La couche cornée, couche la plus superficielle de l'épiderme, assure une fonction de barrière multifonctionnelle vitale pour l'organisme. Le maintien de cette barrière dépend de la fonctionnalité des cellules sous-jacentes, les kératinocytes granuleux, dernière couche de cellules vivantes de l'épiderme. Les kératinocytes granuleux contiennent dans leur cytoplasme de nombreux organites tubulo-vésiculaires sécrétoires, appelés corps lamellaires (CL). Les CL jouent un rôle majeur dans la formation et le maintien de cette barrière en déversant leur contenu (lipides, lipases, protéases, inhibiteurs de protéases, peptides antimicrobiens,...) à la transition couche cornée - couche granuleuse. Le trafic intracellulaire des CL est donc un processus déterminant dans l'homéostasie de la couche cornée. Cependant, les mécanismes moléculaires impliqués dans la régulation de ce trafic ne sont pas élucidés. Précédemment, au laboratoire, une analyse systématique par spectrométrie de masse des protéines associées aux CL a permis d'identifier plusieurs GTPases de la famille Rab et certains de leurs effecteurs. Ces protéines étant des régulateurs majeurs du trafic vésiculaire dans tous les types cellulaires, elles pourraient jouer un rôle dans le routage des CL et, ainsi, dans la fonctionnalité de la barrière épidermique. L'objectif de ma thèse était d'identifier les GTPases Rab et effecteurs impliqués dans la régulation du trafic intracellulaire des CL. Dans un premier temps, j'ai mis en évidence que la GTPase Rab11a est fortement exprimée dans les kératinocytes granuleux où elle est partiellement associée aux CL. J'ai montré que la déplétion de Rab11a dans un modèle tridimensionnel d'épiderme reconstruit in vitro réalisée grâce à la technique d'interférence à l'ARN, induit une diminution de la densité et de la sécrétion des CL. L'extinction de Rab11a entraîne également une baisse du taux de céramides et de cholestérol, une désorganisation des lipides intercornéocytaires et une augmentation de la perméabilité de la couche cornée. Dans les épidermes reconstruits déplétés, les composants des CL non-sécrétés sont adressés vers une voie de dégradation lysosomale. Dans un deuxième temps, j'ai observé que la perte d'expression de Rab11a induit une anomalie de distribution du moteur moléculaire Myosine-Vb, effecteur majeur de Rab11a. J'ai donc analysé les conséquences de la déplétion de Myosine-Vb dans les épidermes reconstruits et montré qu'elle induit un phénotype comparable à celui observé lors de la déplétion de Rab11a. L'ensemble de ces résultats suggèrent fortement que le complexe bipartite Rab11a-Myosine-Vb constitue un régulateur majeur de la biogenèse des CL et ainsi, de l'homéostasie de la barrière épidermique. Mon travail de thèse est la première étape du décryptage des voies moléculaires impliquées dans la biogenèse des CL et contribue à une meilleure compréhension du rôle du trafic intracellulaire dans la constitution de la barrière épidermique. Il pourrait permettre de caractériser les mécanismes physiopathologiques associés à un défaut de trafic des CL. / The stratum corneum, the most superficial layer of the epidermis, provides a multifunctional protective barrier which is vital for the organism. The maintenance of this barrier is directly dependent on the underlying granular keratinocytes which are the last living cells in the epidermis. The granular keratinocytes contain in their cytoplasm numerous tubulovesicular secretory organelles called lamellar bodies (LB). LB play a major role in the establishment and the maintenance of the epidermal barrier by releasing their content (lipids, lipases, proteases, protease inhibitors, antimicrobial peptides,...) at the junction between the stratum corneum and the stratum granulosum. Because of LB importance in the maintenance of the stratum corneum homeostasis, the regulation of their trafficking deserves further study.Previously, in my laboratory, a proteomic characterization of LB by mass spectrometry has identified several Rab family GTPases and some of their effectors. In any cell type, from yeast to human, Rab GTPases are considered as major regulator of vesicular trafficking. Thus, I postulated that these proteins could play a role in the regulation of LB routing in the cytoplasm of granular keratinocytes. In this context, the aim of my thesis was to determine which Rab GTPases and effectors are involved in this process. In a first step, I demonstrated that Rab11a is strongly expressed in granular keratinocytes where it is associated with LB. I showed that Rab11a silencing using RNA interference technique in an in vitro tridimensional model of reconstructed human epidermis induces a decrease of LB density and secretion in granular keratinocytes. The Rab11a depletion also leads to a decrease of ceramide and cholesterol level and a disorganization of intercorneocyte lipids, generating a defective epidermal barrier. In depleted reconstructed epidermis, there is a missorting of non-secreted LB components, driven to the lysosomal degradation pathway. In a second step, I observed that Rab11a silencing affects distribution of its effector, the molecular motor Myosin-Vb. So, I analyzed the consequences of Myosin-Vb depletion in the model of reconstructed epidermis and I demonstrated that the phenotype obtained is similar that of a Rab11a depleted epidermis. Taken together, these results strongly suggest that the bipartite complex Rab11a-Myosin-Vb is able to regulate the biogenesis of LB in granular keratinocytes. Thus, this molecular complex is a crucial regulator of the epidermal barrier homeostasis. My thesis work is a first step in the deciphering of the molecular pathway involved in LB biogenesis. It is a breakthrough in the comprehension that membrane dynamic in the granular keratinocytes is a major regulator of epidermal barrier. It may contribute to a better understanding of pathophysiological mechanisms related to dysregulated LB trafficking in skin diseases.

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...

Introdução: In vivo ou in vitro a exposição de fibroblastos a alta concentração de glicose promove um aumento do estresse oxidativo e consequentemente prejudica a migração celular, assim como a maturação da adesão. Além disso, a elevada concentração de glicose reduz a expressão de diferentes integrinas na superfície celular devido alterações na síntese do receptor e sua reciclagem. Objetivo: Avaliar os efeitos da elevada concentração de glicose no tráfego de vesículas contendo EEA1 (endossomos primários), Rab4 (via rápida da reciclagem), Rab11 (via lenta de reciclagem) e Rab7 (endossomos de degradação) em fibroblastos NIH3T3. Métodos: células foram cultivadas em meio contendo baixa concentração de glicose (LG, 5 mM) ou em alta concentração (HG 25 mM) durante 21 dias antes de realizar os experimentos. EEA1, Rab4, Rab11 e Rab7 expressão e distribuição foram avaliados por western blotting e imunofluorescência, respectivamente. Resultados: Células expostas à alta concentração não apresentaram diferenças na expressão e distribuição das proteínas EEA1 e Rab7, enquanto a expressão de Rab11 foi reduzida em 30%. Conclusão: a alta concentração de glicose altera a via lenta da reciclagem contendo Rab11, afetando potencialmente a reciclagem de integrinas e outros receptores e a sua expressão na superfície celular. / Background: In vivo or in vitro exposure of fibroblasts to high glucose concentrations (HG) promotes oxidative stress and consequently impairs cell migration, also inhibiting adhesion maturation. Additionally, HG reduces the expression of different integrins on the cell surface, potentially due to altered receptor synthesis and recycling. Aim: to evaluate the effects of HG on the trafficking vesicles containing EEA1 (early endosomes), Rab4 (fast recycling pathway) and Rab7 (endocytic degradation pathway) on NIH3T3 fibroblasts. Methods: cells were cultured under low glucose (LG, 5 mM) or HG (25 mM) concentrations during 21 days before the assays. EEA1, Rab4 and Rab7 expression and distribution were evaluated by western blotting and immunofluorescence, respectively. Results: HG did not affect proteins EEA1 and Rab7 expression and distribution, whereas Rab11 expression was reduced by 30%. The number of vesicles containing Rab11 was also significantly reduced in HG cells. Conclusion: high glucose alters the slow recycling endocytic pathway via Rab11, potentially affecting integrins and other receptors synthesis and expression on the cell surface.

Introdução: In vivo ou in vitro a exposição de fibroblastos a alta concentração de glicose promove um aumento do estresse oxidativo e consequentemente prejudica a migração celular, assim como a maturação da adesão. Além disso, a elevada concentração de glicose reduz a expressão de diferentes integrinas na superfície celular devido alterações na síntese do receptor e sua reciclagem. Objetivo: Avaliar os efeitos da elevada concentração de glicose no tráfego de vesículas contendo EEA1 (endossomos primários), Rab4 (via rápida da reciclagem), Rab11 (via lenta de reciclagem) e Rab7 (endossomos de degradação) em fibroblastos NIH3T3. Métodos: células foram cultivadas em meio contendo baixa concentração de glicose (LG, 5 mM) ou em alta concentração (HG 25 mM) durante 21 dias antes de realizar os experimentos. EEA1, Rab4, Rab11 e Rab7 expressão e distribuição foram avaliados por western blotting e imunofluorescência, respectivamente. Resultados: Células expostas à alta concentração não apresentaram diferenças na expressão e distribuição das proteínas EEA1 e Rab7, enquanto a expressão de Rab11 foi reduzida em 30%. Conclusão: a alta concentração de glicose altera a via lenta da reciclagem contendo Rab11, afetando potencialmente a reciclagem de integrinas e outros receptores e a sua expressão na superfície celular. / Background: In vivo or in vitro exposure of fibroblasts to high glucose concentrations (HG) promotes oxidative stress and consequently impairs cell migration, also inhibiting adhesion maturation. Additionally, HG reduces the expression of different integrins on the cell surface, potentially due to altered receptor synthesis and recycling. Aim: to evaluate the effects of HG on the trafficking vesicles containing EEA1 (early endosomes), Rab4 (fast recycling pathway) and Rab7 (endocytic degradation pathway) on NIH3T3 fibroblasts. Methods: cells were cultured under low glucose (LG, 5 mM) or HG (25 mM) concentrations during 21 days before the assays. EEA1, Rab4 and Rab7 expression and distribution were evaluated by western blotting and immunofluorescence, respectively. Results: HG did not affect proteins EEA1 and Rab7 expression and distribution, whereas Rab11 expression was reduced by 30%. The number of vesicles containing Rab11 was also significantly reduced in HG cells. Conclusion: high glucose alters the slow recycling endocytic pathway via Rab11, potentially affecting integrins and other receptors synthesis and expression on the cell surface.

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.

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