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Le trafic membranaire est un processus universel qui est essentiel pour la fonction neuronale dans un large spectre de fonctions. De la croissance neuronale et le développement morphologique à la libération des neurotransmetteurs et la plasticité synaptique, il prend en charge l'activité neuronale et donne d'innombrables questions qui animent la recherche sur la neurobiologie d'aujourd'hui. Notamment, l’exocytose des endosomes de recyclage (ER) dans les compartiments somatodendritiques participe à la transmission synaptique et à la potentialisation synaptique à long terme (PLT). Cependant la machine moléculaire sous-tendant l’exocytose des ER reste encore méconnue. Afin d’identifier les protéines SNAREs vésiculaires (v-SNARE) impliquées dans les différentes formes d’exocytose des ER postsynaptiques, nous avons d'abord imagé les protéines VAMP neuronales fusionnées avec la pHluorine, une GFP mutée sensible au pH dans les neurones de l’hippocampe en culture. Nous avons constaté que seulement VAMP2 et VAMP4, mais pas VAMP7, rapportaient des événements d’exocytose somatodendritique dans les neurones matures. Après avoir identifié ces deux protéines candidates, nous avons utilisé la combinaison de différentes techniques de régulation négative chronique ou aiguë pour désactiver leur fonction et observer les conséquences sur l’exocytose des ER, la transmission synaptique basale ou la PLT. Nos résultats suggèrent que VAMP2 est impliqué dans une forme d’exocytose régulée importante pour la PLT, mais pas l’exocytose constitutive des récepteurs AMPA, qui stabilise la transmission basale. VAMP4 est nécessaire pour l'exocytose constitutive d'une grande partie des endosomes, mais l'implication fonctionnelle de ces endosomes doit encore être explorée, car la régulation négative de VAMP4 ne modifie pas la transmission basale. / Membrane trafficking is a universal process that is essential for neuronal function in a wide spectrum of applications. From neuronal growth and morphological development to neurotransmitter release and synaptic plasticity, it supports neuronal activity and gives countless questions that drive today’s neurobiology research. Notably, the trafficking of recycling endosomes (REs) in somatodendritic compartments participates in synaptic transmission and plasticity, such as long-term synaptic potentiation (LTP). However, the fusion machinery mediating RE exocytosis is still unclear. To identify the vesicular SNAREs (v-SNAREs) involved in different forms of postsynaptic RE exocytosis, we first imaged neuronal VAMP proteins fused with pH-sensitive pHluorin in cultured hippocampal neurons, and found that only VAMP2 and VAMP4, but not VAMP7, underwent somatodendritic exocytosis in mature neurons. After identifying these two candidate proteins, we used a combination of different downregulation techniques to chronically or acutely deactivate their function and observe consequences on REs exocytosis, basal synaptic transmission and LTP. Our results suggest that VAMP2 is involved in activity-regulated exocytosis important for LTP, but not constitutive postsynaptic AMPARs exocytosis, supporting basal transmission. VAMP4 is required for constitutive exocytosis of at least a large proportion of REs, but the functional implication of these endosomes still need to be explored, as VAMP4 downregulation did not alter basal synaptic transmission.

No description available.

Eukaryotic cells are populated by membrane-enclosed organelles possessing discrete molecular and biochemical properties. Communication between organelles is established by shuttling vesicles that transport proteins and other molecules. Vesicles bud from a donor organelle, travel in the cytosol, and are delivered to a target organelle. All these steps are regulated to ensure that cargoes are transported in a specific and directed manner. The focus of this thesis is on the last part of the journey of a vesicle: the process of vesicle targeting. Two phases can be distinguished in this process: vesicle tethering, defined as the first interaction between the shuttling vesicle and the target membrane, and membrane fusion, which is the mixing of the lipid bilayers and of lumen content. Both phases are mediated by a minimal set of molecular components that include one member of the family of Rab GTPases, a vesicle tethering factor, a phosphoinositide lipid, and four SNAREs together with their regulatory proteins. While many studies have investigated the molecular details of how SNAREs mediate membrane fusion, the process of vesicle tethering is less well understood. The overall scope of my study is to describe the molecular details of vesicle tethering and how they can contribute to the general process of vesicle targeting. To address this question I developed an in vitro assay where I reconstitute in vitro the process of vesicle tethering. This bottom-up approach allows the molecular dissection of cellular processes outside of the complex context of the cell. With this assay I have characterized the vesicle tethering abilities of individual proteins involved in vesicle tethering on early endosomes. I show that a minimal vesicle tethering machinery can be formed by the concomitant interaction between one vesicle tethering factor and a phosphoinositide on the membrane of one vesicle, and by a vesicle tethering factor and a Rab GTPase on the membrane of another vesicle. These results provide an explanation for how vesicle tethering contributes to the specificity of vesicle targeting and to the directionality of cargo transport. In particular, specificity of vesicle targeting can arise from the specific interaction between a Rab and a vesicle tethering factor that is an effector of the Rab. I show that the asymmetric distribution of binding sites in the structure of a vesicle tethering factor can generate a heterotypic vesicle tethering reaction that can account for the directionality of cargo transport. The outcome of this thesis emphasizes the role that vesicle tethering factors have in the self-organized system of vesicle trafficking of eukaryotic cells. To identify novel Rab5 effectors implicated in vesicle tethering, I carried out a Rab5-chromatography on mouse liver. Amongst other novel Rab5 effectors, I identify a multi-subunit vesicle tethering complex that was not previously characterized in mammalian cells. The complex, named CORVET, is conserved from yeast to humans and plays a major role in cell physiology since its removal causes embryonic death in mice. I define its subunits composition, determine its subcellular localization, and elucidate its role in cargo transport. This finding reconciles a disharmony between findings in mammals and yeast regarding the molecular machinery responsible for the conversion from early to late endosomes. I also show that the newly identified subunit of the mammalian CORVET complex is the only Rab5 effector to localize to autophagosomes. I hypothesise that it is through the CORVET complex that Rab5 is involved in the formation and maturation of autophagosomes.

SNARE proteins play essential role in most membrane fusions taking place in eukaryotic cell. They are responsible for all fusions that occur across endocytic and secretory pathways. Apart from these processes stand mitochondria and plastids. Fusion of these organelles is directed by specific protein machineries. In this work we review up-to-date information on SNARE mediated membrane fusion and fusion of outer and inner mitochondrial membranes with an emphasis on situation in flagellated protozoan parasite Giradia intestinalis. It was suggested that one of typical SNARE protein in Giardia (GiSec20) is localised to its highly reduced mitochondria called mitosomes. This protein is also essential for surviving of Giardia trophozoites. In this work we show that mitosomal localization of Gisec20 is caused by episomal expression however the protein is localised to endoplasmic reticulum under physiological conditions. Using GFP tag we were able to characterize its targeting signal which showed to be localised in transmembrane domain of GiSec20. This signal targets the protein to mitosomes of G. intestinalis and S. cerevisiae, respectively. Mitosomal localization was prevented by adding 3'UTR to gene sequence and its episomal expression. This suggests existence of targeting mechanism based on information...

Vesicular trafficking, the movement of vesicles between organelles and the plasma membrane for secretion, consists of multiple highly regulated processes. Many protein families function as specificity and regulatory determinants to ensure correct vesicle targeting and timing of trafficking events. The SNARE proteins dock and fuse vesicles to their target membranes. Sec1/Munc18 (SM) proteins regulate membrane fusion through interactions with the SNAREs—SM proteins have been shown to act as both inhibitors and stimulators of SNARE assembly and membrane fusion. However, the details of these SM protein functions are not understood. Constructing a model of SM protein function has been challenging due to the various modes of interactions reported between SM proteins and their SNAREs. SM proteins interact with their cognate SNAREs and SNARE complexes through several distinct modes. The most conserved mode is an interaction with the syntaxin N-peptide; other modes of binding, such as the syntaxin closed conformation, are hypothesized to be specific for specialized cell types. In order to elucidate the general function of SM proteins, I investigated the function of the endosomal SM protein Vps45p by analyzing its interactions with its cognate syntaxin Tlg2p and its role in SNARE assembly. I had two main hypotheses: that the Tlg2p N-peptide does not solely mediate the interaction between Vps45p and Tlg2p; and that Vps45p functions to stimulate SNARE complex assembly. I systematically mapped the interaction between Vps45p and Tlg2p using various Tlg2p truncations containing the different domains of Tlg2p and discovered a second binding site on Tlg2p that corresponds to the closed conformation. The neuronal SM-syntaxin pair interacts in a similar manner, indicating that this interaction mode is conserved. To characterize the closed conformation binding mode further, and determine its relationship to the N-peptide binding mode, I developed a quantitative fluorescent electrophoretic mobility shift assay. Results indicate that these two sites do not bind simultaneously and that the N-peptide binding modulates the closed conformation affinity. Furthermore, I monitored the effect of Vps45p on SNARE complex assembly using size exclusion chromatography. Under the conditions tested, Vps45p did not appear to stimulate SNARE complex assembly. The work presented here addresses several puzzling issues in the field and significantly contributes to the construction of a new mechanistic model for SM protein function. In this new model, the SM protein is recruited to the membrane by its interaction with the syntaxin N-peptide. The SM protein then binds the syntaxin closed conformation thus inhibiting SNARE complex assembly. Upon dissociation of the SM protein from the closed conformation, an event perhaps regulated by the SM protein, syntaxin opens and interacts with the other SNAREs to form a SNARE complex. Fusion ensues, stimulated by the SM protein.

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