt. Sua ativação induziu o perfil de célula progenitora, sugerindo uma nova ferramenta para o mecanismo de reparo retiniano. / Ciliary Body (CB) is a potential source of stem cells in the adult retina, but its activation is still unknown. Rho GTPases play a role in actin-based cytoskeleton reorganization, regulate signaling pathways and gene transcription, cell survival and cell proliferation. In this study we investigated the expression of Rho GTPases in CB cells and their role on cell cycle regulation. The GTPases RhoA, RhoB and Rac1 were present in CB cells and the activation by lysophosphatidic acid (LPA) increased the expression of the progenitor genes Pax6 and Chx10. The inhibition by Clostridium difficile Toxin A increased the proliferation of CB cells and potentiated the proliferative effect of growth factors. The specific inhibition decreased the expression of p21cip, p27kip, p16INK4a and p19INK4d as well as increased Ki67, cyclinA and D1 transcripts. The Wnt pathway study indicated that Rac1 regulated -catenin degradation genes components and Lef1. Taken together, the inactivation of Rho GTPases stimulated the proliferation of progenitor cells located in CB as well as regulate the Wnt signaling pathway. The proteins activation was correlated to progenitor profile induction. These different mechanisms may provide a potential new approach on retinal repair.

Notre système immunitaire détecte les microorganismes via des molécules absentes de l’hôte appelées MAMPs. Mais étant donné que les MAMPs sont exprimés par tous les microorganismes indépendamment de leur potentiel pathogène, ce mécanisme n’explique pas comment le système immunitaire distingue les microorganismes pathogènes des non-pathogènes. De récents travaux ont mis en évidence un mécanisme de détection de l’activité des facteurs de virulence bactériens. En utilisant la drosophile, notre laboratoire a précédemment démontré que l’activation de la Rho GTPase Rac2 par la toxine CNF1 d’Escherichia coli induisait une réponse immunitaire innée conservée au cours de l’évolution chez le mammifère et similaire à l’immunité induite par les effecteurs chez la plante. Par la suite, nous avons évalué l’importance de cette réponse immunitaire au cours de la bactériémie chez la souris et démontré le rôle central de la cytokine IL-1β dans l’élimination des bactéries en réponse à la détection de CNF1. Des expériences in vitro nous ont permis d’identifier les mécanismes moléculaires mis en jeu et l’inflammasome responsable de l’activation de la caspase-1 et du clivage de l’IL-1β. De manière intéressante, CNF1 est toujours co-exprimée avec la toxine hémolysine-α (HlyA) dans les souches pathogènes d’E. coli. En outre, nous avons découvert que l’HlyA bloquait l’élimination des bactéries induite par CNF1 au cours de la bactériémie et inhibait la sécrétion de l’IL-1β. Ici, nous avons rapporté le premier exemple d’immunité induite par une toxine (CNF1) et contrecarrée par une autre (HlyA). / Our immune system detects microorganisms via molecules absent from the host called MAMPs. Since MAMPs are shared by all microorganisms regardless of their pathogenic potential, this mechanism does not explain how the immune system distinguishes between pathogenic and non pathogenic microorganisms. The detection of the activities of pathogen-encoded virulence factors has emerged as a new paradigm of pathogen recognition. Using Drosophila we previously demonstrated that the Escherichia coli CNF1 toxin-induced activation of the Rho GTPase Rac2 is sufficient to initiate a defense signal evolutionarily conserved from flies to mammals and similar to Effector-Triggered Immunity in plants. We further addressed the importance of this innate immune mechanism during bacteremia in mice and demonstrated the central role of the IL-1β cytokine in the clearance of bacteria in response to the detection of CNF1. In vitro experiments allowed us to identify the involved molecular mechanisms and the inflammasome responsible of caspase-1 activation and IL-1β maturation. Interestingly, CNF1 is always co-expressed with α-hemolysin toxin in pathogenic E. coli. In addition, we found that HlyA blocked the elimination of bacteria induced by CNF1 during bacteremia and inhibited the secretion of IL-1β. Here, we have reported the first example of immunity induced by a toxin (CNF1) and counteracted by another (HlyA).

La progression métastatique des cancers est responsable de 90% des décès liés à la maladie. Cette cascade est initiée par l’invasion des cellules cancéreuses du stroma péritumoral, et conduit à leur dissémination dans l’organisme.Mon travail de thèse a eu pour but d’identifier les mécanismes moléculaires et cellulaires régulant l’invasion des cancers colorectaux (CRC), qui est le 2ème cancer le plus répandu dans le monde. Grâce à une analyse réalisée sur des échantillons humains de tumeurs primaires, nous avons révélés que les cellules de CRC utilisent un mode d’invasion collective dans lequel elles gardent une architecture glandulaire spécifique des épithelia. Afin d’étudier les voies de signalisation régulant cette invasion, nous avons utilisé des modèles organotypiques récapitulant l’architecture des glandes de CRC (cystes de Caco-2 et tumoroïdes de xénogreffes derivés de patients) dans des tests d’invasion utilisant du collagen-I. Du fait de son rôle central dans la régulation de la motilité cellulaire, la voie des RhoGTPases était un bon candidat à la régulation de l’invasion collective des CRC. Dans un screen utilisant des siRNA ciblant tous les effecteurs connus des RhoGTPases, seule la déplétion des protéines kinases ROCK a déclenché l’invasion collective dans notre système expérimental. Nous avons démontré que l’inhibition de ROCK2 et non de ROCK1 était suffisante pour induire la formation de cellules leader, permettant la polarisation leader/follower requise pour l’invasion collective. Nos résultats montrent que l’inhibition de ROCK2 déclenche l’invasion collective par l’inhibition de MyosinII combinée à l’activation du facteur d’échange nucléotide guanine (GEF), FARP2, et de RAC1. Notre étude permet donc d’identifier FARP2 comme un nouvel effecteur de ROCK2 et le positionne comme un nouveau médiateur du crosstalk entre RhoA et RAC1 dans la régulation de l’invasion collective des CRC. En conclusion, nous avons décrit une nouvelle voie de signalisation dépendante de ROCK2 contrôlant l’invasion collective de glandes de CRC. De façon intéressante, notre étude révèle un rôle anti-invasive de ROCK2 contredisant son rôle pro-invasive décrit dans l’invasion de cellules individuelles. Cela suggère que ROCK2 assure des rôles distincts en fonction du mode d’invasion adopté par les cellules cancéreuses et remet en question le bénéfice thérapeutique de l’inhibition de ROCK proposé pour bloquer l’invasion des cellules cancéreuses. / Metastatic progression of cancer is responsible for 90% of the disease related death. It is a multi-step process which is initiated by invasion of the peritumoral stroma by cancer cells and which leads to the dissemination of cancer cells in the organism.My PhD work aimed at identifying the molecular and cellular process driving colorectal carcinoma (CRC) invasion, which is the 2nd most frequent cancer worldwide. Our analysis of live and human primary cancer specimen revealed that CRC cells used a collective mode of invasion to disseminate, in which cells retain an epithelium specific -glandular architecture. To investigate the signaling pathways regulating this mode of invasion, we used 3D organotypic models recapitulating the features of CRC glands (Caco-2 cysts and Patient derived Xenografts (PDX) tumoroids) in collagen-I based organotypic invasion assays and in microscopy-based analyses. Because of its central role in the regulation of cell motility, we postulated that RhoGTPases signaling pathways could control the collective of CRC. In a siRNA based- screen targeting all the known effectors of RhoGTPases we found that only ROCK kinases downregulation induced collective invasion in our experimental settings. We demonstrated that ROCK2 but not ROCK1 inhibition was sufficient to promote Leader cell formation, which induced the leader/follower polarization necessary for collective invasion. Our results revealed that ROCK2 inhibition triggered collective invasion through the concomitant inhibition of MyosinII and activation of the guanine nucleotide exchange factor (GEF) FARP2 and the RhoGTPase RAC1. We therefore identify FARP2 as a new effector of ROCK2 and a mediator of the RhoA-RAC1 crosstalk in the regulation of collective invasion. In conclusion our study proposes a new ROCK dependent-signaling pathway in the regulation of collective invasion of highly polarized CRC glands models. Importantly, we found ROCK2 to be an anti-invasive protein which is in contradiction with its described pro-invasive role in single cell invasion. This suggests distinct roles of ROCK which may depend on the mode of invasion adopted by the cells and questions the benefice of proposed ROCK inhibition strategies to block cancer cell invasion.

Indiana University-Purdue University Indianapolis (IUPUI) / Knowledge of the molecular and cellular mechanisms of ischemic injury is necessary for understanding acute kidney injury and devising optimal treatment regimens. The cortical actin cytoskeleton in the proximal tubule epithelial cells of the kidney nephron, playing an important role in both the establishment and maintenance of cell polarity, is drastically disrupted by the onset of ischemia. We found that in LLC-PK cells (a porcine kidney proximal tubule epithelial cell line), cortactin, an important regulator of actin assembly and organization, translocated from the cell cortex to the cytoplasmic regions upon ischemia/ATP-depletion. Meanwhile both the tyrosine phosphorylation level of cortactin and cortactin’s interaction with either F-actin or the actin nucleator Arp2/3 complex were down-regulated upon ischemia/ATP-depletion or inhibition of Src kinase activity. These results suggest that tyrosine phosphorylation plays an important role in regulating cortactin’s cellular function and localization in the scenario of kidney ischemia. The Rho GTPase signaling pathways is also a critical mediator of the effects of ATP depletion and ischemia on the actin cytoskeleton, but the mechanism by which ATP depletion leads to altered RhoA and Rac1 activity is unknown. We propose that ischemia and ATP depletion result in activation of AMP-activated protein kinase (AMPK) and that this affects Rho GTPase activity and cytoskeletal organization (possibly via TSC1/2 complex and/or mTOR complex). We found that AMPK was rapidly activated (≤5 minutes) by ATP depletion in S3 epithelial cells derived from the proximal tubule in mouse kidney, and there was a corresponding decrease in RhoA and Rac1 activity. During graded ATP-depletion, we found intermediate levels of AMPK activity at the intermediate ATP levels, and that the activity of RhoA and Rac1 activity correlated inversely with the activity of AMPK. Activation of AMPK using two different drugs suppressed RhoA activity, and also led to morphological changes of stress fibers. In addition, the inhibition of AMPK activation partially rescued the disruption of stress fibers caused by ATP-depletion. This evidence supports our hypothesis that the activation of AMPK is upstream of the signaling pathways that eventually lead to RhoA inactivation and cytoskeletal dysregulation during ATP-depletion.

Neutrophils migrate rapidly towards a site of inflammation and mediate bacterial killing through highly regulated pathways that involve the phagocytosis of bacteria and the generation of reactive oxygen species by the NADPH oxidase complex. The Rac small GTPases have prominent roles in the regulation of neutrophil signaling pathways but the research strategies used to analyze their functions in live cells have been limited, since neutrophils are terminally differentiated and difficult to manipulate genetically. In this thesis, I describe a novel high efficiency protocol for transiently transfecting neutrophils that allowed me to investigate the roles of Rac1 and Rac2 in neutrophils in a completely new way, in real time. Using this technique, I show that a bacterial protein known to inhibit chemotaxis in vitro, selectively inhibits Rac1 activation downstream of fMLP stimulation and inhibits neutrophils polarization. Further dissecting the roles of Rac isoforms, I used various approaches to show that Rac1 and Rac2 differentially regulate free-barbed end (FBE) formation downstream of the fMLP receptor. Rac1 is responsible for ~30% of FBE whereas Rac2 is the regulator of FBE formation (~70%) through the activation of cofilin and Arp2/3. Finally, these observations led to the analysis of the mechanisms underlying the Rac1 and Rac2 functions. I show that membrane charge determines Rac1 and Rac2 differential localization during phagocytosis and chemotaxis iii based on their different aminoacid residues in the polybasic domain. This mechanism depends on lipid metabolism and the accumulation of negatively charged lipids at cellular membranes. During chemotaxis, neutrophils have a polarized accumulation of negatively charged lipids at the leading edge membrane that selectively recruit Rac1. In contrast, the lipid metabolism that occurs at the phagosome membrane decreases its negativity and selectively recruits Rac2. All together, this thesis describes the study of primary neutrophil functions from a new angle and adds some valuable information to the comprehension of effective neutrophil activation based on the analysis of Rac isoforms.

Small GTPases are important proteins that affect many cellular processes. In my work I compare the five most important protein families of small GTPases - Arf, Rab, Ran, Ras and Rho to identify amino acids responsible for major functional differences between different protein families. To compare them, I have used the structural data from the PDB database and sequences from the UniProt database. I have discovered previously undescribed groups of amino acids specific for each protein family of small GTPases with the help of programs ConSurf and Sca5. I also carried out a pilot study of the applicability of B-factors as indicators of bond strength in the protein structure on the example of small GTPases. The first results are not entirely conclusive, but they do not exclude the applicability of B-factors as indicators of bond strength either. Powered by TCPDF (www.tcpdf.org)

Notch signaling controls normal and pathological angiogenesis through transcriptional regulation of a wide network of target genes. Despite intensive studies of the endothelial Notch function, a comprehensive list of Notch-regulated genes, especially direct transcriptional targets, has not been assembled in endothelial cells (ECs). Here we uncovered novel EC Notch targets that are rapidly regulated by Notch signaling using several unbiased in vivo and in vitro screening approaches that captured genes regulated within 6 hours or less of Notch signal activation. We used a gamma-secretase inhibitor in neonates to profile Notch targets in the brain endothelium using the RiboTag technique, allowing for isolation of endothelial specific mRNA from a complex tissue without disrupting cell-cell contact. We used two types of primary cultured endothelial cells to define ligand-specific Notch targets by tethered-ligand stimulation. The identified Notch targets were validated by determining their regulation within one to two hours of EGTA-mediated Notch activation. By comparing significantly regulated genes in each of the screens, we assembled a comprehensive database of potential Notch targets in endothelial cells. Of particular interest, we uncovered G protein pathway related genes as potential novel Notch targets. We focused on a novel candidate target passing selection criteria after all screens, a small GTPase RND1. RND1(Rho GTPase1) regulates cytoskeleton arrangement through Rho and Ras signaling. RND1 was validated as an endothelial Notch target in multiple endothelial cell types. In Human Umbilical Vein Endothelial Cells (HUVECs) we established angiogenic activity for RND1 that included regulation of cell migration towards VEGF and function in sprouting angiogenesis. We established that Notch and RND1 suppressed Ras activation but had no effects on Rho activation in HUVECs. These results demonstrate that RND1 expression is regulated by Notch signaling in endothelium and suggest that RND1 functions downstream of Notch in sprouting angiogenesis, revealing an unexplored role of endothelial Notch in regulating G protein pathways.

Thesis (Ph. D. in Cancer Biology)--Vanderbilt University, May 2007. / Title from title screen. Includes bibliographical references.

Upon activation by the platelet-derived growth factor receptor (PDGFR), phosphatidylinositol 3'-kinase (PI3K) converts phosphatidylinositol 4,5-bisphosphate to phosphatidylinositol 3,4,5-trisphosphate to activate the PI3K/Akt cellular survival signalling pathway within cells. The p85 subunit of PI3K has also been shown to have GTPase activating protein (GAP) activity towards Rab proteins involved in receptor endocytosis and trafficking, specifically Rab5 and Rab4. Rab5 is responsible for regulating the fusion of vesicles containing activated receptors to traffic them to intracellular early/sorting endosomes. Rab4 is responsible for regulating the exit of receptors to a recycling pathway back to the plasma membrane. The p85 RabGAP activity is responsible for deactivating Rab5 and Rab4 function by accelerating their GTPase activity, resulting in the inactive conformation of Rab5 and Rab4, and decreased vesicle fusion events during receptor trafficking. The work in this thesis was performed to understand how p85 interacts with, and regulates, Rab5 and Rab4. Glutathione S-transferase pulldown experiments showed the p85 protein was able to interact with Rab5 through its BH domain and another unidentified domain. Cells expressing a p85-R274A mutant defective for RabGAP activity displayed increased PDGFR activation and decreased degradation. To understand the mechanism of decreased PDGFR degradation, PDGFR immunoprecipitation experiments showed the PDGFR was ubiquitinated, a signal needed for multi-vesicular body sorting. Biotinylation experiments showed the PDGFR was being more rapidly endocytosed and then sequestered within the cell. Immunofluorescence experiments showed cells expressing the p85-R274A mutant clearly altered PDGFR trafficking during receptor endocytosis. These results suggest the PDGFR was not spending longer periods of time on the cell surface to continue signalling and was not lacking the modification needed to be sorted to a degradative pathway. The defective trafficking observed in p85-R274A expressing cells, over time, may block PDGFR trafficking, which prevents normal PDGFR dephosphorylation and degradation, and could be attributed to a lack of sufficient cytosolic Rab5-GDP and Rab4-GDP required to associate with new membranes and facilitate additional vesicle fusion events. The lack of lysosomal targeting allows the receptor to be sequestered in cells, but still have the ability to signal as the receptor would not be targeted to multi-vesicular bodies where signalling is abolished.