L'activation constitutive de FGFR3 par mutation ou translocation est l'un des évènements les plus fréquents dans le cancer de la vessie. Une dérégulation de RAB25,une protéine impliquée dans le processus de recyclage des récepteurs de surface, a été montrée dans différents cancers. Des données du transcriptome des cancers de vessie ont montré que RAB25 est surexprimé dans les tumeurs présentant des altérations de FGFR3. L'objectif de cette thèse a été d'étudier l'implication possible de RAB25, des protéines de la même famille, RAB11A et RAB11B, et leur effecteurs RAB11FIP2 et MYO5B dans 1) la tumorigénèse des tumeurs altérées pour FGFR3 et 2) le trafic et la signalisation de FGFR3. Nos résultats montrent que l'extinction de ces protéines par des siARNs induit une diminution significative de la viabilité cellulaire des cellules exprimant des formes constitutivement activées de FGFR3. Les effets de la déplétion de RAB25 et RAB11 sur le recyclage de FGFR3, sur les voies de signalisation de FGFR3 et sur l'expression des gènes cibles de FGFR3 suggèrent que le recyclage de FGFR3 régulé par RAB25 et RAB11 peut prolonger le signal de FGFR3 et peut fournir une plateforme pour la signalisation de FGFR3. Nous avons également comparé la distribution cellulaire des formes sauvage et muté (S249C) de FGFR3 portant une étiquette GFP dans des cellules HeLa. Les deux formes de FGFR3 se trouvent dans plusieurs compartiments intracellulaires mais FGFR3 muté se localise préférentiellement dans le compartiment de recyclage. Ce projet nous a permis de mieux caractériser la trafic de FGFR3 dans le cancer de la vessie et son lien avec la signalisation et l'activité de FGFR3. / Activation of FGFR3 by point mutation, translocation and overexpression is one of the most frequent events in bladder cancer. The dysfunction of RAB25, a GTPase involved in endocytic recycling of transmembrane receptor, has been shown in many cancers. Gene expression data in bladder cancer indicates that RAB25 expression is significant higher in tumors carrying altered FGFR3. The thesis project aimed to investigate the potential role of RAB25, proteins from the same family (RAB11A and RAB11B) and their effectors RAB11FIP2 and MYO5B in 1) the tumorigenesis of tumors carrying altered FGFR3 and 2) the trafficking and the signaling of FGFR3. Our results demonstrate that depletion of these proteins by siRNA significantly reduces cell viability in cells expressing constitutively activated forms of FGFR3. The effects of RAB25 and RAB11 silencing on FGFR3 trafficking and signaling and the expression of FGFR3 target genes suggest that the RAB11- and RAB25-mediated recycling can sustain the signaling by protecting altered FGFR3 from the degradation pathway, and can provide a platform for FGFR3 signaling We also compared the subcellular distribution of wild type and mutant (S249C) forms of FGFR3. These two forms localize to different compartments including early endosomes, late endosomes and recycling compartments. The S249C FGFR3 mutant preferentially localizes to the endocytic recycling compartment. Our findings shed light to the molecular mechanisms underlying the relationships between the trafficking and signaling of FGFR3 in the context of bladder cancer.

RhoB est une petite GTPase rapidement activée par les facteurs de croissance et les stress cellulaires, qui régule des processus biologiques fondamentaux comme la migration, l'angiogenèse, la réparation de l'ADN, l'apoptose ainsi que la réponse à des thérapeutiques anticancéreuses. L'activité des petites GTPases est finement régulée par leur localisation subcellulaire. Cependant, l'activation de RhoB en cellules vivantes n'avait jamais été investiguée. Ce travail a permis d'adapter et de valider une méthode innovante d'analyse des interactions protéine-protéine par complémentation split-GFP tripartite, pour la détection sensible et spécifique de l'activation des petites GTPases en cellules vivantes. Nous avons ensuite développé un modèle cellulaire optimisé par la combinaison de la technologie split-GFP tripartite et d'un intracorps anti-GFP amplificateur de fluorescence, pour détecter la régulation de l'activation de RhoB avec une haute résolution spatiale. Ce biosenseur a mis en évidence la translocation de la forme active de RhoB en réponse au sérum à partir des endosomes pour s'accumuler au niveau de la membrane plasmique, révélant ainsi une nouvelle plateforme de signalisation membranaire de RhoB. Ce biosenseur permettra d'analyser le profil d'activation de RhoB et d'autres petites GTPases, sous d'autres stimulations ou dans différents contextes cellulaires, et d'identifier leurs partenaires et les modulateurs de leur activation. / RhoB is a small GTPase that is rapidly activated in response to growth factors and cellular stress. It regulates fundamental biological processes such as cell migration, angiogenesis, DNA repair, apoptosis and response to anticancer therapies. Small GTPases activity is tightly regulated by their subcellular localization. However, RhoB activation had never been investigated in living cells. In this work, we have adapted and validated an innovative method of protein-protein interactions analysis using tripartite split-GFP complementation, for the sensitive and specific detection of small GTPases activation in living cells. Then, we developed an optimized cellular model by combining the tripartite split-GFP technology with an anti-GFP intrabody fluorescence-enhancer to detect the regulation of RhoB activation with high spatial resolution. This biosensor highlighted the translocation of active RhoB from endosomes to accumulate at the plasma membrane upon serum stimulation, revealing a novel membrane signaling platform of RhoB. Future studies based on this biosensor will enable the analysis of RhoB activation profile and other small GTPases upon various stimuli or in different cellular contexts, as well as the identification of the GTPases partners and activation modulators.

and keywords: During metastasis, cancer cells can invade the extracellular matrix using various strategies. When invading individually, they employ either the amoeboid invasion mode, during which the cell body dynamically deforms by enhanced contractility to squeeze through pores within the matrix, or protease dependent mesenchymal migration that takes advantage of the possibility to digest the surrounding matrix. Cells migrating in one mode can actively switch to the other by mesenchymal-amoeboid (MAT) or amoeboid-mesenchymal transitions (AMT). This enables escape mechanisms and considerably complicates anti-metastatic treatment. It is well known that Rho GTPases are master regulators of cytoskeleton re-arrangements and thus, unsurprisingly, play a major role in both invasion modes and can directly drive the transitions. However, upstream activation of these pathways is still largely unclear. This thesis aimed to optimize 3D conditions suitable for studying plasticity of cell invasion in vitro, establish AMT and MAT in melanoma cells based on manipulation of Rho GTPases and verify novel candidates regulating cell invasion plasticity based on previous RNA sequencing of cells before and after MAT. Last, by synthesis of published data, results from sequencing and new findings presented in this...

Chez l’ensemble des organismes, la capacité des cellules à se polariser est une propriété nécessaire à la croissance, à la division, à la mobilité, à la différenciation ou encore au trafic intracellulaire. Les GTPases de la famille Rho jouent des rôles prépondérants dans la régulation de ces mécanismes. Chez la levure Saccharomyces cerevisiae, nous étudions la manière dont le régulateur RhoGAP Rgd1p des protéines Rho3 et Rho4 est lui-même régulé au cours de la croissance polarisée. Nous avons montré que la protéine Rgd1 est localisée au fond du bourgeon lors de la croissance isotropique du bourgeon puis sous forme d’un anneau au cou du bourgeon lors de la cytocinèse. Nous avons également montré que le trafic intracellulaire, notamment l’exocytose et les phosphoinosotides tels que le PdtIns(4)P et le PdtIns(4,5)P2 ont un rôle majeur dans la régulation spatio-temporelle de la protéine Rgd1. Ces mécanismes permettraient d’acheminer la protéine RhoGAP au niveau des sites de croissances afin d’agir sur les protéines Rho3 et Rho4. / In all organisms, the ability of cells to polarize is a property necessary for growth, cell division, mobility, cell differentiation or intracellular trafficking. GTPases of the Rho family play central roles in the regulation of these mechanisms. In the yeast Saccharomyces cerevisiae, we study how the regulator RhoGAP Rgd1p of Rho3 and Rho4 proteins may be itself regulated during polarized growth. We showed that the protein Rgd1 is located at the bud tip during isotropic growth of the bud and form a ring at the bud neck during cytokinesis. We also showed that the intracellular traffic, especially the exocytosis and phosphoinosotides as the PdtIns(4)P and PdtIns(4,5)P2 have a major role in the spatio-temporal regulation of the Rgd1 protein. These mechanisms would deliver the RhoGAP protein at growth sites to act on Rho3 and Rho4 proteins.

Les GTPases de la famille Rho sont les principaux régulateurs du remodelage du cytosquelette d’actine lors de la migration et l’invasion cellulaire. En particulier, deux membres de cette famille sont importants dans ce processus : les GTPases RhoA et Rac1. En effet, il existe une balance d’activité de ces GTPases, responsables respectivement de la contraction cellulaire et de la formation d’extensions cytoplasmiques, des étapes clefs de la migration. L’objectif de ce travail de thèse a été d’étudier la régulation de ces protéines dans la migration et l’invasion cellulaire. Pour cela, plusieurs stratégies ont été entreprises. Tout d’abord, une étude structure/fonction de la protéine p190RhoGAP-A (p190A), un des régulateurs majeurs de la GTPase RhoA, a été réalisée. Cette étude a permis de mettre en évidence un domaine, appelé PLS pour « protrusion localization sequence », permettant à cette protéine de se localiser au niveau des extensions membranaires appelées « replis membranaires » et « lamellipodes » où RhoA est régulée localement. D’autre part, un mutant délété de ce domaine, appelé PLSp190A, ne peux pas se localiser au niveau de ces structures et a un impact négatif sur leur formation et la migration cellulaire. De plus, l’analyse de ce mutant a révélé que le domaine PLS était impliqué dans la régulation négative de p190A. Ainsi, nous avons mis en évidence un nouveau domaine de p190A responsable de sa localisation intracellulaire et de sa fonction. La deuxième partie de ce travail de thèse a été consacrée à la mise en place d’un outil de mesure de l’activité des GTPases Rho par la technologie Alphascreen. Ce test a permis de mesurer l’activité de Rac1 in vitro et in cellulo mais a également été appliqué à un crible en vue d’identifier de nouvelles molécules régulatrices de Rac1. Ainsi, ce travail de thèse, en abordant par plusieurs angles la régulation des GTPases de la famille Rho, a permis d’apporter des informations et des outils pour la compréhension des mécanismes complexes régissant la capacité des cellules à se mouvoir dans leur environnement. / RhoGTPases are major regulators of the actin cytoskeleton during cell migration and invasion. Particularly, the two members of the RhoGTPase family, RhoA and Rac1 play important roles in these processes. Indeed, a reciprocal balance between these GTPases’activity that leads to cell contraction and cell protrusion formation, determines cell movement. The aim of this PhD thesis was to study the regulation of RhoA and Rac1 during cell migration and invasion. To this end, various strategies were undertaken.We first performed a structure/function analysis of p190RhoGAP-A (p190A), a major negative regulator of RhoA. This led to the identification of a protrusion localization sequence (“PLS”) necessary and sufficient for p190A targeting to actin-based structures. A p190A mutant deleted of the PLS domain (PLS), does not localize to ruffles and lamellipodia, where RhoA is locally regulated during cell migration. This analysis also revealed that the PLS is required for the negative regulation of p190A activity. Finally, p190APLS expression has a dominant negative effect on the formation of actin protrusions and cell migration. Thus, we identified a novel functional domain of p190A required for its proper subcellular localization and functions. The second part of this PhD thesis was focused on the design of an Alphascreen technology-based assay to study GTPases activity. This assay allowed the measurement of Rac1 activity in vitro and in cellulo. Moreover, we used this assay to screen for new regulators of Rac1 activity. In conclusion, this work provides new insights and new tools for the understanding of RhoGTPase involvement in cell migration.

In this work, the evolutionary biophysics approach is applied to the two of the largest protein superfamilies present in human genomes, namely P-loop fold nucleoside triphosphatases (P-loop NTPases) and G-protein coupled receptors (GPCRs). This approach combines comparative analysis of protein structures and sequences with molecular modeling techniques in order to reveal not only the conservation of particular residues among proteins within each superfamily but also their role in the fundamental mechanisms underlying common functions. The study of the hydrolysis activation mechanism in P-loop NTPases started with the molecular dynamics simulations of Mg-NTP complexes (Mg-ATP and Mg-GTP) in the presence of K+, NH4+, and Na+ ions. These simulations showed that in the presence of large cations (K+ and NH4+), the conformation of the phosphate chain of ATP and GTP is extended, with large distances between alpha- and gamma-phosphates. This conformation is similar to the shape of ATP and GTP molecules (or their analogs) in the crystal structures of various P-loop NTPases. To clarify the role of monovalent cations in P-loop NTPases, MD simulations were conducted for two cation-dependent GTPases: tRNA modification GTPase MnmE and translation factor EF-Tu. MD simulations of Mg-GTP/EF-Tu complex bound to the tRNA and ribosome fragment in the presence of K+ ions have shown consistent binding of a potassium ion from the solution between alpha- and gamma-phosphates (AG site), similar to the cation binding in MnmE and other cation-dependent P-loop GTPases. In both proteins, binding of K+ ion in the AG site led to the rotation of gamma-phosphate, making this group more eclipsed with alpha-phosphate. The new rotated position of gamma-phosphate was stabilized by a novel H-bond with the backbone nitrogen of the K-3 residue (relative to the ubiquitously conserved Lys) of the P-loop motif. The activation mechanism observed in MD simulations of MnmE and EF-Tu could be envisioned as basic for P-loop NTPases, as these cation-dependent proteins are among the most ancient members of the P-loop superfamily. This mechanism was used as a basis for extensive comparative analysis of representative proteins from all major classes of P-loop NTPases. Based on the established conservation and presence of the key features in active sites of P-loop NTPases, the chain of events where rotation of gamma-phosphate triggers the nucleophilic attack and gamma-phosphate cleavage has been proposed as the basic universal activation mechanism of NTP hydrolysis in P-loop NTPases. The second part of this work explores the activation of GPCRs as sodium-translocating receptors. Crystal structures of the novel Na-pumping microbial rhodopsin along with the recent avalanche of GPCR structures provided the basis for comparative structure analysis, focused on investigating the similarities in the Na-binding sites of the two superfamilies. Structure superposition of GPCRs and microbial rhodopsins (MRs) based on comparison of their Na-binding sites was used to produce structure-guided sequence alignments of the two superfamilies. The only residue universally conserved between the two superfamilies was Trp in the helix 6/F (Trp6.48 in GPCRs). In both families, the signaling mechanism directly involves this residue, which is likely to be an ancient feature inherited from the common ancestor of MRs and GPCRs – the Na-pumping light-activated rhodopsin. The similarity of GPCRs with light-activated sodium pumps endorses the suggestion that GPCRs may also function as Na+ ion translocators. A model of GPCR activation accompanied by translocation of Na+ was constructed to demonstrate how this mechanism can explain the voltage sensitivity of certain Class A GPCRs. Two modes of activation were modeled – one where Na+ ion is transported into the cytoplasm and the one where Na+ ion is expelled to the intracellular space. The two modes quantitatively describe the behavior of voltage-activated and voltage-suppressed GPCRs, respectively. Finally, further structure scrutiny and rotamer analysis provided a plausible pathway of Na+ transmembrane translocation through the helical bundle of GPCRs.

No description available.

Aims: Oxidative stress and inflammation contribute to atherogenesis. Rac1 GTPase regulates pro-oxidant NADPH oxidase activity, reactive oxygen species (ROS) formation, actin cytoskeleton organization and monocyte adhesion. We investigated the vascular effects of pharmacological inhibition of Rac1 GTPase in mice. Methods and Results: We treated wild-type and apolipoprotein E-deficient (ApoE−/−) mice with Clostridium sordellii lethal toxin (LT), a Rac1 inhibitor, and assessed vascular oxidative stress, expression and activity of involved proteins, endothelial function, macrophage infiltration, and atherosclerosis development. LT-treated wild-type mice displayed decreased vascular NADPH oxidase activity and ROS production. Therapeutic LT doses had no impact on behavior, food intake, body weight, heart rate, blood pressure, vascular and myocardial function, differential blood count, and vascular permeability. ApoE−/− mice were fed a cholesterol-rich diet and were treated with LT or vehicle. LT treatment led to decreased aortic Rac1 GTPase activity, NADPH oxidase activity and ROS production, but had no impact on expression and membrane translocation of NADPH oxidase subunits and RhoA GTPase activity. LT-treated mice showed improved aortic endothelium-dependent vasodilation, attenuated atherosclerotic lesion formation and reduced macrophage infiltration of atherosclerotic plaques. Concomitant treatment of cholesterol-fed ApoE−/− mice with LT, the specific synthetic Rac1 inhibitor NSC 23766 or simvastatin comparably reduced aortic Rac1 activity, NADPH oxidase activity, oxidative stress, endothelial dysfunction, atherosclerosis development, and macrophage infiltration. Conclusions: These findings identify an important role of the small GTPase Rac1 in atherogenesis and provide a potential target for anti-atherosclerotic therapy.

No description available.

No description available.