A família das septinas caracteriza-se pela capacidade de ligar nucleotídeos de guanina e de se associarem formando filamentos. Diversas funções biológicas têm sido reportadas para esses filamentos e sua dissociação pode estar relacionada a patologias. A septina 12 humana expressa especificamente em testículos, foi identificada em filamentos que compõem o annulus do espermatozoide, cuja integridade está relacionada com a morfologia deste. Embora muitos estudos tenham sido reportados, vários aspectos das bases moleculares e fisiológicas de sua função e automontagem permanecem desconhecidos. Neste trabalho, procurou-se obter informações estruturais para o domínio de ligação ao nucleotídeo da SEPT12 (SEPT12G), do mutante SEPT12GT89M e do heterodímero SEPT7-SEPT12. A expressão destas proteínas foi realizada em células de E. coli da linhagem Rosetta(DE3) pelos vetores de expressão pET28a(+) e pETDuet-1. As etapas de purificação foram cromatografia de afinidade e exclusão molecular. A proteína SEPT12G foi submetida à avaliação do estado oligomérico, fluorescência intrínseca, ensaios de conteúdo de nucleotídeo, atividade GTPásica e transição térmica. O heterodímero SEPT7-SEPT12 foi submetido à avaliação do estado oligomérico e conteúdo de nucleotídeo. Ensaios de cristalização foram realizados para todas as proteínas. A coleta de dados realizada na linha I24 do Diamond Light Source (Didcot, Inglaterra) resultou em conjuntos de dados de alta resolução para a SET12G, SEPT12GT89M e baixa resolução para a SEPT7NGc. Os estudos biofísicos mostraram que a SEPT12G foi obtida em sua forma nativa ou, seja, capaz de ligar e hidrolisar o nucleotídeo GTP e que o heterodímero obtido apresentou ambas as proteínas. As estruturas cristalográficas foram resolvidas e permitiram realizar observações importantes para o grupo I das septinas humanas (SEPT3, SEPT9 e SEPT12). Para a SEPT12 pôde-se observar como a mudança que ocorre no motivo G4 pode alterar a estabilidade da interface G. No contexto do grupo I esta estrutura permitiu concluir que todas as proteínas deste subgrupo podem formar duas interfaces NC, dos tipos aberta e fechada. Além disso, reforçou a observação da orientação diferencial da hélice α5\', cuja função ainda não está esclarecida, mas sem dúvidas é um diferencial que caracteriza este grupo, possivelmente relacionado com a ancoragem da região polibásica na conformação aberta. A estrutura cristalográfica do mutante SEPT12T89M permitiu observar que a mutação levou a uma alteração na primeira esfera de coordenação do íon Mg2+, mudança que interrompe o mecanismo do switch universal e deixa a proteína catalíticamente inativa. Por fim, o estudo cristalográfica do complexo entre a SEPT12 e SEPT7 não foi possível, uma vez que todas as tentativas resultaram em cristais contendo apenas a SEPT7, o que pode ser consequência da precipitação da SEPT12 ou da condição de cristalização utilizada, que desestabiliza o heterodímero. / The septin family of proteins is characterized by their ability to bind guanine nucleotides and associate into filaments. Several biological functions have been reported for these filaments and their dissociation may be related to pathologies. Human septin is 12 specifically expressed in testes and has been identified in filaments that form the sperm annulus, whose integrity is related to its morphology. Although many studies have been reported, the molecular and physiological bases of septin filament function and self-assembly have yet to be completely elucidated. This study aims to obtain structural information for the nucleotide binding domain of SEPT12 (SEPT12G), the SEPT12GT89M mutant and the SEPT7-SEPT12 heterodimer. Expression of these proteins was performed in E. coli Rosetta(DE3) strain using the pET28a (+) and pETDuet-1 expression vectors. Purification was performed by affinity and size exclusion chromatography. The SEPT12G protein was submitted to an evaluation of its oligomeric state, intrinsic fluorescence, nucleotide content, GTPase activity and thermal transition. The oligomeric state and nucleotide content of SEPT7-SEPT12 was also evaluated. Crystallization assays were performed for all proteins. Data collection on line I24 of the Diamond Light Source (Didcot, England) resulted in high-resolution data sets for SET12G and SEPT12GT89M but only low resolution data for the SEPT7NGc. Biophysical studies showed that SEPT12G was obtained in its native form or, in other words, capable of binding and hydrolyzing GTP and that the purified heterodimer presented both proteins. The crystallographic structures were solved by molecular replacement allowing the identification of features characteristics of the group I septins (SEPT3, SEPT9 and SEPT12). The structure also confirmed that all the proteins of this group are able to form two different NC interfaces: open and closed. In addition, it reinforced the observation that the α5\' helix assumes a different orientation, whose function has not yet been clarified, but without doubt is a characteristic of this group which may be related to anchoring the polybasic regions whilst in the open conformation. The SEPT12T89M mutant crystal structure shows that the first shell coordination of the Mg2+ ion is altered, leading to an interruption of the universal switch mechanism and a consequent lack of catalytic activity. Finally, structural studies of the interaction between SEPT12 and SEPT7 were not possible, since all attempts resulted in crystals containing only SEPT7. This may be a consequence of SEPT12 precipitation or the crystallization condition used, destabilizing the heterodimeric interface.

La protéine Rnd3 est un membre atypique de la famille des Rho GTPases. Dénuée d’activité GTPasique, elle est ainsi constitutivement sous forme active et liée au GTP. La régulation de cette protéine ne passe donc pas par le cycle classique des Rho GTPases mais par d’autres mécanismes transcriptionnels, post-transcriptionnels ou encore traductionnels. Dans le carcinome hépatocellulaire (CHC), Rnd3 est significativement sous-exprimée, et cette sous-expression procure un avantage invasif aux hépatocytes. Ce projet de thèse avait pour objectif de déterminer plus précisément les mécanismes à la base de la régulation de Rnd3 dans les cellules tumorales, et notamment les cellules dérivées de carcinome hépatocellulaire. Les travaux de cette thèse ont été divisés en deux axes principaux. Dans une première partie, la régulation de Rnd3 par la β-caténine a été étudiée. En effet, la β-caténine est retrouvée mutée dans plus d’un tiers des CHC, et la présence de mutations activatrices de la β-caténine corrèle avec un faible niveau d’expression de Rnd3 dans les CHC. L’établissement d’un modèle original dans les cellules de CHC, HepG2, a permis d’étudier indépendamment l’implication de la β-caténine sauvage et la β-caténine mutée dans la régulation d’expression de Rnd3. Ce modèle a permis de mettre en évidence une régulation différentielle de Rnd3 par les deux formes de la β-caténine, la forme sauvage régulant Rnd3 au niveau transcriptionnel, et la forme mutée régulant Rnd3 au niveau post-transcriptionnel. La deuxième partie de ce travail, qui constitue la partie principale du projet, s’est intéressée à la régulation de Rnd3 par la voie de mécanotransduction MRTF/SRF. L’activation de cette voie de signalisation est très intimement reliée à l’organisation du cytosquelette d’actine, et cette voie régule en retour l’expression de nombreux gènes impliqués dans la dynamique de l’actine. Les résultats obtenus ont permis de déterminer Rnd3 comme une nouvelle cible directe de la voie MRTF/SRF dans les cellules tumorales, et placent Rnd3 au centre d’une boucle de régulation de cette voie de mécanotransduction. L’ensemble des résultats obtenus au cours de ce projet de thèse ont permis de mieux caractériser la régulation de l’expression de Rnd3 dans les cellules tumorales. / Rnd3 protein is an atypical member of the Rho GTPase family, devoid of GTPase activity and constitutively active and bound to GTP. Rnd3 regulation does not occur through the classical GTPase cycle but is achieved at transcriptional, posttranscriptional or translational level. Rnd3 is underexpressed in hepatocellular carcinoma (HCC), and this down-regulation increases HCC cell invasion and is linked to HCC progression. The aim of this thesis project was to better decipher the mechanisms involved in Rnd3 expression in tumor cells, and particularly in HCC cells. In a first part, Rnd3 regulation by β-catenin was studied. β-catenin is found mutated in one third of HCC, and activating β-catenin mutations in human HCC correlates with the lowest levels of Rnd3. An original model established in HepG2 cells allowed the study of the involvement of WT β-catenin versus mutated β-catenin in the regulation of Rnd3 expression.Interestingly, our results demonstrated that both forms of ß-catenin independently regulate Rnd3 mRNA expression. The WT β-catenin regulates Rnd3 at the transcriptional level, whereas the mutated β-catenin acts through the 3’UTR of Rnd3 mRNA. The second and main part of this thesis project was the study of the regulation of Rnd3 expression by the mechanotransduction pathway MRTF/SRF. The activation of this signaling pathway is tightly regulated by actin cytoskeleton, and the MRTF/SRF pathway directs in return the expression of a huge number of genes involved in actin dynamics. Our results uncovered Rnd3 as a new direct target of MRTF/SRF pathway in tumor cells. Indeed, upon actin dynamics changes, MRTF/SRF is able to bind Rnd3 promoter in order to favor its expression. As Rnd3 also acts as a regulator of the actin cytoskeleton, our results highlight Rnd3 at the center of a feedback loop of the MRTF/SRF mechanotransduction pathway. Taken together, all of the results obtained helped to better decipher the mechanisms of Rnd3 regulation in tumor cells.

Durant la division cellulaire, la cellule subit des changements importants dans sa forme et son adhésion qui dépendent de l'efficacité du remodelage du cytosquelette d'actine. Ce processus est localement et temporellement régulé pour assurer le bon déroulement de la cytokinèse, l'étape finale de la division cellulaire. Il est contrôlé par les GTPases de la famille Rho via le remodelage du cytosquelette d'actine. Les Rho-GTPases fonctionnent comme des interrupteurs moléculaires, passant d'une forme au repos (liée au GDP) à une forme active (liée au GTP). La forme au repos interagit avec des facteurs d'échange, les GEFs (Guanine nucleotide Exchange Factors) qui déplacent le GDP et permet la fixation du GTP. Le retour à la forme inactive se fait par hydrolyse du GTP en GDP, stimulée par les protéines GAPs (GTPase Activating Proteins). RhoA est un régulateur positif de la cytokinèse, activée spécifiquement à l'équateur de la cellule, et qui promeut l'assemblage et la constriction de l'anneau d'actomyosine. En contraste, Rac1 a été proposée pour réguler négativement ce processus et doit être inactivée spécifiquement à l'équateur de la cellule pour le bon déroulement de la cytokinèse. Ainsi, une GAP de Rac1, MgcRacGAP, qui est localisé sur le fuseau central de microtubules, inactive Rac1 à l'équateur de la cellule. La déplétion de MgcRacGAP induit des défauts de cytokinèse qui peuvent être sauvés en co-déplétant Rac1. Cependant, le Rho-GEF activant Rac1 durant la division cellulaire n'a pas encore été identifié. Pour identifier un GEF régulant l'activité de Rac1 dans les cellules en division, nous avons réalisé une approche de « screening » par siRNA dans les cellules HeLa. Les Rac-GEFs sont déplétés par siRNA seul ou en combinaison avec un siRNA ciblant MgcRacGAP, dans le but d'identifier lesquels sont capables de sauver le nombre de cellules multinuclées induit par la déplétion de MgcRacGAP. De façon intéressante, la co-déplétion de MgcRacGAP et du Rho-GEF Trio, un GEF caractérisé principalement pour son rôle dans la croissance et le guidage axonal, entraîne une forte diminution du nombre de cellules multinuclées. Par la suite, nous démontrons que ce sauvetage du phénotype passe par la voie Trio-Rac1 en utilisant des mutants GEFs inactifs de Trio et un inhibiteur spécifique de l'activation de Rac1 par Trio. Ces résultats et le rôle de MgcRacGAP dans l'inactivation de Rac1 en cytokinèse, suggèrent que la déplétion de Trio pourrait sauver les défauts de cytokinèse induits par la déplétion de MgcRacGAP en diminuant l'activité de Rac1. Cela suggère aussi que Trio pourrait être un GEF de Rac1 dans les cellules en division. Pour directement tester si Trio pouvait fonctionner comme un GEF de Rac1 dans les cellules en division, la quantité de Rac1 a été mesurée par « pull-down assay » dans des cellules synchronisées en mitose. Comparé aux cellules traitées avec un siRNA contrôle, la déplétion de Trio réduit de moitié la quantité de Rac1 activée dans les cellules en mitose, démontrant que Trio active Rac1 en mitose. De plus, la déplétion de Trio induit des défauts de remodelage du cytosquelette d'actine dans les cellules en anaphase. De façon intéressante, la déplétion de Trio phénocopie la déplétion de Rac1 et de son effecteur Arp2/3, en accord avec un rôle de la voie Trio-Rac1 dans le contrôle du remodelage du cytosquelette d'actine dans les cellules en division. L'ensemble de ce travail a permis d'identifier pour la première fois un GEF contrôlant l'activité de Rac1 dans les cellules en division dont l'activité s'oppose à la fonction de MgcRacGAP en cytokinèse. Nous proposons ainsi un modèle dans lequel Trio contrôle l'activation de Rac1 et le remodelage du cytosquelette d'actine au cortex cellulaire dans les cellules en division. Dans notre modèle, MgcRacGAP s'oppose à l'action de Trio en inhibant localement et temporellement l'activation de Rac1 au plan de division, assurant ainsi le bon déroulement de la cytokinèse. / During cell division, cells undergo dramatic changes in shape and adhesion that depend on efficient actin cytoskeleton remodeling. This process has to be locally and temporally regulated to accurately ensure cytokinesis, the final stage of cell division. The small GTPases Rac1 and RhoA play an essential role in this process by controlling F-actin cytoskeleton remodeling. GTPases oscillate between an inactive, GDP-bound state and an active, GTP-bound state. They are activated by Guanine-nucleotide Exchange Factors (GEFs), which stimulate the GDP-to-GTP exchange, while they are turned off by GTPase-Activating Proteins (GAPs) which catalyse the hydrolysis of GTP. RhoA is a positive regulator of cytokinesis specifically activated at the division plane, which promotes the assembly and constriction of the actomyosin network. In contrast, Rac1 has been proposed to negatively regulate this process and has to be inactivated at the division plane for cytokinesis to occur properly. A central spindle localized GAP, MgcRacGAP, component of the centralspindlin complex, controls Rac1 inactivation at the cleavage plane. Depletion of Rac1 can suppress the cytokinesis failure induced by MgcRacGAP depletion. However, the Rho-GEF that activates Rac1 during cell division has not been identified yet. To identify a GEF regulating Rac1 activity in dividing cells, we performed a siRNA screening approach in HeLa cells. Rac-GEFs were depleted by siRNA alone or in combination with MgcRacGAP siRNAs, in order to identify the ones able to rescue the multinucleated cells induced by MgcRacGAP depletion. Importantly, co-depletion of MgcRacGAP and Rho-GEF Trio, a GEF characterized primarily for its role in axon outgrowth and guidance resulted in a strong decrease in the number of multinucleated cells. Then, we demonstrate that this rescue is mediated by the Trio-Rac1 pathway, using GEF dead mutants of Trio and a specific inhibitor of Rac1 activation by Trio. These data and the fact that MgcRacGAP was recently described to be essential for Rac1 inactivation in cytokinesis, suggest that Trio depletion could rescue the cytokinesis failure induced by MgcRacGAP depletion by decreasing Rac1 activity. It therefore suggests that Trio could be a GEF of Rac1 in dividing cells. To directly test if Trio could function as a GEF of Rac1 in dividing cells, the amount of activated Rac1 was monitored by pull down assay in synchronized mitotic cells. Compared to control siRNA-treated cells, Trio depletion reduced by half the amount of activated Rac1 in mitotic cells, showing that Trio activates Rac1 in mitosis. Strikingly, Trio depletion led to defects in F-actin cytoskeleton remodeling in anaphase cells. Indeed, the F-actin staining at the cortex was significantly reduced in Trio-depleted cells compared to control cells. Interestingly, Trio depletion phenocopied the depletion of Rac1, consistent with a role for the Trio-Rac1 pathway in controlling F-actin remodeling in dividing cells.Overall, this work identifies for the first time a GEF controlling Rac1 activation in dividing cells that counteracts MgcRacGAP function in cytokinesis. Based on these observations, we propose a model in which Trio functions as a GEF of Rac1 during cell division. Trio, which is expressed throughout the cell cycle, activates Rac1 to control F-actin cytoskeleton remodeling at the cell cortex of dividing cells. MgcRacGAP therefore counteracts the action of Trio by locally and temporally inhibiting Rac1 activation at the division plane, subsequently ensuring accurate cytokinesis.

Les petites GTPases de la famille Arf sont des régulateurs majeurs du trafic cellulaire. Ils participent à presque tous les aspects de ce processus. Il existe cinq familles d'ArfGEFs chez les eucaryotes, responsables de l'activation des Arfs au sein des membranes. Ces Arfs GEFs partagent un domaine Sec7 catalytique conservé mais aussi des domaines de régulation qui interviennent soit dans des interactions avec des protéines ou des interactions avec des membranes. L'un des moyens d'étudier la fonction des Arfs et leur régulation par les ArfGEFs repose sur l'utilisation de l'inhibition par de petiotes molécules chimiques. La BFA est le premier inhibiteur d'ArfGEF à avoir été identifié et a permis d'identifier de nombreuses fonctions du trafic. D'autres composés ont été identifiés par différentes méthodes de criblage mais leur spécificité in vitro reste peu caractérisée, ce qui peut être un obstacle à une bonne interprétation des observations que l'on en fait en Biologie cellulaire. L'objectif de ce travail a été d'évaluer in vitro la spécificité de chaque petite molécule sur un ensemble de famille d'ArfGEFs en solution et pour la première en présence de membranes artificielles. / Small GTPases of the Arf family are major regulators of almost every aspect of membrane traffic in cells. In eukaryotes, five subfamilies of guanine nucleotide exchange factors (ArfGEFs) activate them on different membranes by combining a conserved Sec7 domain, which stimulate the GDP/GTP exchange, and distinct regulatory and membrane binding domains (reviewed in [1]). The identification of the natural compound Brefeldin A as the first known GEF inhibitor, which inhibits Arf functions at the Golgi, established the Arf machinery as model systems to investigate the druggability of small GTPases and their GEFs in diseases. Chemical compounds of unrelated structure have been reported by us and others to inhibit ArfGEF subfamilies. Some of them, recently discovered, remain poorly characterized in vitro, which hampers their use as relevant biological tools. Here, we compared the efficiency and specificity of these inhibitors towards representative members of the major ArfGEF subfamilies using highly purified recombinant proteins reconstituted in artificial membranes.

No description available.

No description available.

Engineered protein systems have been created to impart new functions, or “re-program” mammalian cells for applications including cancer and HIV/AIDS therapies. The successful development of mammalian cells for re-programming will depend on having well-defined, modular systems. Migration is a particularly important cell function that will determine the efficiency and efficacy of many re-programming applications in vivo, and Rho proteins are responsible for regulation of cell migration natively. While there have been several reports of photo-activated Rho proteins, no strategy has been developed such that Rho proteins and cell migration can be controlled by a variety of extracellular stimuli that may be compatible with signaling in large organisms. Here, several methods are described for engineering Ca2+-sensitive Rho proteins so that the large, natural toolbox of Ca2+-mobilizing proteins can use the Ca2+ intermediate to activate Rho proteins in response to a variety of exogenous stimuli, including chemicals, growth factors, and light. First, an unreported calmodulin binding site was identified in RhoA. This knowledge was used to create a tandem fusion of RhoA and calmodulin that mediated Ca2+-sensitive bleb retraction in response to a variety of Ca2+-elevating chemicals. Ca2+-mobilizing modules including channelrhodopsin-2 and nicotinic acetylcholine receptor α4 were used for light- and acetylcholine-dependent bleb retraction. Second, a more robust morphology switch was created by embedding a calmodulin binding site into RhoA to enable Ca2+-responsive bleb formation. A wider range of Ca2+-mobilizing modules were also used here including LOVS1K/Orai1 and vascular endothelial growth factor 2. Combining Ca2+-mobilizing and Ca2+-responsive modules increased amoeboid-like cell migration in wound closure and transwell assays. Finally, the embedded peptide design was applied to Rac1 and Cdc42 to enable control of new morphologies and migration modes. The modular Ca2+ control over Rho proteins developed here is an important contribution to cell re-programming because it shows that control over cell migration can be rewired in a way that is flexible and tunable.

Thesis (M. Phil.)--University of Hong Kong, 2010. / Includes bibliographical references (leaves 77-100). Also available in print.

Engineered protein systems have been created to impart new functions, or “re-program” mammalian cells for applications including cancer and HIV/AIDS therapies. The successful development of mammalian cells for re-programming will depend on having well-defined, modular systems. Migration is a particularly important cell function that will determine the efficiency and efficacy of many re-programming applications in vivo, and Rho proteins are responsible for regulation of cell migration natively. While there have been several reports of photo-activated Rho proteins, no strategy has been developed such that Rho proteins and cell migration can be controlled by a variety of extracellular stimuli that may be compatible with signaling in large organisms. Here, several methods are described for engineering Ca2+-sensitive Rho proteins so that the large, natural toolbox of Ca2+-mobilizing proteins can use the Ca2+ intermediate to activate Rho proteins in response to a variety of exogenous stimuli, including chemicals, growth factors, and light. First, an unreported calmodulin binding site was identified in RhoA. This knowledge was used to create a tandem fusion of RhoA and calmodulin that mediated Ca2+-sensitive bleb retraction in response to a variety of Ca2+-elevating chemicals. Ca2+-mobilizing modules including channelrhodopsin-2 and nicotinic acetylcholine receptor α4 were used for light- and acetylcholine-dependent bleb retraction. Second, a more robust morphology switch was created by embedding a calmodulin binding site into RhoA to enable Ca2+-responsive bleb formation. A wider range of Ca2+-mobilizing modules were also used here including LOVS1K/Orai1 and vascular endothelial growth factor 2. Combining Ca2+-mobilizing and Ca2+-responsive modules increased amoeboid-like cell migration in wound closure and transwell assays. Finally, the embedded peptide design was applied to Rac1 and Cdc42 to enable control of new morphologies and migration modes. The modular Ca2+ control over Rho proteins developed here is an important contribution to cell re-programming because it shows that control over cell migration can be rewired in a way that is flexible and tunable.

Thesis (Ph.D.)--University of California, San Francisco, 2004. / Includes bibliographical references. Also available online.