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Accueil > Agenda > Les séminaires Jean Roche > Voie d’exocytose insensible à la neurotoxine tétanique : fonctions avant et (...)

Voie d’exocytose insensible à la neurotoxine tétanique : (...)

Mercredi 12 octobre 2006,11h, salle Lissitzky.


1 : Med Sci (Paris). 2005 Oct ;21(10):789-790.

Tetanus neurotoxin-mediated cleavage of cellubrevin impairs epithelial cell migration and integrin-dependent cell adhesion.

[Article in French]

Proux-Gillardeaux V, Galli T.

Trafic membranaire et Morphogenese neuronale et epitheliale, Equipe Avenir Inserm, Institut Jacques Monod, 2, place Jussieu, 75005 Paris, France. thierry

2 : Biophys J. 2005 Sep 16 ; [Epub ahead of print]

Weak Effect of Membrane Diffusion on the Rate of Receptor Accumulation at Adhesive Contacts.

Thoumine O, Saint-Michel E, Dequidt C, Falk J, Rudge R, Galli T, Faivre-Sarrailh C, Choquet D.

CNRS 5091-Universite Bordeaux 2.

To assess if membrane diffusion could affect the kinetics of receptor recruitment at adhesive contacts, we transfected neurons with GFP-tagged IgCAMs of varying length (25-180 kD), and measured the lateral mobility of single Quantum Dots bound to those receptors at the cell surface. The diffusion coefficient varied within a physiological range (0.1-0.5 microm2/s), and was inversely proportional to the size of the receptor. We then triggered adhesive contact formation by placing anti-GFP coated microspheres on growth cones using optical tweezers, and measured surface receptor recruitment around microspheres by time-lapse fluorescence imaging. The accumulation rate was rather insensitive to the type of receptor, suggesting that the long range membrane diffusion of IgCAMs is not a limiting step in the initiation of neuronal contacts.

3 : Proc Natl Acad Sci U S A. 2005 May 3 ;102(18):6362-7. Epub 2005 Apr 25.

Tetanus neurotoxin-mediated cleavage of cellubrevin impairs epithelial cell migration and integrin-dependent cell adhesion.

Proux-Gillardeaux V, Gavard J, Irinopoulou T, Mege RM, Galli T.

Membrane Traffic in Neuronal and Epithelial Morphogenesis, Institut National de la Sante et de la Recherche Medicale Avenir Team, Centre National de la Recherche Scientifique, Universites Paris 6 et 7, 75005 Paris, France.

A role for endocytosis and exocytosis in cell migration has been proposed but not yet demonstrated. Here, we show that cellubrevin (Cb), an early endosomal v-SNARE, mediates trafficking in the lamellipod of migrating epithelial cells and partially colocalizes with markers of focal contacts. Expression of tetanus neurotoxin, which selectively cleaves Cb, significantly reduced the speed of migrating epithelial cells. Furthermore, expression of tetanus neurotoxin enhanced the adhesion of epithelial cells to collagen, laminin, fibronectin, and E-cadherin ; altered spreading on collagen ; and impaired the recycling of beta1 integrins. These results suggest that Cb-dependent membrane trafficking participates in cell motility through the regulation of cell adhesion.

4 : Traffic. 2005 May ;6(5):366-73.

The tetanus neurotoxin-sensitive and insensitive routes to and from the plasma membrane : fast and slow pathways ?

Proux-Gillardeaux V, Rudge R, Galli T.

Team ’Avenir’ INSERM Membrane Traffic in Neuronal & Epithelial Morphogenesis Institut Jacques Monod CNRS/Univ. Paris 6/Univ. Paris 7 UMR7592, 2, place Jussieu, F-75251 Paris Cedex 05, France.

Intracellular membrane trafficking in eukaryotes involves the budding of vesicles from a donor compartment, their translocation, and subsequent fusion with a target membrane. This last step has been shown to involve SNARE proteins, classified into two categories, vesicular (v)-SNAREs and target (t)-SNAREs. It is the pairing of v- and t-SNAREs that is responsible for bringing the lipid bilayers together for membrane fusion. Key to the discovery of SNAREs is the sensitivity of their neuronal synaptic prototypes, which mediate the release of neurotransmitters, to clostridial neurotoxins. In this review, we focus on tetanus neurotoxin-sensitive and tetanus neurotoxin-insensitive v-SNAREs, in particular synaptobrevin and cellubrevin, both tetanus neurotoxin-sensitive and Tetanus neurotoxin-Insensitive Vesicle-Associated Membrane Protein (TI-VAMP, also called VAMP7). The brevins are characterized by an RD sequence in the middle of their SNARE motif whereas TI-VAMP has an RG sequence. These two categories of exocytic v-SNAREs define two important routes to and from the plasma membrane : one sensitive, the other insensitive to tetanus neurotoxin. We also discuss the central role of the endosomal system that could be considered, as already suggested for Rab proteins, as a mosaic of v-SNAREs, thus raising the question of whether or not these two routes can merge, and if so, how and where.

5 : Genome Res. 2005 Mar ;15(3):376-84. Epub 2005 Feb 14.

Protein interaction mapping : a Drosophila case study.

Formstecher E, Aresta S, Collura V, Hamburger A, Meil A, Trehin A, Reverdy C, Betin V, Maire S, Brun C, Jacq B, Arpin M, Bellaiche Y, Bellusci S, Benaroch P, Bornens M, Chanet R, Chavrier P, Delattre O, Doye V, Fehon R, Faye G, Galli T, Girault JA, Goud B, de Gunzburg J, Johannes L, Junier MP, Mirouse V, Mukherjee A, Papadopoulo D, Perez F, Plessis A, Rosse C, Saule S, Stoppa-Lyonnet D, Vincent A, White M, Legrain P, Wojcik J, Camonis J, Daviet L.

Hybrigenics, 75014 Paris, France.

The Drosophila (fruit fly) model system has been instrumental in our current understanding of human biology, development, and diseases. Here, we used a high-throughput yeast two-hybrid (Y2H)-based technology to screen 102 bait proteins from Drosophila melanogaster, most of them orthologous to human cancer-related and/or signaling proteins, against high-complexity fly cDNA libraries. More than 2300 protein-protein interactions (PPI) were identified, of which 710 are of high confidence. The computation of a reliability score for each protein-protein interaction and the systematic identification of the interacting domain combined with a prediction of structural/functional motifs allow the elaboration of known complexes and the identification of new ones. The full data set can be visualized using a graphical Web interface, the PIMRider (, and is also accessible in the PSI standard Molecular Interaction data format. Our fly Protein Interaction Map (PIM) is surprisingly different from the one recently proposed by Giot et al. with little overlap between the two data sets. Analysis of the differences in data sets and methods suggests alternative strategies to enhance the accuracy and comprehensiveness of the post-genomic generation of broad-scale protein interaction maps.

6 : Sci STKE. 2005 Jan 18 ;2005(267):tr2.

A model for fast-track exocytosis of synaptic vesicles.

Galli T, Haucke V.

Membrane Traffic and Neuronal Plasticity Group, INSERM U536, Institut du Fer-a-moulin, 75005 Paris, France. thierry>

The animation illustrates one model for "kiss-and-run" or fast-track release of neurotransmitter through a calcium-triggered transient pore formed between the synaptic vesicle and plasma membrane. The vesicles remain attached to the plasma membrane throughout the cycle. The animation illustrates a role for SNARE proteins and dynamin in the formation and closure of the pore. The animation would be useful in both neurobiology courses, but also any cell biology course that explores calcium-regulated secretion events.

7 : Sci STKE. 2005 Jan 18 ;2005(267):tr1.

Calcium-triggered exocytosis and clathrin-mediated endocytosis of synaptic vesicles.

Galli T, Haucke V.

Membrane Traffic and Neuronal Plasticity Group, INSERM U536, Institut du Fer-a-moulin, 75005 Paris, France. thierry

The animation illustrates the recruitment, calcium-triggered exocytosis, and clathrin-mediated endocytosis of synaptic vesicles. The animation shows not only the proteins involved in synaptic vesicle fusion and recycling, but also changes in the lipid composition that are important for targeting of proteins to sites of membrane internalization. The animation would be useful in both neurobiology courses, but also any cell biology course that explores clathrin-mediated endocytic and calcium-regulated secretion events.

8 : Med Sci (Paris). 2005 Jan ;21(1):9-10.

[Activation-induced polarized recycling targets T cell receptors to the immunological synapse]

[Article in French]

Alcover A, Thoulouze MI, Galli T.

9 : Sci STKE. 2004 Dec 21 ;2004(264):re19.

Cycling of synaptic vesicles : how far ? How fast !

Galli T, Haucke V.

Membrane Traffic and Neuronal Plasticity Group, INSERM U536, Institut du Fer-a-moulin, 75005 Paris, France. thierry>

Synaptic transmission is based on the regulated exocytotic fusion of synaptic vesicles filled with neurotransmitter. In order to sustain neurotransmitter release, these vesicles need to be recycled locally. Recent data suggest that two tracks for the cycling of synaptic vesicles coexist : a slow track in which vesicles fuse completely with the presynaptic plasma membrane, followed by clathrin-mediated recycling of the vesicular components, and a fast track that may correspond to the transient opening and closing of a fusion pore. In this review, we attempt to provide an overview of the components involved in both tracks of vesicle cycling, as well as to identify possible mechanistic links between these two pathways.

10 : Trends Biochem Sci. 2004 Dec ;29(12):682-8.

Longins and their longin domains : regulated SNAREs and multifunctional SNARE regulators.

Rossi V, Banfield DK, Vacca M, Dietrich LE, Ungermann C, D’Esposito M, Galli T, Filippini F.

Molecular Biology and Bioinformatics Unit (MOLBINFO), Department of Biology, University of Padua, 35131 Padua, Italy.

Longins are the only R-SNAREs that are common to all eukaryotes and are characterized by a conserved N-terminal domain with a profilin-like fold called a longin domain (LD). These domains seem to be essential for regulating membrane trafficking and they mediate unexpected biochemical functions via a range of protein-protein and intramolecular binding specificities. In addition to the longins, proteins involved in the regulation of intracellular trafficking, such as subunits of the adaptor and transport protein particle complexes, also have LD-like folds. The functions and cellular localization of longins are regulated at several levels and the longin prototypes TI-VAMP, Sec22 and Ykt6 show different distributions among eukaryotes, reflecting their modular and functional diversity. In mammals, TI-VAMP and Ykt6 are crucial for neuronal function, and defects in longin structure or function might underlie some human neurological pathologies.

11 : EMBO J. 2004 Oct 27 ;23(21):4166-76. Epub 2004 Oct 7.

TI-VAMP/VAMP7 is required for optimal phagocytosis of opsonised particles in macrophages.

Braun V, Fraisier V, Raposo G, Hurbain I, Sibarita JB, Chavrier P, Galli T, Niedergang F.

Membrane and Cytoskeleton Dynamics group, UMR144 CNRS-Institut Curie, rue d’Ulm 75248 Paris, France.

Phagocytosis relies on extension of plasmalemmal pseudopods generated by focal actin polymerisation and delivery of membranes from intracellular pools. Here we show that compartments of the late endocytic pathway, bearing the tetanus neurotoxin-insensitive vesicle-associated membrane protein (TI-VAMP/VAMP7), are recruited upon particle binding and undergo exocytosis before phagosome sealing in macrophages during Fc receptor (FcR)-mediated phagocytosis. Expression of the dominant-negative amino-terminal domain of TI-VAMP or depletion of TI-VAMP with small interfering RNAs inhibited phagocytosis mediated by Fc or complement receptors. In addition, inhibition of TI-VAMP activity led to a reduced exocytosis of late endocytic vesicles and this resulted in an early blockade of pseudopod extension, as observed by scanning electron microscopy. Therefore, TI-VAMP defines a new pathway of membrane delivery required for optimal FcR-mediated phagocytosis.

12 : Mol Biol Cell. 2004 Dec ;15(12):5565-73. Epub 2004 Oct 6.

Insulin and hypertonicity recruit GLUT4 to the plasma membrane of muscle cells by using N-ethylmaleimide-sensitive factor-dependent SNARE mechanisms but different v-SNAREs : role of TI-VAMP.

Randhawa VK, Thong FS, Lim DY, Li D, Garg RR, Rudge R, Galli T, Rudich A, Klip A.

Programme in Cell Biology, The Hospital for Sick Children, Toronto, Ontario M5G 1X8, Canada.

Insulin and hypertonicity each increase the content of GLUT4 glucose transporters at the surface of muscle cells. Insulin enhances GLUT4 exocytosis without diminishing its endocytosis. The insulin but not the hypertonicity response is reduced by tetanus neurotoxin, which cleaves vesicle-associated membrane protein (VAMP)2 and VAMP3, and is rescued upon introducing tetanus neurotoxin-resistant VAMP2. Here, we show that hypertonicity enhances GLUT4 recycling, compounding its previously shown ability to reduce GLUT4 endocytosis. To examine whether the canonical soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) mechanism is required for the plasma membrane fusion of the tetanus neurotoxin-insensitive GLUT4 vesicles, L6 myoblasts stably expressing myc-tagged GLUT4 (GLUT4myc) were transiently transfected with dominant negative N-ethylmaleimide-sensitive factor (NSF) (DN-NSF) or small-interfering RNA to tetanus neurotoxin-insensitive VAMP (TI-VAMP siRNA). Both strategies markedly reduced the basal level of surface GLUT4myc and the surface gain of GLUT4myc in response to hypertonicity. The insulin effect was abolished by DN-NSF, but only partly reduced by TI-VAMP siRNA. We propose that insulin and hypertonicity recruit GLUT4myc from partly overlapping, but distinct sources defined by VAMP2 and TI-VAMP, respectively.

13 : Biol Cell. 2004 May ;96(4):251-6.

VAMP subfamilies identified by specific R-SNARE motifs.

Rossi V, Picco R, Vacca M, D’Esposito M, D’Urso M, Galli T, Filippini F.

Department of Biology, University of Padua, 35131 Padova, Italy.

In eukaryotes, interactions among the alpha-helical coiled-coil domains (CCDs) of soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) play a pivotal role in mediating the fusion among vesicles and target membranes. Surface residues of such CCDs are major candidates to regulate the specificity of membrane fusion, as they may alter local charge at the interaction layers and surface of the fusion complex, possibly modulating its formation and/or the binding of non-SNARE regulatory factors. Based on alternate patterns in surface residues, we have identified two motifs which group vesicular SNAREs in two novel subfamilies : RG-SNAREs and RD-SNAREs. The RG-SNARE CCD is common to all members of the widely conserved family of long VAMPs or longins and to yeast and non-neuronal VAMPs, possibly mediating "basic" fusion mechanisms ; instead, only synaptobrevins from Bilateria share an RD-SNARE CCD, which is likely to mediate interactions to specific, yet unknown, regulatory factors and/or be the landmark of rapid fusion reactions like that mediating the release of neurotransmitters.

14 : Immunity. 2004 May ;20(5):577-88.

Activation-induced polarized recycling targets T cell antigen receptors to the immunological synapse ; involvement of SNARE complexes.

Das V, Nal B, Dujeancourt A, Thoulouze MI, Galli T, Roux P, Dautry-Varsat A, Alcover A.

Unite de Biologie des Interactions Cellulaires, Centre National de la Recherche Scientifique, Unite de Recherche Associee-2582, 75724 Paris Cedex 15, France.

The mechanism by which T cell antigen receptors (TCR) accumulate at the immunological synapse has not been fully elucidated. Since TCRs are continuously internalized and recycled back to the cell surface, we investigated the role of polarized recycling in TCR targeting to the immunological synapse. We show here that the recycling endosomal compartment of T cells encountering activatory antigen-presenting cells (APCs) polarizes towards the T cell-APC contact site. Moreover, TCRs in transit through recycling endosomes are targeted to the immunological synapse. Inhibition of T cell polarity, constitutive TCR endocytosis, or recycling reduces TCR accumulation at the immunological synapse. Conversely, increasing the amount of TCRs in recycling endosomes before synapse formation enhanced their accumulation. Finally, we show that exocytic t-SNAREs from T cells cluster at the APC contact site and that tetanus toxin inhibits TCR accumulation at the immunological synapse, indicating that vesicle fusion mediated by SNARE complexes is involved in TCR targeting to the immunological synapse.

15 : Traffic. 2004 May ;5(5):371-82.

A mutant impaired in SNARE complex dissociation identifies the plasma membrane as first target of synaptobrevin 2.

Martinez-Arca S, Arold S, Rudge R, Laroche F, Galli T.

Membrane Traffic and Neuronal Plasticity, INSERM U536, Institut du Fer-a-Moulin, 75005, Paris, France.

Membrane fusion depends on the formation of a complex of four SNARE motifs, three that bear a central glutamine and are localized in the target membrane (t-SNARE) and one that bears an arginine and is localized in the donor vesicle (v-SNARE). We have characterized the arginine 56 to proline mutant (R56P) of synaptobrevin-2 (Sb). SbR56P was blocked at the plasma membrane in association with the endogenous plasma membrane t-SNARE due to an inhibition of SNARE complex dissociation, suggesting that the plasma membrane is its first target. Cell surface blockade of SbR56P could be rescued by coexpression of synaptophysin, a partner of Sb. Sb was blocked at the plasma membrane but SNARE complexes were unaffected in cells expressing defective dynamin, indicating that the phenotype of SbR56P was not due to an internalization defect. When expressed in neurons, SbR56P localized both to axonal and dendritic plasma membranes, showing that both domains are initial targets of Sb. The R56P mutation affects a highly conserved position in v-SNAREs, and might thus provide a general tool for identifying their first target membranes.

16 : J Biol Chem. 2004 May 7 ;279(19):20471-9. Epub 2004 Mar 1.

Identification of SNAREs involved in synaptotagmin VII-regulated lysosomal exocytosis.

Rao SK, Huynh C, Proux-Gillardeaux V, Galli T, Andrews NW.

Section of Microbial Pathogenesis and Department of Cell Biology, Yale University School of Medicine, Boyer Center for Molecular Medicine, 295 Congress Avenue, New Haven, CT 06511, USA.

Ca2+-regulated exocytosis of lysosomes has been recognized recently as a ubiquitous process, important for the repair of plasma membrane wounds. Lysosomal exocytosis is regulated by synaptotagmin VII, a member of the synaptotagmin family of Ca2+-binding proteins localized on lysosomes. Here we show that Ca2+-dependent interaction of the synaptotagmin VII C(2)A domain with SNAP-23 is facilitated by syntaxin 4. Specific interactions also occurred in cell lysates between the plasma membrane t-SNAREs SNAP-23 and syntaxin 4 and the lysosomal v-SNARE TI-VAMP/VAMP7. Following cytosolic Ca2+ elevation, SDS-resistant complexes containing SNAP-23, syntaxin 4, and TI-VAMP/VAMP7 were detected on membrane fractions. Lysosomal exocytosis was inhibited by the SNARE domains of syntaxin 4 and TI-VAMP/VAMP7 and by cleavage of SNAP-23 with botulinum neurotoxin E, thereby functionally implicating these SNAREs in Ca2+-regulated exocytosis of conventional lysosomes.

17 : Neuron. 2004 Feb 19 ;41(4):599-610.

SNAP-25 modulation of calcium dynamics underlies differences in GABAergic and glutamatergic responsiveness to depolarization.

Verderio C, Pozzi D, Pravettoni E, Inverardi F, Schenk U, Coco S, Proux-Gillardeaux V, Galli T, Rossetto O, Frassoni C, Matteoli M.

Department of Medical Pharmacology, CNR Institute of Neuroscience, Center of Excellence for Neurodegenerative Diseases, University of Milano, Via vanvitelli 32, Milano 20129, Italy.

SNAP-25 is a component of the SNARE complex implicated in synaptic vesicle exocytosis. In this study, we demonstrate that hippocampal GABAergic synapses, both in culture and in brain, lack SNAP-25 and are resistant to the action of botulinum toxins type A and E, which cleave this SNARE protein. Relative to glutamatergic neurons, which express SNAP-25, GABAergic cells were characterized by a higher calcium responsiveness to depolarization. Exogenous expression of SNAP-25 in GABAergic interneurons lowered calcium responsiveness, and SNAP-25 silencing in glutamatergic neurons increased calcium elevations evoked by depolarization. Expression of SNAP-25(1-197) but not of SNAP-25(1-180) inhibited calcium responsiveness, pointing to the involvement of the 180-197 residues in the observed function. These data indicate that SNAP-25 is crucial for the regulation of intracellular calcium dynamics and, possibly, of network excitability. SNAP-25 is therefore a multifunctional protein that participates in exocytotic function both at the mechanistic and at the regulatory level.

18 : Biol Cell. 2003 Oct ;95(7):419-24.

The cell outgrowth secretory endosome (COSE) : a specialized compartment involved in neuronal morphogenesis.

Alberts P, Galli T.

Membrane Traffic and Neuronal Plasticity, INSERM U536, 17, rue du Fer a Moulin, 75005 Paris, France.

The role of intracellular membrane trafficking in cellular morphogenesis is still unclear. We propose here a prominent function of a recently identified compartment that we propose to call the cell outgrowth secretory endosome (COSE), the exocytosis of which is controlled by the v-SNARE TIVAMP and by cell-cell adhesion.

19 : Mol Biol Cell. 2003 Oct ;14(10):4207-20. Epub 2003 Jun 27.

Cross talk between tetanus neurotoxin-insensitive vesicle-associated membrane protein-mediated transport and L1-mediated adhesion.

Alberts P, Rudge R, Hinners I, Muzerelle A, Martinez-Arca S, Irinopoulou T, Marthiens V, Tooze S, Rathjen F, Gaspar P, Galli T.

Membrane Traffic and Neuronal Plasticity, Institut National de la Sante et de la Recherche Medicale U536, F-75005 Paris, France.

The membrane-trafficking pathway mediated by tetanus neurotoxin-insensitive vesicle-associated membrane protein (TI-VAMP) in neurons is still unknown. We show herein that TI-VAMP expression is necessary for neurite outgrowth in PC12 cells and hippocampal neurons in culture. TI-VAMP interacts with plasma membrane and endosomal target soluble N-ethylmaleimide-sensitive factor attachment protein receptors, suggesting that TI-VAMP mediates a recycling pathway. L1, a cell-cell adhesion molecule involved in axonal outgrowth, colocalized with TI-VAMP in the developing brain, neurons in culture, and PC12 cells. Plasma membrane L1 was internalized into the TI-VAMP-containing compartment. Silencing of TI-VAMP resulted in reduced expression of L1 at the plasma membrane. Finally, using the extracellular domain of L1 and N-cadherin immobilized on beads, we found that the silencing of TI-VAMP led to impaired L1- but not N-cadherin-mediated adhesion. Furthermore, TI-VAMP- but not synaptobrevin 2-containing vesicles accumulated at the site of the L1 bead-cell junction. We conclude that TI-VAMP mediates the intracellular transport of L1 and that L1-mediated adhesion controls this membrane trafficking, thereby suggesting an important cross talk between membrane trafficking and cell-cell adhesion.

20 : Proc Natl Acad Sci U S A. 2003 Jul 22 ;100(15):9011-6. Epub 2003 Jul 9.

A dual mechanism controlling the localization and function of exocytic v-SNAREs.

Martinez-Arca S, Rudge R, Vacca M, Raposo G, Camonis J, Proux-Gillardeaux V, Daviet L, Formstecher E, Hamburger A, Filippini F, D’Esposito M, Galli T.

Membrane Traffic and Neuronal Plasticity, Institut National de la Sante et de la Recherche Medicale U536, Institut du Fer-a-Moulin, 75005 Paris, France.

SNARE [soluble NSF (N-ethylmaleimide-sensitive factor) attachment protein receptor] proteins are essential for membrane fusion but their regulation is not yet fully understood. We have previously shown that the amino-terminal Longin domain of the v-SNARE TI-VAMP (tetanus neurotoxin-insensitive vesicle-associated membrane protein)/VAMP7 plays an inhibitory role in neurite outgrowth. The goal of this study was to investigate the regulation of TI-VAMP as a model of v-SNARE regulation. We show here that the Longin domain (LD) plays a dual role. First, it negatively regulates the ability of TI-VAMP and of a Longin/Synaptobrevin chimera to participate in SNARE complexes. Second, it interacts with the adaptor complex AP-3 and this interaction targets TI-VAMP to late endosomes. Accordingly, in mocha cells lacking AP-3 delta, TI-VAMP is retained in an early endosomal compartment. Furthermore, TI-VAMPc, an isoform of TI-VAMP lacking part of the LD, does not interact with AP-3, and therefore is not targeted to late endosomes ; however, this shorter LD still inhibits SNARE-complex formation. These findings support a mechanism controlling both localization and function of TI-VAMP through the LD and clathrin adaptors. Moreover, they point to the amino-terminal domains of SNARE proteins as multifunctional modules responsible for the fine tuning of SNARE function.

21 : Dev Cell. 2003 Jul ;5(1):161-74.

Retroviral genomic RNAs are transported to the plasma membrane by endosomal vesicles.

Basyuk E, Galli T, Mougel M, Blanchard JM, Sitbon M, Bertrand E.

IGMM-CNRS UMR5535, Universite Montpellier II, IFR 24, 1919, route de Mende, 34293 Cedex 5, Montpellier, France.

The viral genomes of alpha- and gamma-retroviruses follow an outbound route through the cytoplasm before assembling with the budding particle at the plasma membrane. We show here that murine leukemia virus (MLV) RNAs are transported on lysosomes and transferrin-positive endosomes. Transport on transferrin-positive vesicles requires both Gag and Env polyproteins. In the presence of Env, Gag is rerouted from lysosomes to transferrin-positive endosomes, and virion production becomes highly sensitive to drugs poisoning vesicular and endosomal traffic. Vesicular transport of the RNA does not require prior endocytosis, indicating that it is recruited directly from the cytosol. Viral prebudding complexes containing Env, Gag, and retroviral RNAs are thus formed on endosomes, and subsequently routed to the plasma membrane. This may allow retroviruses to hijack the endosomal machinery as part of their biosynthetic pathway. More generally, tethering to vesicles may provide an efficient mechanism for directed RNA transport.

22 : J Cell Sci. 2003 Jul 1 ;116(Pt 13):2805-16. Epub 2003 May 20.

Ectopic expression of syntaxin 1 in the ER redirects TI-VAMP- and cellubrevin-containing vesicles.

Martinez-Arca S, Proux-Gillardeaux V, Alberts P, Louvard D, Galli T.

Membrane Traffic and Neuronal Plasticity, INSERM U536, Institut du Fer-a-Moulin, 75005 Paris, France.

SNARE proteins are key mediators of membrane fusion. Their function in ensuring compartmental specificity of membrane fusion has been suggested by in vitro studies but not demonstrated in vivo. We show here that ectopic expression of the plasma membrane t-SNARE heavy chain syntaxin 1 in the endoplasmic reticulum induces the redistribution of its cognate vesicular SNAREs, TI-VAMP and cellubrevin, and its light chain t-SNARE SNAP-23. These effects were prevented by co-expressing nSec1. Expression of syntaxin 1 alone impaired the cell surface expression of TI-VAMP and cellubrevin but not the recycling of transferrin receptor. TI-VAMP, cellubrevin and SNAP-23 associated in vivo with exogenous syntaxin 1. Redistribution of TI-VAMP in the ER of syntaxin-1-expressing cells was microtubule dependent and impaired the trafficking of CD63, a cargo of TI-VAMP-containing vesicles. We conclude that the destination of v-SNAREs is driven by their specific interaction with cognate t-SNAREs. Our in vivo data provide strong support for the theory that highly specific v-SNARE-t-SNARE interactions control compartmental specificity of membrane fusion.

23 : Biochem J. 2003 Feb 15 ;370(Pt 1):213-21.

D53 is a novel endosomal SNARE-binding protein that enhances interaction of syntaxin 1 with the synaptobrevin 2 complex in vitro.

Proux-Gillardeaux V, Galli T, Callebaut I, Mikhailik A, Calothy G, Marx M.

Regulations Cellulaires et Oncogenese, UMR 146 du CNRS, Institut Curie, Centre Universitaire, 91405 Orsay Cedex, France.

Synaptobrevin 2 (Sb2), syntaxin1 (Stx1), and synaptosomal-associated protein of 25 kDa (SNAP-25) are the main components of the soluble N -ethylmaleimide-sensitive fusion protein attachment protein receptor (SNARE) complex involved in fusion of synaptic vesicles with the presynaptic plasma membrane. We report the characterization of D53, a novel SNARE-binding protein preferentially expressed in neural and neuro-endocrine cells. Its two-dimensional organization, established by the hydrophobic cluster analysis, is reminiscent of SNARE proteins. D53 contains two putative helical regions, one of which includes a large coiled-coil domain involved in the interaction with Sb2 in vitro. Following subcellular fractionation, endogenous D53 was specifically detected in the membrane-containing fraction of PC12 cells, where it co-immunoprecipitated with Sb2. Analysis by confocal microscopy showed that, in these cells, endogenous D53 co-localized partially with the transferrin receptor in early endosomes. In vitro assays revealed that binding properties of D53 to Stx1 and Sb2 are comparable with those of SNAP-25. Furthermore, D53 forms Sb2/Stx1/D53 complexes in vitro in a manner similar to SNAP-25. We propose that D53 could be involved in the assembly or disassembly of endosomal SNARE complexes by regulating Sb2/Stx interaction.

24 : J Cell Biol. 2002 Feb 18 ;156(4):653-64. Epub 2002 Feb 11.

Early/recycling endosomes-to-TGN transport involves two SNARE complexes and a Rab6 isoform.

Mallard F, Tang BL, Galli T, Tenza D, Saint-Pol A, Yue X, Antony C, Hong W, Goud B, Johannes L.

UMR144 Curie/CNRS, Institut Curie, F-75248 Paris Cedex 05, France.

The molecular mechanisms underlying early/recycling endosomes-to-TGN transport are still not understood. We identified interactions between the TGN-localized putative t-SNAREs syntaxin 6, syntaxin 16, and Vti1a, and two early/recycling endosomal v-SNAREs, VAMP3/cellubrevin, and VAMP4. Using a novel permeabilized cell system, these proteins were functionally implicated in the post-Golgi retrograde transport step. The function of Rab6a’ was also required, whereas its closely related isoform, Rab6a, has previously been implicated in Golgi-to-endoplasmic reticulum transport. Thus, our study shows that membrane exchange between the early endocytic and the biosynthetic/secretory pathways involves specific components of the Rab and SNARE machinery, and suggests that retrograde transport between early/recycling endosomes and the endoplasmic reticulum is critically dependent on the sequential action of two members of the Rab6 subfamily.

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