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La synapse est un assemblage macromoléculaire dynamique : les protéines synaptiques sont constamment et rapidement échangées par un mouvement diffusif entre l'assemblage synaptique et la région extrasynaptique, alors que l'assemblage reste stable. Par ailleurs, dans le compartiment présynaptique, les cycles d'endo- et d'exocytose des vésicules synaptiques imposent une réorgani- sation dynamique de la membrane plasmique. La syntaxin1A est une protéine membranaire impliquée dans l'exocytose. Quelles sont les caractéristiques de la diffusion latérale de la syntaxin et ses implications pour la transmission sy- naptique ? Nous avons étudié la diffusion latérale de la syntaxin à l'échelle de la popu- lation (méthode de retour de fluorescence après photoblanchiment, FRAP) et à l'échelle de la molécule individuelle (suivi de particule unique, SPT). Nous avons montré que la syntaxin diffuse rapidement dans la membrane plasmique et présente des périodes d'immobilisation transitoire, qui reflètent des inter- actions moléculaires impliquées dans la formation du complexe exocytotique. Enfin, nous avons construit un modèle computationnel qui réconcilie les des- criptions de la diffusion latérale de la syntaxin à l'échelle de la population et de la molécule individuelle. Ce modèle a permis de caractériser les cinétiques des interactions entre la syntaxin et ses partenaires, qui conduisent à sa stabilisa- tion à la synapse.

Melanosome biogenesis is a highly regulated endosomal maturation process wherein structural fibers harbouring immature melanosomes acquires its biosynthetic proteins through the secretory pathway and finally matures into a functional organelle. These processes were shown to be dependent on several cytosolic protein complexes such as AP (adaptor protein)-1, AP-3, BLOC (biogenesis of lysosome-related organelles complex)-1, -2 and -3; in addition to kinesin motor KIF13A and Rab GTPases 7, 32 or 38. Mutations in the subunits of these complexes or Rab38 result into defective melanosome maturation leading to occulocutaneous albinism, a clinical phenotype commonly observed in Hermansky-Pudlak syndrome (HPS). Moreover, molecular function of these complexes in regulating the biogenesis of melanosome is partially known. The delivery of cargo to maturing melanosomal membranes requires fusion machinery that includes Rab GTPases, tethering factors and SNARE (soluble N-ethylmaleimide sensitive factor attachment protein receptor) proteins. However, the SNAREs involved in the transport of cargo to melanosomes is poorly understood. In this study entitled as “understanding the SNARE dynamics during melanosome biogenesis” we focus on functional role of endosomal Qa-SNARE protein, Syntaxin 13 (formally called STX12, herein referred to as STX13) in the organelle biogenesis and its transport in and out of melanosome. Moreover, these studies show that STX13-mediated cargo transport require a melanosomal membrane localized R-SNARE VAMP7 and these SNAREs are interdependent on each other in regulating their steady state distribution. In addition, this study illustrated the possible mechanism of SNARE recycling which occurs indirectly through AP-3 complex. Thus, these studies underscore the STX13‟s role in cargo transport to maturating melanosomes and its trafficking routes to and from the melanosomes. Chapter-I describes the literature review on melanosome biogenesis; Chapter-II lists the experimental procedures used in this study and Chapter-III to V focuses on results and discussion, segregated into three sections. Chapter-III: Screening and identification of endosomal SNAREs involved in the trafficking of melanosomal proteins. Our preliminary RNAi screen for SNAREs involved in melanosome biogenesis revealed STX13 as one of the Qa-SNARE affecting pigmentation and cargo transport. STX13, a recycling endosomal SNARE has been reported to interact with pallidin, a subunit of BLOC-1; however the functional role of this interaction in pigment formation is unknown. In addition, previous studies from our lab have shown that STX13 colocalize with endosomal Rab11 and partially with EEA1- or Rab5-positive organelles in melanocytes. Together, these observations insinuated us to characterize the functional role of STX13 in melanosome biogenesis. Upon STX13 inactivation, wild type mouse melanocytes showed hypopigmentation due to mistargeting of cargo such as TYRP1 and TYR to lysosomes. Knockdown of STX13 dramatically decrease the population of immature and mature melanosomes. Moreover, STX13 associate with the melanosome cargo on endosomal tubular structures. In addition, deletion of regulatory domain in STX13 increases the cargo transport to melanosomes due to its increased SNARE activity. This is possibly due to loss in intracellular regulation of SNARE occur through multiple factors such as SM (Sec1p/Munc18) proteins. Together this data suggests that STX13 mediates cargo transport to melanosomes from recycling endosomes. Chapter-IV: Functional characterization of the SNAREs involved in melanosomal maturation. Several in vitro studies have shown that a set of four SNAREs such as Qa, Qb, Qc (or Qbc) and R control the membrane fusion event duing the cargo transport. Additionally, this process is further regulated by SM proteins in in vivo. Electron microscopic studies in melanocytes have shown that melanosomal proteins were delivered to the melanosomal membrane through recycling endosomal tubular domains. Moreover, our RNAi screen show that STX13 possibly acts as Qa-SNARE in mediating the fusion events between melanosomal membranes and the endosomal tubular or vesicular intermediates. However, the role of other SNAREs for this membrane transport is unknown. It has been shown that the expression of VAMP family SNAREs such as VAMP3, VAMP7 and VAMP8 increased with melanogenesis upon differentiation of melanoma cells. VAMPs belong to the class of R-SNAREs, in which VAMP7 is known to interact with VARP (abbreviation) and AP-3 (mediates the trafficking of TYR) separately, and these molecules are known to regulate the cargo transport to melanosomes. However, the precise role of VAMP7 in pigment granule maturation is unknown. Therefore, we set out to characterize the functional role of VAMP7 in melanosome biogenesis. VAMP7 has been shown to localizes to multiple sub-cellular compartments and regulate the several transport steps in other cell types. Our study found that GFP-epitope tagged either human or rat VAMP7 localize to melanosomes at steady state in wild type mouse melanocytes. Knockdown of VAMP7 causes hypopigmentation of melanocytes and misroutes the cargo to lysosomes. Further, the inactivation of VAMP7 in melanocytes phenocopies the STX13 depletion, suggesting both the SNAREs are required for the melanosome biogenesis. In addition, knockdown of STX13 target the VAMP7 to lysosomes; while inactivation of VAMP7 affect the localization of STX13 to recycling tubular structures. Subsequently, the dominant active mutants of STX13 were not able to rescue the pigmentation or cargo transport defects in VAMP7 knockdown melanocytes. Together, the data suggests that STX13 functions from recycling endosomes and VAMP7 on melanosome membrane for the transport of cargo to melanosomes Chapter-V: Understanding the mechanism of STX13 recycling during melanosome biogenesis. At steady state, SNAREs are localized to the membranes of specific organelles where they mediate or regulate the membrane fusion. During this process, three or two Q-SNAREs on one membrane (in a trans-SNARE complex, possibly formed by Qa, Qb, Qc or Qbc) interact with a R-SNARE on another member to form a SNAREpin complex. Post-fusion, SNAREs are disassembled by SNAP and NSF proteins and then recycled back to the original compartment for next round of fusion. Here, we address the mechanism of post-fusion recycling of STX13 from melanosomes to endosomes. Previous studies have shown that STX13 mislocalize to melanosomes in AP-3-deficient melanocytes, suggesting a role for AP-3 in recycling the SNARE from melanosomes. Bioinformatic analysis of the N-terminal region of STX13 revealed the presence of two canonical adaptor binding motifs 3YGP6L and KETNE80L81L, resembling the tyrosine-based (YXXø) and dileucine-based motif [DE]XXXL[LI], recognized by several adaptor proteins. Point mutagenesis of these motifs in STX13 had no effect on their steady state distribution indicating that STX13 possibly uses non-canonical residues for its recycling. Further, deletion of the N-terminal region (either 1-129 or 14-129 aa) in STX13 redistributes the SNARE to melanosomes. Moreover, the activity and the trafficking of recycling defective STX13 mutants are dependent on another HPS complex, BLOC-2 and the SNARE, VAMP7. Absence of 1-129 region in STX13 or mutations in the subunits of AP-3 perturbs the steady state localization of STX13 suggesting an indirect role for AP-3 in recycling of STX13 to endosome via non canonical motifs present in its 1-129 aa region.

La v-ATPase est une pompe à protons. Elle permet l’acidification d’organelles, ce qui est indispensable à de nombreux processus cellulaires. Cette enzyme est composée de 14 sous-unités différentes, organisées en deux domaines, le domaine catalytique V1 et le domaine membranaire V0. La sous-unité a du domaine V0 est essentielle au transport des protons. Il en existe 4 isoformes (a1 à a4) et des variants d’épissage (a1-I à a1-IV pour a1) permettant à la v-ATPase d’être adressée vers différents compartiments et donc d’être impliquée dans différents processus. Deux projets visant à étudier cette sous-unité ont été réalisés.En plus de son rôle dans le transport des protons, il a été montré que le domaine V0 de la v-ATPase est impliqué dans des évènements de trafic membranaire, tel que l’exocytose de vésicules de sécrétion. Ce rôle semble nécessiter des interactions avec les protéines SNAREs. J’ai montré, pendant la première partie de ma thèse, que les sous-unités flag-a1-I et flag-a1-IV sont toutes deux adressées aux granules de sécrétion de cellules neurosécrétrices et interagissent avec les protéines SNAREs VAMP2 et syntaxine-1. De façon intéressante la syntaxine-1 semble interagir préférentiellement avec la sous-unité a1-I qui dans les neurones est l’isoforme adressée aux terminaisons nerveuses. Les sous-unités a1-IV ne diffèrent d’a1-I que par l’ajout de 7 acides aminés dans sa moitié N-terminale. Le domaine d’interaction de la sous-unité a avec la syntaxine-1 semble donc être localisé dans cette région.Dans la deuxième partie de ma thèse, j’ai mis en évidence et étudié l’expression de la sous-unité rénale a4 dans des gliomes humains. Ces tumeurs sont les tumeurs cérébrales les plus fréquentes et sont en général associées à un mauvais pronostic. L’OMS distingue, en fonction de paramètres histologiques, les astrocytomes (de grade I à IV), les oligodendrogliomes et les gliomes mixtes (chacun de grade II ou III). Cette classification est controversée, notamment à cause de son manque de reproductibilité, et la prise en compte de marqueurs moléculaires semble s’imposer comme une solution pour la renforcer.J’ai quantifié par RT-PCR quantitative l’expression du gène ATP6V0A4 (codant la sous-unité a4) dans 188 prélèvements de gliomes humains. Nous avons ainsi montré que l’expression de la sous-unité a4 peut être utilisée comme marqueur diagnostique des oligodendrogliomes anaplasiques (35 % l’expriment). Dans un prélèvement, la présence de la codélétion 1p/19q et l’expression de a4, tout deux marqueurs indépendants des oligodendrogliomes, permettra le renforcement du diagnostique oligodendrogliome anaplasique. De plus a4 est fréquemment exprimée par les astrocytomes pilocytiques (70%), où elle est associée à la duplication en tandem de la région chromosomique 7q34 située à proximité directe du gène ATP6V0A4. Enfin une observation prometteuse est que l’expression de a4 pourrait être un marqueur de mauvais pronostic pour les patients atteints d’oligodendrogliome anaplasique ne présentant pas la co-délétion 1p/19q, observation qui devra être confirmée sur une plus grande cohorte de patients. / Vacuolar type H+-ATPase is a proton pump, which acidifies numerous organelles, crucial for many cellular processes. This enzyme is composed of 14 different subunits organized in two domains, a catalytic V1 domain and a V0 membrane domain. The a-subunit of V0 is essential for proton transport. There are 4 isoforms of a (a1 to a4) and splicing variants (a1-I to a1-IV for the a1 subunit). v-ATPases containing different a-subunit isoforms are localized in different compartments allowing v-ATPase to participate in different processes. The a-subunits were studied in this work in two distinct projects.Besides its role in proton pumping, V0 domain of v-ATPase is implicated in organelles trafficking events, like vesicles exocytosis. This role seems to require interactions of V0 with SNARE proteins. During my thesis work, I showed that flag-a1-I and flag-a1-IV are both targeted to secretion granules in PC12 neurosecretory cells. These subunits interact with the SNARE proteins VAMP2 and syntaxin-1. Interestingly, syntaxin-1 seems to preferentially interact with the a1-I subunit, isoform which in neurons is sorted to nerve terminals. The only difference between a1-I and a1-IV subunits is the addition of 7 amino acids in the N-terminal half of a1-IV. So syntaxin-1 probably interacts with a1-I at this location. In a second project, I studied the expression of the renal a4-subunit in human gliomas. These tumors are the most frequent brain tumors and are generally associated with a poor prognosis. Based on histological parameters,WHO distinguishes, astrocytomas (grade I to IV), oligodendrogliomas and oligoastrogliomas (each of grade II or III). This classification suffers of a lack of reproducibility, which could be overcome by the identification of specific molecular markers.In the present work, by real time quantitative PCR, ATP6V0A4 gene (encoding the renal a4) expression was quantified in 188 human glioma biopsies. We established a4 expression as a new marker of grade III oligodendrogliomas (35 % express it), independent of the 1p/19q codeletion, an established marker of oligodendrogliomas. Moreover, a4 is expressed in 70% of pilocytic astrocytomas, in which it is associated with the tandem duplication of 7q34, localized at direct proximity of the ATP6V0A4 gene. Of promising interest is the observation that a4 expression could be considered as a bad prognostic marker for patients with 1p/19q non-deleted oligodendrogliomas, an observation that should be confirmed on larger cohorts of patients.

Indiana University-Purdue University Indianapolis (IUPUI) / In response to glucose, pancreatic islet beta cells secrete insulin in a biphasic manner, and both phases are diminished in type 2 diabetes. In beta cells, cortical F-actin beneath the plasma membrane (PM) prevents insulin granule access to the PM and glucose stimulates remodeling of this cortical F-actin to allow trafficking of insulin granules to the PM. Glucose stimulation activates the small GTPase Cdc42, which then activates p21-activated kinase 1 (PAK1); both Cdc42 and PAK1 are required for insulin secretion. In conjunction with Cdc42-PAK1 signaling, the SNARE protein Syntaxin 4 dissociates from F-actin to allow SNARE complex formation and insulin exocytosis. My central hypothesis is that, in the pancreatic beta cell, glucose signals through a Cdc42-PAK1-mediated pathway to remodel the F-actin cytoskeleton to mobilize insulin granules to SNARE docking sites at the PM to evoke glucose stimulated second phase insulin secretion. To investigate this, PAK1 was inhibited in MIN6 beta cells with IPA3 followed by live-cell imaging of F-actin remodeling using the F-actin probe, Lifeact-GFP. PAK1 inhibition prevented normal glucose-induced F-actin remodeling. PAK1 inhibition also prevented insulin granule accumulation at the PM in response to glucose. The ERK pathway was implicated, as glucose-stimulated ERK activation was decreased under PAK1-depleted conditions. Further study showed that inhibition of ERK impaired insulin secretion and cortical F-actin remodeling. One of the final steps of insulin secretion is the fusion of insulin granules with the PM which is facilitated by the SNARE proteins Syntaxin 4 on the PM and VAMP2 on the insulin granule. PAK1 activation was also found to be critical for Syntaxin 4-F-actin complex dynamics in beta cells, linking the Cdc42-PAK1 signaling pathway to SNARE-mediated exocytosis. Syntaxin 4 interacts with the F-actin severing protein Gelsolin, and in response to glucose Gelsolin dissociates from Syntaxin 4 in a calcium-dependent manner to allow Syntaxin 4 activation. Disrupting the interaction between Syntaxin 4 and Gelsolin aberrantly activates endogenous Syntaxin 4, elevating basal insulin secretion. Taken together, these results illustrate that signaling to F-actin remodeling is important for insulin secretion and that F-actin and its binding proteins can impact the final steps of insulin secretion.

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Proper neuronal function requires the preservation of appropriate neural excitability. An adaptive increase in neural excitability after exposure to agents that depress neuronal signaling blunts the sedative drug effects upon subsequent drug exposure. This adaptive response to drug exposure leads to changes in drug induced behaviors such as tolerance, withdrawal and addiction. Here I use Drosophila melanogaster to study the cellular and neuronal components which mediate behavioral tolerance to the anesthetic benzyl alcohol. I demonstrate that rapid tolerance to benzyl alcohol is a pharmacodynamic mechanism independent of drug metabolism. Furthermore, tolerance is a cell autonomous response which occurs in the absence of neural signaling. Using genetic and pharmacological manipulations I find the synapse to play an important role in the development of tolerance. In addition, the neural circuits that regulate arousal and sleep also alter benzyl alcohol sensitivity. Beyond previously described transcriptional mechanisms I find a post-translational role of the Ca2+-activated K+-channel, slowpoke in the development of tolerance. Finally, I explore a form of juvenile onset tolerance, which may have origins that differ from rapid tolerance. The implications of this study go beyond tolerance in Drosophila melanogaster to benzyl alcohol and can shed light on human drug tolerance, withdrawal and addiction. / text

Dans l'introduction, diverses voies de trafic intracellulaire et endocytose seront discutées. Je familiarise le lecteur avec des protéines inactivant les ribosomes, en mettant l'accent sur la structure, l'endocytose, et le trafic intracellulaire de la toxine bactérienne Shiga toxin (STX). STx et la ricine suivent la voie rétrograde pour exercer leur effet toxique sur les cellules. Ils sont respectivement, une menace maladie infectieuse pour la santé humaine et des outils potentiels pour le bioterrorisme pour lequel aucun antidote n’existe actuellement. D'un criblage à haut débit, Retro-1 et Retro-2 avaient déjà été identifiés comme de puissants inhibiteurs de la voie rétrograde à l'interface des endosomes précoces-TGN, et Retro-2 a été démontré pour protéger les souris contre la ricine. Parmi les facteurs de trafic analysés, seule la protéine SNARE syntaxine-5 a été ré- localisée dans les cellules traitées avec Rétro - 2. / In the introduction, various endocytic and intracellular trafficking pathways will be discussed. I acquaint the reader with ribosome-inactivating proteins, with emphasis on the structure, endocytosis, and intracellular trafficking of the bacterial toxin Shiga toxin (STx). STx and ricin follow the retrograde route to exert their toxic effect on cells. They are respectively, an infectious disease threat to human health and potential tools for bioterrorism for which no antidote currently exists. From a high throughput screening, Retro-1 and Retro-2 had previously been identified as potent inhibitors of the retrograde route at the early endosomes-TGN interface, and Retro-2 was demonstrated to protect mice against ricin. Of the trafficking factors analyzed, only the SNARE protein syntaxin-5 was re-localized in Retro-2 treated cells. Yet, whether syntaxin-5 is the direct target of Retro-2 and whether its re-localization was directly responsible for retrograde transport inhibition remained to be established.