Aux synapses Fibres Parallèles - Cellules de Purkinje, le récepteur mGluR4 est le seul mGluR du groupe III à moduler la neurotransmission en inhibant les influx calciques qui régulent la libération de glutamate. Dans des systèmes hétérologues, il a été montré que mGluR4 était lié à des protéines G de type Gi/o couplées négativement à l'adénylate cyclase (AC). Afin de rester au plus proche des interactions physiologiques, nous avons débuté notre étude par la définition du réceptosome des récepteurs mGluR4 natifs dans le cervelet de rat. Nous avons identifié 184 partenaires putatifs du récepteur. Afin de confirmer ces interactions, mais aussi de recenser d'autres interacteurs éventuels, nous avons réalisé une approche complémentaire et indépendante de chromatographie d'affinité. Nombre de protéines ont été retrouvées par cette deuxième approche, en particulier des protéines appartenant aux familles de l'exocytose et du trafic cellulaire. Nos résultats suggèrent que le contrôle de la neurotransmission par mGluR4 pourrait s'effectuer, au moins partiellement, par une interaction avec ce type de protéines. D'autre part, nos approches biochimiques n'ont pas mis en évidence de protéines de la voie AC, mais au contraire plusieurs protéines identifiées appartiennent à la voie Phospholipase C/ Protein Kinase C (PLC/PKC). Ces résultats biochimiques corroborent certains résultats fonctionnels du laboratoire et ouvrent de nouvelles pistes quant à la modulation négative de la neurotransmission par les récepteurs mGluR4 natifs dans le cervelet / At Purkinje Cell - Parallel Fiber synapses, mGluR4 receptors are the only glutamatergic metabotropic receptors of group III to modulate glutamatergic transmission by inhibiting calcium presynaptic influx controlling glutamate release. In heterologous systems, mGluR4 has been shown to activate G proteins of type Gi/o that would be negatively linked to adenylate cyclase (AC). In order to conserve most of physiological interactions, we first studied the receptosome of native mGluR4 in rat cerebellum. We identified 184 putative partners of the receptor. Moreover, in order to confirm these interactions, but also to find other partners, we decided to perform an independent and complementary approach of chromatography affinity. Numerous proteins have been found by this method, particularly proteins belonging to exocytosis and cellular trafficking families. Our results suggest that a partial control of neurotransmission could be due to interaction of mGluR4 with these kinds of proteins. On the other hand, biochemical approaches did not reveal interactions of mGluR4 with some proteins belonging to AC pathway, but with proteins of PLC/PKC pathway. These results are consistent with some functional studies of our lab and gave the way for elucidating the native molecular mechanisms of the cerebellar neurotransmission modulation by mGluR4.

De récentes études ont montré dans les cellules chromaffines que la libération des granules de sécrétion est temporellement et spatialement couplée au processus d’endocytose. Nous avons proposé l’hypothèse que la membrane du granule préserve son intégrité au sein de la membrane plasmique durant l’exocytose avant d’être internalisée ainsi avec ses composants. Cependant, les mécanismes moléculaires de ce processus d’endocytose compensatrice sont encore inconnus. Ainsi, mon projet de thèse vise a répondre à la question suivante : Quels sont les différents mécanismes déclenchant et régulant l’exocytose et l’endocytose compensatrice? Les propriétés physiques des lipides jouent des rôles fondamentaux dans le trafic membranaire. Ils servent de système d’échafaudage pour maintenir la machinerie spécifique à des endroits précis de la membrane plasmique. Par exemple, la formation de microdomaines de gangliosides et de PIP2 au niveau des sites d’exocytose ou encore le mélange de lipides au sein de la bicouche lipidique représentent des processus attractifs pour permettre cette fonction au cours des événements d’exo-endocytose dans les cellules neuroendocrines. De plus, en raison de leur implication importante dans les processus d’exo-endocytose ou dans le remodelage des lipides, l’annexine A2, la synaptotagmine 1, l’oligophrénine1 et la scramblase 1 doivent être considérées comme des signaux potentiels pour le déclenchement de l’endocytose de la membrane granulaire. Au cours de mon doctorat, je me suis intéressée à étudier comment l’exocytose et l’endocytose compensatrice sont régulées par la scramblase1 et l’oligophrénine1 dans les cellules chromaffines de la glande surrénale. / Recent studies in neuroendocrine chromaffin cells have suggested that the secretory granule release is temporally and spatially coupled to a compensatory endocytic process. Hence, we hypothesized that the secretory granule membrane would preserve its integrity within the plasma membrane after exocytosis before being retrieved as such along with its components. However, the underlying molecular mechanisms of this compensatory endocytic process are largely unknown today. Therefore my thesis project is aiming to address the following specific question: What are the different mechanisms triggering and regulating exocytosis and the compensatory endocytosis? Physical properties of lipids play fundamental roles in membrane trafficking. They act as a scaffolding system to maintain specific machinery at restricted site of the plasma membrane. For example, the formation of ganglioside- and PIP2-enriched microdomains at the exocytic sites or the phospholipid scrambling across the bilayer plasma membrane, represent attractive processes to fulfill this function during exo- endocytosis events in neuroendocrine cells. Moreover, in view to their important implication in exo-endocytotic processes or lipid remodeling, annexin-A2, synaptotagmin- 1, oligophrenin-1 and phospholipid scramblase-1 have to be considered as potential signal-triggers of the granule endocytosis. During my PhD, I focused in investigating how exocytosis and compensatory endocytosis are regulated by PLSCR-1 and OPHN1 in adrenal chrommaffin cells.

The nasal mucosa provides a non-invasive route for drug administration to the systemic circulation and potentially directly to the CNS. Nanoparticles made from biodegradable polymers, including PLGA, are of great interest for use in drug delivery systems due to their relative safety and ease of surface modification. Owing to their small size, nanoparticles may provide enhanced targeting and transport through the nasal mucosa. An improved understanding of the mechanisms and pathways of nanoparticle transfer across the nasal mucosa is needed to design effective new nasal delivery systems. This study focuses on the preparation of PLGA nanoparticles in various diameters and with varying surface characteristics followed by the in vitro investigation of the mechanisms of endocytosis and exocytosis of PLGA nanoparticles in the nasal mucosa. PLGA nanoparticles (60 nm or 125 nm) containing the lipophilic fluorescent dye, Nile Red, were prepared using a surfactant-free nanoprecipitation method. In one investigation, the inherent negative surface charge of 60 nm PLGA nanoparticles was modified to a positive charge using a 5th generation polyamidoamine dendrimer (PAMAM) during preparation of nanoparticles. In addition, 60 nm PLGA nanoparticle surfaces were coated by adding 5 % (w/v) bovine serum albumin (BSA) to the nanoparticle dispersion and allowing protein adsorption on the particle surface. Nile Red-loaded PLGA nanoparticles were transported into the epithelial layer and reached the sub-mucosal connective tissues, yet only < 5% of the PLGA nanoparticle load was transferred into the nasal mucosa. Total uptake was size dependent, where the uptake of 60 nm unmodified PLGA nanoparticles was significantly higher than uptake of 125 nm nanoparticles. The amount of Nile Red measured in the tissues after expose to the 125 nm nanoparticles was double the amount from the 60 nm nanoparticles due to differences in the carrying capabilities of the 60 and 125 nm PLGA nanoparticles. Modification of the nanoparticle surface with PAMAM or BSA decreased the uptake of 60 nm PLGA nanoparticles into the nasal mucosa. Endocytic mechanisms involved in the uptake of PLGA nanoparticles were studied using chemical inhibitors. Nanoparticle uptake in the nasal respiratory mucosa involved energy-dependent processes utilizing multiple known mechanisms, including clathrin-mediated endocytosis and macropinocytosis. In the olfactory mucosa, significant energy-independent nanoparticle uptake was also observed. In order to investigate how nanoparticles exit epithelial cells for further distribution to distant tissues, the exocytosis of 60 nm Nile Red-loaded PLGA nanoparticles was evaluated using three different epithelial cell line models, RPMI-2650 (nasal), Calu-3 (lung) and MDCK-II wild type (kidney) cells. Following a 30 min exposure to a 60 nm PLGA nanoparticles dispersion, nanoparticle exocytosis into a protein-free medium was evaluated for additional 30 or 60 min. Only a limited number of NP (~ 20 % of the endocytosed NP) underwent exocytosis into the medium after 60 min, while the majority of the internalized nanoparticles remained within the cells. The measurable transfer of PLGA nanoparticles into the nasal mucosal tissues indicates that they may be useful drug carriers for nasal administration. However, the limited exocytosis of 60 nm NP and the resulting potential for intracellular accumulation may raise toxicity concerns and result in potential cellular injury. While PLGA nanoparticles provide promising drug delivery systems for nasal administration, only with careful design of the nanoparticles, including their size and surface characteristics, will efficient and effective, safe drug delivery be accomplished.

L’exocytose vésiculaire est une voie physiologique majeure de la communication intercellulaire. Dans ce contexte, le TIRFM (microscopie de fluorescence par réflexion totale interne) et l’ampérométrie sont aujourd'hui les deux méthodes analytiques les plus fréquemment utilisées dans l’étude de l’exocytose. En raison de la complémentarité de ces deux techniques d’analyse pour le suivi de la sécrétion exocytotique, leur combinaison pour suivre la sécrétion exocytotique a d'abord été réalisée par notre groupe en 2011. Ce couplage a permis un enregistrement simultané des signaux fluorescents et ampérométriques avec une bonne résolution spatiale et temporelle. L'inconvénient majeur de ce travail reste le chargement indépendant des sondes optique et électrochimique dans les vésicules de sécrétion, ce qui entraîne la détection d’évènements « orphelins » ampérométriques ou optiques ainsi que la faible efficacité de détection des évènements couplés. Par conséquent, dans cette thèse, nous avons tenté de mettre à profit une sonde unique à la fois fluorescente et électroactive pour suivre l’exocytose par la méthodologie couplée TIRFM/ampérométrie. Ainsi, un analogue de neurotransmetteurs monoamine primaire, la 4-(2-amino-éthyl)-6-chloro-7-hydroxy-2H-1-benzopyran-2-one (nommé 1 dans ce travail), a été synthétisé.1 présente une fluorescence forte, stable et pH-dépendante. Lorsque cette entité est excitée à 405 nm, son intensité de fluorescence est presque doublée de pH 5 (valeur intra-vésiculaire) à 7 (valeur milieu extracellulaire). De plus, des études en voltammétrie ont pu mettre en évidence que 1 est oxydable sur électrode de carbone vitreux, microélectrode à fibre de carbone et ITO (oxyde d’indium dopé à l’étain), montrant ainsi une bonne électroactivité. La pénétration cellulaire dans les vésicules de cellules BON N13 a également été démontrée, prouvant la spécificité de l’interaction entre 1 et ces vésicules équipées d’un transporteur de monoamines primaires (VMAT). L’utilisation de 1 comme sonde unique optique et électrochimique pour le suivi de l'exocytose a ensuite été validée séparément dans des cellules BON N13 par TIRFM et ampérométrie. L’enregistrement simultané par fluorescence et électrochimie en utilisant 1 comme sonde double a ensuite été réalisé dans un microdispositif constitué d’électrodes ITO conductrice et transparente. Nos résultats basés sur la sonde unique 1 montrent qu’elle semble plus adaptée que toutes les stratégies antérieures impliquant deux sondes indépendantes. Les résolutions spatiale et temporelle de cette méthode combinée ont permis d'analyser des sécrétions d’exocytose de cellules marquées par 1. Une analyse ultérieure de ces signaux couplés optique et électrochimique sera à même d’étudier la corrélation entre le comportement du pore de fusion (dynamique d'ouverture/de fermeture, stabilité..) détecté par ampérométrie et le mouvement d'une vésicule en trois dimensions (ancrage, amarrage, fusion puis retrait dans le cytoplasme) détecté par TIRFM. / Vesicular exocytosis is a ubiquitous way for intercellular communications. TIRFM (total internal reflection fluorescence microscopy) and amperometry are nowadays the two most frequently used analytical methods with complementary features for its investigation. The combination of these two analytical techniques to track exocytotic secretions was firstly achieved by our group in 2011 and this new technique was demonstrated to show both high temporal and spatial resolutions by simultaneously recording the fluorescent and amperometric signals. The major disadvantage of this former work was the independent loading of optical and electrochemical probes to the secretory vesicles, which resulted in 'sightless' amperometric or optical signals as well as low coupling efficiency. Therefore, in this thesis, we attempted to develop a unique probe with dual fluorescent/electrochemical characteristics to track exocytotic process by TIRFM/amperometry coupling technique. This is why an analog of biogenic monoamine neurotransmitters, 4-(2-aminoethyl)-6-chloro-7-hydroxy-2H-1-benzopyran-2-one hydrochloride (named as 1 in this work) was synthesized. 1 exhibited bright, stable, pH-dependent fluorescence. When excited at 405 nm, its fluorescence intensity was almost doubled with the increase of pH values from 5 (similar to that in the vesicular lumen) to 7 (similar to the extracellular medium). Furthermore, in voltammetry, 1 was demonstrated to be easily electrooxidized on GCE (glassy carbon electrode), CFE (carbon fiber electrode) and ITO (indium tin oxide) electrodes surfaces, showing good electroactivity. 1 was also shown to penetrate easily into the vesicles of BON N13 cells within 1 hour incubation, testifying its specific affinity with these VMAT-equipped (vesicular monoamine transporter) vesicles. The applications of 1 as optical and electrochemical probes for exocytosis monitoring were then separately validated in BON N13 cells by TIRFM and amperometry measurements, respectively. Simultaneous recording of fluorescent and amperometric information by using 1 dual probes loaded cells was subsequently acquired in a microfabricated device constituted by conductive and transparent ITO electrodes. Our results based on the unique probe 1 for electrochemical and fluorescent detection of exocytotic release seemed more adapted than all the previous works involving independent probes. The high spatial and temporal resolutions of this combined method also allowed analyzing consecutive exocytotic secretions as well as overlapped events in 1-stained cells. Further analysis of these two signals with complementary information will shed more light on the correlation of the fusion pore behavior (opening/closure dynamics, stability…) measured by amperometry and the motion of a secretory vesicle in three dimensions (tethering, docking, fusion and retrieval) detected by TIRFM.

Upon vascular injury, activated blood platelets fuse their granules to the plasma membrane and release cargo to regulate the vascular microenvironment, a dynamic process central to platelet function in many critical processes including hemostasis, thrombosis, immunity, wound healing, angiogenesis etc. This granule- plasma membrane fusion is mediated by a family of membrane proteins- Soluble N-ethyl maleimide Attachment Receptor Proteins(SNAREs). SNAREs that reside on vesicle (v-SNAREs) /Vesicle-Associated Membrane Proteins(VAMPs) interact with target/t-SNAREs forming a trans-bilayer complex that facilitates granule fusion. Though many components of exocytic machinery are identified, it is still not clear how it could be manipulated to prevent occlusive thrombosis without triggering bleeding. My work addresses this question by showing how the rates and extents of granule secretion could be regulated by various v-SNAREs. We also show that the granule cargo decondensation is an intermediate to secretion that also contributes to rates of cargo release. Platelets contain four major VAMP isoforms (-2, -3, -7, and -8), however, VAMP-8 and -7 play a primary role while VAMP-2 and -3 are ancillary in secretion. To exploit this heterogeneity in VAMP usage, platelet-specific V-2/3-/- and V-2/3/8-/- mouse models were generated and characterized to understand how secretion influences hemostasis. We found that each VAMP isoform differentially contributes by altering the rates and extents of cargo release. The loss of VAMP-2 and -3 had a minimal impact while the loss of VAMP-2, -3 and -8 significantly reduced the granule secretion. Platelet activation and aggregation were not affected though the spreading was reduced in V-2/3/8-/- platelets indicating the importance of secretion in spreading. Though coagulation pathways were unaltered, PS exposure was reduced in both V-2/3-/- and V-2/3/8-/- platelets suggesting diminished procoagulant activity. In vivo experiments showed that V-2/3/8-/- animals bled profusely upon tail transaction and failed to form occlusive thrombus upon arterial injury while V-2/3-/- animals did not display any hemostatic deficiency. These data suggest that about 40-50% reduction in secretion provides protection against thrombosis without compromising hemostasis and beyond 50% secretion deficiency, the animals fail to form functional thrombi and exhibit severe bleeding. Additionally, detailed structural analysis of activated platelets suggests that the post-stimulation cargo dissolution depends on an agonist concentration and stimulation duration. This process is VAMP-dependent and represents intermediate steps leading to a full exodus of cargo. Moreover, we also show that VAMP-8 is important for compound fusion events and regulates fusion pore size. This is a first comprehensive report that shows how manipulation of the exocytic machinery have an impact on secretion and ultimately on hemostasis. These animals will be instrumental in future investigations of platelet secretion in many other vascular processes.

Coupling the small GTPase Rab3 to the Synaptic Vesicle Cycle

No description available.

No description available.

<p>Due to its close proximity to the gastrointestinal tract, the human urinary tract is</p><p>subjected to constant barrage by gut-­associated bacteria. However, for the most part, this tract has resisted infection by various microbes. The impregnability of the urinary tract to microbial colonization is attributable to the ability of the bladder to promptly sense and mount robust responses to microbial challenge. A powerful mechanism for the elimination of invading bacteria was recently described in bladder epithelial cells, involving non-­lytic ejection of intracellular bacteria back into the extracellular milieu. In spite of the effectiveness of this defense strategy, much of the underlying mechanisms surrounding how this powerful cellular defense activity detects intracellular UPEC and shuttles them from their intracellular location to the plasma membrane of BECs to be exported remains largely a mystery.</p><p> Here, we describe uropathogenic E.coli (UPEC) expelled from infected bladder</p><p>epithelium cells (BECs) within membrane-­bound vesicles as a distinct cellular defense</p><p>response. Examination of the intracellular UPEC revealed that intracellular bacteria were</p><p>initially processed via autophagy, the conventional degradative pathway, then delivered</p><p>into multivesicular bodies (MVBs) and encapsulated in nascent intraluminal vesicle membrane. We further show the bacterial expulsion is triggered when intracellular UPEC follow the natural degradative trafficking pathway and reach lysosomes and attempt to neutralize its pH to avoid degradation. This pathogen-­mediated activity is detected by mucolipin TRP channel 3 (TRPML3), a transient receptor potential cation channel localized on lysosomes, which spontaneously initiates lysosome exocytosis resulting in expulsion of exosome-­encased bacteria. These studies reveal a cellular default system for lysosome homeostasis and also, how it is coopted by the autonomous defense program to clear recalcitrant pathogens.</p> / Dissertation

The focus of this thesis is to characterise the interactions between GLUT4-related SNARE proteins – syntaxin4, SNAP23 and VAMP2 – and a regulatory protein, Munc18c. GLUT4 is the primary insulin-regulated glucose transporter and is presentin fat and muscle cells. GLUT4 is held in intracellular pools of vesicles until it is transported to the cell surface upon insulin stimulation. Insulin initiates a cellular signalling cascade via the insulin receptor on the cell membrane, which in turn stimulates GLUT4 vesicles to move to the cell surface where they fuse to the plasmamembrane via SNARE proteins. SNAREs are membrane-anchored proteins present on both vesicle and target membranes that form a tight complex which brings themembranes together for fusion. Fusion of vesicles to the target membrane releases the vesicular cargo.SNARE-mediated membrane fusion is a conserved mechanism that controls many other vesicle fusion processes such as neurotransmitter release and yeast vesicular trafficking. However, the regulation of the SNARE mechanism is not fully understood. SNAREs can interact with many other proteins that could act as regulatory factors,and studies have focused primarily on a group of effector proteins called Sec1p/Munc18 (SM) proteins. SM proteins were discovered and characterised because they bind to one type of SNARE protein, syntaxin. The SM protein that interacts with the GLUT4-related SNARE, syntaxin4, is Munc18c.The aim of this thesis was to investigate Munc18c interactions with SNARE proteins, principally syntaxin4, using biochemical techniques with purified recombinant proteins. This work was carried out in several stages including: 1) development of methods to produce and purify GLUT4-related SNARE proteins, SNARE complexes and Munc18c, 2) development of an assay to quantify Munc18c interactions with binding partners using surface plasmon resonance, 3) investigation into interactions between Munc18c and SNARE ternary complex, 4) characterising Munc18c interactions with syntaxin4, and 5) developing a method to produce selenomethionine-containing Munc18c in a baculovirus system to be used in structural studies. The methods and outcomes of these experiments are described inthis thesis. There were two major outcomes from this work. Firstly, Munc18c interacts with SNARE ternary complex, and secondly, Munc18c requires only the N-terminal 29residues of syntaxin4 for an interaction to occur. These results were determined using pulldown assays with purified proteins, as well as other chromatographic methods to show that protein complexes were formed. The steps taken to develop these binding assays are also discussed. Initial crystallisation conditions forMunc18c-HIS and a peptide consisting of syntaxin4 residues 1-20 have been identified using crystallisation screens. The interactions determined for Munc18c binding to Sx4 are in direct contrast to those of neuronal SM protein, Munc18a, and its interaction with neuronal SNARE proteins - Munc18a does not bind to its ternary complex and binds to the entire cytoplasmic domain of Sx1a. Rather, the Munc18c:Sx4 interactions are similar to that for the yeast SM protein, Sly1p, which can interact with both its SNARE ternary complex and with its syntaxin via the Nterminal residues. Another interesting outcome of this research was that syntaxin4 binds to metals (cobalt and nickel). This finding represents the first reported for a syntaxin interacting with metals. Preliminary results indicate that un-tagged syntaxin4 can bind to cobalt resin, and to nickel immobilised on a chip. This interesting and novel property of syntaxin4 binding was serendipitously discovered while investigating conditions for the Munc18c assay. Overall, I have shown that Munc18c, the SM protein involved in GLUT4 trafficking, interacts with SNARE proteins in a different manner to its mammalian counterpart inneurons, Munc18a, and is more like Sly1p, a yeast ER-Golgi SM protein. Munc18c interacts with SNARE complexes and only the N-terminal residues of syntaxin4.These interactions demonstrate that the regulatory mechanism for SNARE-mediated fusion is conserved between yeast and mammals. This finding has several implications for the role of Munc18c in the exocytosis of GLUT4-containing vesicles. Munc18c could act at several stages in the fusion process via syntaxin4 binding.These interactions could involve binding to other proteins (such as synip or tomosyn), conformational switching of syntaxin4 or interaction with metal ions to induce conformational changes in the proteins. Finally, these studies of GLUT4 exocytosis contribute to our understanding of glucose transport disorders such as Type 2 diabetes and could one day pave the way for the design of therapeutic agents.