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Thierry Galli

Publications and source records attributed to Thierry Galli.

28 records · Page 2Linked to original sources
PubMed2004

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

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.

Animals↗
PubMed2004

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

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.

Antigen-Presenting Cells↗
PubMed2004

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

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.

Animals↗
PubMed2003

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

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.

Adaptor Protein Complex 3↗
PubMed2003

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

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.

Animals↗
PubMed2003

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

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.

Antigens, CD↗
PubMed2003

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

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.

Amino Acid Sequence↗
PubMed2003

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

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.

3T3 Cells↗
PubMed2002

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

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.

Animals↗