Les exosomes sont des vésicules membranaires de 30 à 100 nm de diamètre, formées dans les endosomes multivésiculaires et sécrétées par la plupart des cellules. Les propriétés biophysiques et biochimiques des exosomes ainsi que les mécanismes permettant leur biogénèse et sécrétion ont fait l’objet de nombreuses études. Cependant, ces derniers sont encore méconnus, limitant l'analyse des fonctions des exosomes in vivo. Au moins deux mécanismes ont été proposés pour la biogénèse des exosomes : un mécanisme nécessiterait l’action de protéines impliquées dans le tri endosomal, les ESCRT (« endosomalsorting complex required for transport »). Un autre mécanisme serait indépendant de leur fonction. La sécrétion des exosomes, une fois générés dans les endosomes, requiert la petite GTPase, Rab27a, comme montré dans un modèle cellulaire humain. Mes travaux de thèse ont porté sur l’étude des mécanismes moléculaires impliqués dans la biogénèse et la sécrétion des exosomes. Une première étude visant à analyser la fonction de Rab27a dans des cellules murines, m’a permis de mettre en évidence l’existence de différentes populations d’exosomes, dont la sécrétion dépend ou non de Rab27a. Une deuxième étude a eu pour objectif d’analyser l’implication des ESCRT dans la biogénèse des exosomes dans des cellules HeLa CIITA. Le criblage d’une librairie d’ARN d’interférence dirigés contre les différentes protéines ESCRT, a permis l’identification de 7 molécules potentiellement impliquées dans cette voie : HRS, STAM1, TSG101, leur inactivation induisant la diminution de la sécrétion des exosomes. L’inactivation de CHMP4C, VPS4B,VTA1 et ALIX, au contraire, l’augmente. L’inhibition de l’expression de ces candidats suivie de l’analyse des exosomes sécrétés a démontré l’hétérogénéité des vésicules sécrétées, et une modification de leur taille et de leur composition protéique par rapport aux cellules contrôle. Plus particulièrement, l’inactivation d’ALIX induit une augmentation de lasécrétion d‘exosomes de plus grande taille, et l’enrichissement sélectif en molécules de CMH de classe II. En accord, j’ai montré que les cellules inactivées pour ALIX, aussi bien des cellules HeLa que des cellules dendritiques humaines ont une plus forte expression de CMH de classe II à la surface et dans des compartiments intracellulaires. Ces résultats suggèrent l’implication de certains membres de la famille ESCRT dans la voie de biogenèse et sécrétion des exosomes, ainsi qu’un rôle potentiel d’Alix dans le trafic des molécules CMH de classe II, et dans la modulation de la composition protéique des exosomes. / Exosomes are small membrane vesicles with sizes ranging from 30 to 100 nm in diameter, which are formed in multivesicular endosomes and secreted by most cell types. Numerous studies have focused on the biophysical and biochemical properties of exosomes, as well as the mechanisms of biogenesis and secretion of these vesicles. However, these aspects are not fully understood, which limits the analysis of the functions of exosomes in vivo. At least two mechanisms have been proposed for the biogenesis of exosomes : one would rely on the function of proteins involved in endosomal sorting, the ESCRT family (for “endosomal sorting complex required for transport”). Another mechanism would be independent of their activity. Once exosomes are formed in endosomes, their secretion requires the small GTPase RAB27A, as shown in a human cell line. The objective of my PhD project was to gain insights into the molecular mechanisms that drive exosome biogenesis and secretion. A first study performed to analyze the function of Rab27a in murine cells allowed me to show the existence of different populations of exosomes, dependent or not on Rab27a for their secretion. A second study was aimed at analyzing the involvement of ESCRT proteins in exosome biogenesis in HeLa-CIITA cells. Seven molecules potentially involved in this process were identified on the basis of the screening of an RNA interference library directed against the different ESCRT proteins: the inactivation of HRS, STAM1 and TSG101 induced a decrease in exosome secretion, whereas the down regulation of CHMP4C, VPS4B, VTA1 and ALIX increased it. Gene expression of the different candidate proteins was inhibited and exosomes secreted by these cells were analyzed: we showed the heterogeneity of the secreted vesicles, as well as an alteration of their size and protein composition, as compared to control cells. In particular, the inactivation of ALIX induced an increase in the secretion of larger vesicles, and the selective enrichment of these vesicles in MHC class II molecules. Accordingly, I showed that both HeLa-CIITA and human primary dendritic cells inactivated for ALIX possess a higher expression of MHC class II molecules at the cell surface and in intracellular compartments. These results suggest that some members of the ESCRT family are involved in the exosome biogenesis and secretion pathway, and propose a potential role of ALIX in the trafficking of MHC class II molecules and in the modulation of the protein composition of exosomes.

Indiana University-Purdue University Indianapolis (IUPUI) / Glucose homeostasis is maintained through the coordinated actions of insulin secretion from pancreatic beta cells and insulin action in peripheral tissues. Dysfunction of insulin action yields insulin resistance, and when coupled with altered insulin secretion, results in type 2 diabetes (T2D). Exocytosis of intracellular vesicles, such as insulin granules and glucose transporter (GLUT4) vesicles is carried out by similar SNARE (soluble NSF attachment receptor) protein isoforms and Munc18 proteins. An additional regulatory protein, Doc2b, was implicated in the regulation of these particular exocytosis events in clonal cell lines, but relevance of Doc2b in the maintenance of whole body glucose homeostasis in vivo remained unknown. The objective of my doctoral work was to delineate the mechanisms underlying regulation of insulin secretion and glucose uptake by Doc2b in effort to identify new therapeutic targets within these processes for the prevention and/or treatment of T2D. Towards this, mice deficient in Doc2b (Doc2b-/- knockout mice) were assessed for in vivo alterations in glucose homeostasis. Doc2b knockout mice were highly susceptible to preclinical T2D, exhibiting significant whole-body glucose intolerance related to insulin secretion insufficiency as well as peripheral insulin resistance. These phenotypic defects were accounted for by defects in assembly of SNARE complexes. Having determined that Doc2b was required in the control over whole body glycemia in vivo, whether Doc2b is also limiting for these mechanisms in vivo was examined. To study this, novel Doc2b transgenic (Tg) mice were engineered to express ~3 fold more Doc2b exclusively in pancreas, skeletal muscle and fat tissues. Compared to normal littermate mice, Doc2b Tg mice had improved glucose tolerance, related to concurrent enhancements in insulin mumsecretion from beta cells and insulin-stimulated glucose uptake in the skeletal muscle. At the molecular level, Doc2b overexpression promoted SNARE complex assembly, increasing exocytotic capacities in both cellular processes. These results unveiled the concept that intentional elevation of Doc2b could provide a means of mitigating two primary aberrations underlying T2D development.

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