CNS myelination by oligodendrocytes requires directed transport of myelin membrane components and a timely and spatially controlled membrane expansion. In this study, we show the functional ...involvement of the R-soluble N-ethylmaleimide-sensitive factor attachment protein receptor (R-SNARE) proteins VAMP3/cellubrevin and VAMP7/TI-VAMP in myelin membrane trafficking. VAMP3 and VAMP7 colocalize with the major myelin proteolipid protein (PLP) in recycling endosomes and late endosomes/lysosomes, respectively. Interference with VAMP3 or VAMP7 function using small interfering RNA-mediated silencing and exogenous expression of dominant-negative proteins diminished transport of PLP to the oligodendroglial cell surface. In addition, the association of PLP with myelin-like membranes produced by oligodendrocytes cocultured with cortical neurons was reduced. We furthermore identified Syntaxin-4 and Syntaxin-3 as prime acceptor Q-SNAREs of VAMP3 and VAMP7, respectively. Analysis of VAMP3-deficient mice revealed no myelination defects. Interestingly, AP-3δ-deficient mocha mice, which suffer from impaired secretion of lysosome-related organelles and missorting of VAMP7, exhibit a mild dysmyelination characterized by reduced levels of select myelin proteins, including PLP. We conclude that PLP reaches the cell surface via at least two trafficking pathways with distinct regulations: (1) VAMP3 mediates fusion of recycling endosome-derived vesicles with the oligodendroglial plasma membrane in the course of the secretory pathway; (2) VAMP7 controls exocytosis of PLP from late endosomal/lysosomal organelles as part of a transcytosis pathway. Our in vivo data suggest that exocytosis of lysosome-related organelles controlled by VAMP7 contributes to myelin biogenesis by delivering cargo to the myelin membrane.
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.
Vesicular (v)- and target (t)-SNARE proteins assemble in SNARE complex to mediate membrane fusion. Tetanus neurotoxin-insensitive vesicular-associated membrane protein (TI-VAMP/VAMP7), a vesicular ...SNARE expressed in several cell types including neurons, was previously shown to play a major role in exocytosis involved in neurite growth in cultured neurons. Here we generated a complete constitutive knock-out by deleting the exon 3 of Vamp7. Loss of TI-VAMP expression did not lead to any striking developmental or neurological defect. Knock-out mice displayed decreased brain weight and increased third ventricle volume. Axon growth appeared normal in cultured knock-out neurons. Behavioral characterization unraveled that TI-VAMP knock-out was associated with increased anxiety. Our results thus suggest compensatory mechanisms allowing the TI-VAMP knock-out mice to fulfill major developmental processes. The phenotypic traits unraveled here further indicate an unexpected role of TI-VAMP-mediated vesicular traffic in anxiety and suggest a role for TI-VAMP in higher brain functions.
Epithelial cells are associated laterally in a sheet, with the apical surface facing the lumina, often covered by microvilli, and the basolateral side underlying connective tissue. The assembly of ...tight junctions plays a key role in the formation of structurally and functionally distinct basolateral and apical plasma membrane domains.
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
.
Synaptic vesicle (SV) exocytosis mediating neurotransmitter release occurs spontaneously at low intraterminal calcium concentrations and is stimulated by a rise in intracellular calcium. Exocytosis ...is compensated for by the reformation of vesicles at plasma membrane and endosomes. Although the adaptor complex AP-3 was proposed to be involved in the formation of SVs from endosomes, whether its function has an indirect effect on exocytosis remains unknown. Using mocha mice, which are deficient in functional AP-3, we identify an AP-3-dependent tetanus neurotoxin-resistant asynchronous release that can be evoked at hippocampal mossy fiber (MF) synapses. Presynaptic targeting of the tetanus neurotoxin-resistant vesicle soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) tetanus neurotoxininsensitive vesicle-associated membrane protein (TI-VAMP) is lost in mocha hippocampal MF terminals, whereas the localization of synaptobrevin 2 is unaffected. In addition, quantal release in mocha cultures is more frequent and more sensitive to sucrose. We conclude that lack of AP-3 results in more constitutive secretion and loss of an asynchronous evoked release component, suggesting an important function of AP-3 in regulating SV exocytosis at MF terminals.
The rigidity of the cell environment can vary tremendously between tissues and in pathological conditions. How this property may affect intracellular membrane dynamics is still largely unknown. Here, ...using atomic force microscopy, we show that cells deficient in the secretory lysosome v-SNARE VAMP7 are impaired in adaptation to substrate rigidity. Conversely, VAMP7-mediated secretion is stimulated by more rigid substrate and this regulation depends on the Longin domain of VAMP7. We further find that the Longin domain binds the kinase and retrograde trafficking adaptor LRRK1 and that LRRK1 negatively regulates VAMP7-mediated exocytosis. Conversely, VARP, a VAMP7- and kinesin 1-interacting protein, further controls the availability for secretion of peripheral VAMP7 vesicles and response of cells to mechanical constraints. LRRK1 and VARP interact with VAMP7 in a competitive manner. We propose a mechanism whereby biomechanical constraints regulate VAMP7-dependent lysosomal secretion via LRRK1 and VARP tug-of-war control of the peripheral pool of secretory lysosomes.
Display omitted
•VAMP7 is important for mechano-adaptation to the rigidity of cellular environment•Substrate rigidity regulates VAMP7 exocytosis•LRRK1 and VARP compete for VAMP7 binding•LRRK1 and VARP generate a tug-of-war for the transport of VAMP7
Cell Biology; Functional Aspects of Cell Biology; Biomechanics
Amphiphysin 1, an endocytic adaptor concentrated at synapses that couples clathrin-mediated endocytosis to dynamin-dependent fission, was also shown to have a regulatory role in actin dynamics. Here, ...we report that amphiphysin 1 interacts with N-WASP and stimulates N-WASP- and Arp2/3-dependent actin polymerization. Both the Src homology 3 and the N-BAR domains are required for this stimulation. Acidic liposome-triggered, N-WASP-dependent actin polymerization is strongly impaired in brain cytosol of amphiphysin 1 knock-out mice. FRET-FLIM analysis of Sertoli cells, where endogenously expressed amphiphysin 1 co-localizes with N-WASP in peripheral ruffles, confirmed the association between the two proteins in vivo. This association undergoes regulation and is enhanced by stimulating phosphatidylserine receptors on the cell surface with phosphatidylserine-containing liposomes that trigger ruffle formation. These results indicate that actin regulation is a key function of amphiphysin 1 and that such function cooperates with the endocytic adaptor role and membrane shaping/curvature sensing properties of the protein during the endocytic reaction.
SNARE soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein (SNAP) receptor proteins control the membrane-fusion events of eukaryotic membrane-trafficking pathways. Specific vesicular ...and target SNAREs operate in specific trafficking routes, but the degree of specificity of SNARE functions is still elusive. Apical fusion requires the polarized distribution at the apical surface of the t-SNARE syntaxin 3, and several v-SNAREs including TI-VAMP and VAMP8 operate at the apical plasma membrane in polarized epithelial cells. It is not known, however, whether specific v-SNAREs are involved in direct and indirect routes to the apical surface. Here, we used RNAi to assess the role of two tetanus-neurotoxin-insensitive v-SNAREs, TI-VAMP/VAMP7 and VAMP8, in the sorting of raft- and non-raft-associated apical markers that follow either a direct or a transcytotic delivery, respectively, in FRT or Caco2 cells. We show that TI-VAMP mediates the direct apical delivery of both raft- and non-raft-associated proteins. By contrast, sorting by means of the transcytotic pathway is not affected by TI-VAMP knockdown but does appear to be regulated by VAMP8. Together with the specific role of VAMP3 in basolateral transport, our results demonstrate a high degree of specificity in v-SNARE function in polarized cells.
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-3δ, 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.
PDF
