Tetanus Neurotoxin-insensitive Vesicle-associated Membrane Research Articles

Syntaxin-1/TI-VAMP SNAREs interact with Trk receptors and are required for neurotrophin-dependent outgrowth.

SNARE proteins are essential components of the machinery that regulates vesicle trafficking and exocytosis. Their role is critical for the membrane-fusion processes that occur during neurotransmitter release. However, research in the last decade has also unraveled the relevance of these proteins in membrane expansion and cytoskeletal rearrangements during developmental processes such as neuronal migration and growth cone extension and attraction. Neurotrophins are neurotrophic factors that are required for many cellular functions throughout the brain, including neurite outgrowth and guidance, synaptic formation, and plasticity. Here we show that neurotrophin Trk receptors form a specific protein complex with the t-SNARE protein Syntaxin 1, both in vivo and in vitro. We also demonstrate that blockade of Syntaxin 1 abolishes neurotrophin-dependent growth of axons in neuronal cultures and decreases exocytotic events at the tip of axonal growth cones. 25-kDa soluble N-ethylmaleimide-sensitive factor attachment protein and Vesicle-associated membrane protein 2 do not participate in the formation of this SNARE complex, while tetanus neurotoxin-insensitive vesicle-associated membrane protein interacts with Trk receptors; knockdown of this (v) SNARE impairs Trk-dependent outgrowth. Taken together, our results support the notion that an atypical SNARE complex comprising Syntaxin 1 and tetanus neurotoxin-insensitive vesicle-associated membrane protein is required for axonal neurotrophin function.

Role of tetanus neurotoxin insensitive vesicle-associated membrane protein in membrane domains transport and homeostasis.

Biological membranes in eukaryotes contain a large variety of proteins and lipids often distributed in domains in plasma membrane and endomembranes. Molecular mechanisms responsible for the transport and the organization of these membrane domains along the secretory pathway still remain elusive. Here we show that vesicular SNARE TI-VAMP/VAMP7 plays a major role in membrane domains composition and transport. We found that the transport of exogenous and endogenous GPI-anchored proteins was altered in fibroblasts isolated from VAMP7-knockout mice. Furthermore, disassembly and reformation of the Golgi apparatus induced by Brefeldin A treatment and washout were impaired in VAMP7-depleted cells, suggesting that loss of VAMP7 expression alters biochemical properties and dynamics of the Golgi apparatus. In addition, lipid profiles from these knockout cells indicated a defect in glycosphingolipids homeostasis. We conclude that VAMP7 is required for effective transport of GPI–anchored proteins to cell surface and that VAMP7-dependent transport contributes to both sphingolipids and Golgi homeostasis.

The Coffin-Lowry Syndrome-Associated Protein RSK2 Regulates Neurite Outgrowth through Phosphorylation of Phospholipase D1 (PLD1) and Synthesis of Phosphatidic Acid

More than 80 human X-linked genes have been associated with mental retardation and deficits in learning and memory. However, most of the identified mutations induce limited morphological alterations in brain organization and the molecular bases underlying neuronal clinical features remain elusive. We show here that neurons cultured from mice lacking ribosomal S6 kinase 2 (Rsk2), a model for the Coffin-Lowry syndrome (CLS), exhibit a significant delay in growth in a similar way to that shown by neurons cultured from phospholipase D1 (Pld1) knock-out mice. We found that gene silencing of Pld1 or Rsk2 as well as acute pharmacological inhibition of PLD1 or RSK2 in PC12 cells strongly impaired neuronal growth factor (NGF)-induced neurite outgrowth. Expression of a phosphomimetic PLD1 mutant rescued the inhibition of neurite outgrowth in PC12 cells silenced for RSK2, revealing that PLD1 is a major target for RSK2 in neurite formation. NGF-triggered RSK2-dependent phosphorylation of PLD1 led to its activation and the synthesis of phosphatidic acid at sites of neurite growth. Additionally, total internal reflection fluorescence microscopy experiments revealed that RSK2 and PLD1 positively control fusion of tetanus neurotoxin insensitive vesicle-associated membrane protein (TiVAMP)/VAMP-7 vesicles at sites of neurite outgrowth. We propose that the loss of function mutations in RSK2 that leads to CLS and neuronal deficits are related to defects in neuronal growth due to impaired RSK2-dependent PLD1 activity resulting in a reduced vesicle fusion rate and membrane supply.

TI‐VAMP/VAMP7 is the SNARE of secretory lysosomes contributing to ATP secretion from astrocytes

ATP is the main transmitter stored and released from astrocytes under physiological and pathological conditions. Morphological and functional evidence suggest that besides secretory granules, secretory lysosomes release ATP. However, the molecular mechanisms involved in astrocytic lysosome fusion remain still unknown. In the present study, we identify tetanus neurotoxin-insensitive vesicle-associated membrane protein (TI-VAMP, also called VAMP7) as the vesicular SNARE which mediates secretory lysosome exocytosis, contributing to release of both ATP and cathepsin B from glial cells. We also demonstrate that fusion of secretory lysosomes is triggered by slow and locally restricted calcium elevations, distinct from calcium spikes which induce the fusion of glutamate-containing clear vesicles. Downregulation of TI-VAMP/VAMP7 expression inhibited the fusion of ATP-storing vesicles and ATP-mediated calcium wave propagation. TI-VAMP/VAMP7 downregulation also significantly reduced secretion of cathepsin B from glioma. Given that sustained ATP release from glia upon injury greatly contributes to secondary brain damage and cathepsin B plays a critical role in glioma dissemination, TI-VAMP silencing can represent a novel strategy to control lysosome fusion in pathological conditions.

A Signaling Mechanism Coupling Netrin-1/Deleted in Colorectal Cancer Chemoattraction to SNARE-Mediated Exocytosis in Axonal Growth Cones

Directed cell migration and axonal guidance are essential steps in neural development. Both processes are controlled by specific guidance cues that activate the signaling cascades that ultimately control cytoskeletal dynamics. Another essential step in migration and axonal guidance is the regulation of plasmalemma turnover and exocytosis in leading edges and growth cones. However, the cross talk mechanisms linking guidance receptors and membrane exocytosis are not understood. Netrin-1 is a chemoattractive cue required for the formation of commissural pathways. Here, we show that the Netrin-1 receptor deleted in colorectal cancer (DCC) forms a protein complex with the t-SNARE (target SNARE) protein Syntaxin-1 (Sytx1). This interaction is Netrin-1 dependent both in vitro and in vivo, and requires specific Sytx1 and DCC domains. Blockade of Sytx1 function by using botulinum toxins abolished Netrin-1-dependent chemoattraction of axons in mouse neuronal cultures. Similar loss-of-function experiments in the chicken spinal cord in vivo using dominant-negative Sytx1 constructs or RNAi led to defects in commissural axon pathfinding reminiscent to those described in Netrin-1 and DCC loss-of-function models. We also show that Netrin-1 elicits exocytosis at growth cones in a Sytx1-dependent manner. Moreover, we demonstrate that the Sytx1/DCC complex associates with the v-SNARE (vesicle SNARE) tetanus neurotoxin-insensitive vesicle-associated membrane protein (TI-VAMP) and that knockdown of TI-VAMP in the commissural pathway in the spinal cord results in aberrant axonal guidance phenotypes. Our data provide evidence of a new signaling mechanism that couples chemotropic Netrin-1/DCC axonal guidance and Sytx1/TI-VAMP SNARE proteins regulating membrane turnover and exocytosis.

Autophagosome Precursor Maturation Requires Homotypic Fusion

SummaryAutophagy is a catabolic process in which lysosomes degrade intracytoplasmic contents transported in double-membraned autophagosomes. Autophagosomes are formed by the elongation and fusion of phagophores, which can be derived from preautophagosomal structures coming from the plasma membrane and other sites like the endoplasmic reticulum and mitochondria. The mechanisms by which preautophagosomal structures elongate their membranes and mature toward fully formed autophagosomes still remain unknown. Here, we show that the maturation of the early Atg16L1 precursors requires homotypic fusion, which is essential for subsequent autophagosome formation. Atg16L1 precursor homotypic fusion depends on the SNARE protein VAMP7 together with partner SNAREs. Atg16L1 precursor homotypic fusion is a critical event in the early phases of autophagy that couples membrane acquisition and autophagosome biogenesis, as this step regulates the size of the vesicles, which in turn appears to influence their subsequent maturation into LC3-positive autophagosomes.

Role of Varp, a Rab21 exchange factor and TI‐VAMP/VAMP7 partner, in neurite growth

The vesicular soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) tetanus neurotoxin-insensitive vesicle-associated membrane protein (TI-VAMP/VAMP7) was previously shown to mediate an exocytic pathway involved in neurite growth, but its regulation is still largely unknown. Here we show that TI-VAMP interacts with the Vps9 domain and ankyrin-repeat-containing protein (Varp), a guanine nucleotide exchange factor (GEF) of the small GTPase Rab21, through a specific domain herein called the interacting domain (ID). Varp, TI-VAMP and Rab21 co-localize in the perinuclear region of differentiating hippocampal neurons and transiently in transport vesicles in the shaft of neurites. Silencing the expression of Varp by RNA interference or expressing ID or a form of Varp deprived of its Vps9 domain impairs neurite growth. Furthermore, the mutant form of Rab21, defective in GTP hydrolysis, enhances neurite growth. We conclude that Varp is a positive regulator of neurite growth through both its GEF activity and its interaction with TI-VAMP.

Subcellular localization of the carbohydrate Lewis x adhesion structure in hippocampus cell cultures

The Lewis x (Le x) epitope (Gal(β1–4)[Fuc(α1–3)]GlcNAc-R) has been associated with the development of the central nervous system of diverse species including human and rodents. In this work, Le x has been found in the tetanus neurotoxin insensitive vesicle-associated membrane protein (TI-VAMP) compartment of rat hippocampus neurons in culture, at 7 days in vitro (DIV), when neurite extension is abundant. The TI-VAMP compartment is known to be associated with neurite outgrowth. Le x was found predominantly in neurites but also in somata and in growth cones. Abundant Le x-carrier glycoproteins specific to neurons have been identified at this stage of differentiation. At a later stage of differentiation, at 14 DIV, Le x appeared in extrasynaptic sites of GABAergic neurons, and in synaptic sites of glutamatergic neurons.

Role of HRB in Clathrin-dependent Endocytosis

Human immunodeficiency virus Rev-binding protein (HRB), also called human Rev-interacting protein (hRIP) or Rev/Rex activation domain binding (RAB) is a partner of the tyrosine kinase substrate EPS15, and it has been recovered in the AP-2 interactome. EPS15 and AP-2 are involved in endocytosis, but the function of HRB in this process is still unknown. Here we identified HRB as a partner of the vesicular SNARE tetanus neurotoxin-insensitive vesicle-associated membrane protein (TI-VAMP, also called VAMP7) in yeast two-hybrid screens and using biochemical assays. In HeLa cells, HRB localized both in the nucleus and in the cytoplasm. In the cytoplasm, HRB colocalized with clathrin-, AP-2-, EPS15-, and transferrin receptor-containing vesicles. We did not see significant colocalization between HRB and TI-VAMP in HeLa cells, and we saw partial colocalization with green fluorescent protein-TI-VAMP in stably expressing Madin-Darby canine kidney cells. Nevertheless using a pHLuorin-tagged TI-VAMP construct, we found that HRB and TI-VAMP colocalize close to the plasma membrane after 5 min of anti-green fluorescent protein antibody uptake. These results suggest that TI-VAMP and HRB may interact only during the early stages of endocytosis. Furthermore uptake experiments followed by fluorescence-activated cell sorting showed that the endocytosis of fluorescent transferrin and pHLuorin-TI-VAMP is strongly reduced in HRB knockdown cells. Altogether these results suggest that HRB is involved in clathrin-dependent endocytosis and recruits TI-VAMP in this process.

What is the function of neuronal AP‐3?

Neurotransmission requires the proper organization and rapid recycling of synaptic vesicles. Rapid retrieval has been suggested to occur either by kiss-and-stay or kiss-and-run mechanisms, whereas classical recycling is mediated by clathrin-dependent endocytosis. Molecular coats are key components in the selection of cargos, AP-2 (adaptor protein 2) playing a prominent role in synaptic vesicle endocytosis. Another coat protein, AP-3, has been implicated in synaptic vesicle biogenesis and in the generation of secretory and lysosomal-related organelles. In the present review, we will particularly focus on the recent data concerning the recycling of synaptic vesicles and the function of AP-3 and the v-SNARE (vesicular soluble N-ethylmaleimide-sensitive fusion protein-attachment protein receptor) TI-VAMP (tetanus neurotoxin-insensitive vesicle-associated membrane protein) in these processes. We propose that AP-3 plays an important regulatory role in neurons which contributes to the basal and stimulated exocytosis of synaptic vesicles.

Expression of the Longin domain of TI‐VAMP impairs lysosomal secretion and epithelial cell migration

TI-VAMP (tetanus neurotoxin-insensitive vesicle-associated membrane protein; also called VAMP7) belongs to the Longin subfamily of v-SNAREs (vesicular soluble N-ethylmaleimide-sensitive fusion protein-attachment protein receptors). The regulatory N-terminal extension, called the Longin domain, of TI-VAMP has been shown previously to have a dual biochemical function: it inhibits the capacity of TI-VAMP to form SNARE complexes and it binds to the delta subunit of the AP-3 (adaptor protein 3) complex in early endosomes, thereby targeting TI-VAMP to late endosomes. We have generated MDCK (Madin-Darby canine kidney) cell lines expressing the Longin domain of TI-VAMP coupled to GFP (green fluorescent protein) in a doxycycline-dependent manner. As expected, AP-3delta (AP-3 delta subunit) is not properly localized in Longin-expressing cells. We have shown that the expression of the Longin domain impairs lysosomal secretion, as determined by the release of a pre-internalized fluorescent fluid-phase marker and by electron microscopy of the membrane-associated released particles. Membrane repair following mechanical wounding, a process requiring lysosomal secretion, is also impaired in cells expressing the Longin domain. Furthermore, cell migration, assessed by wound healing of MDCK monolayers, is also inhibited. The results of the present study suggest that the expression of the Longin domain of TI-VAMP regulates lysosomal secretion of epithelial cells and provide molecular evidence for a role of the late endocytic system in cell migration.

Increased levels of fucosyltransferase IX and carbohydrate Lewisx adhesion determinant in human NT2N neurons

The expression of the fucosylated carbohydrate Lewis(x) (Le(x)) determinant (Gal(beta1-4)[Fuc(alpha1-3)]GlcNAc-R) has been found in glycoproteins, proteoglycans, and glycolipids from the nervous system. Evidence suggests its association with cell-cell recognition, neurite outgrowth, and neuronal migration during central nervous system development. In the present work, we detected increased levels of Le(x) in differentiated human NT2N neurons cultured in vitro. To identify which fucosyltransferase (FUT) synthesized the Le(x) in NT2N neurons, RT-PCR, FUT substrate specificity and Western blot analysis were carried out. Strong activity toward acceptors Galbeta4GlcNAc-O-R and Fucalpha2Galbeta4GlcNAc-O-R [R = -(CH(2))(3)NHCO(CH(2))(5)NH-biotin], together with strong FUT9 detection by Western blot and presence of transcripts showed that FUT9 was the enzyme associated with Le(x) biosynthesis in NT2N neurons. Le(x) was detected at the plasma membrane of NT2N neurons, in lysosomes marked with lysosomal-associated membrane protein 1 (LAMP-1), and it was found for the first time to colocalize with the tetanus neurotoxin-insensitive vesicle-associated membrane protein (TI-VAMP) that defines the TI-VAMP exocytic compartment that is involved in neurite outgrowth. Furthermore, incubation with anti-Le(x) monoclonal antibody L5 led to impaired adhesion of NT2N neurons to the surface matrix and inhibited neurite initiation. In conclusion, FUT9 and its product Le(x) are detected specifically in human NT2N neurons and our results indicate that they underlie cell differentiation, cell adhesion, and initiation of neurite outgrowth in those neurons.

Loss of AP-3 function affects spontaneous and evoked release at hippocampal mossy fiber synapses

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 neurotoxin-insensitive 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.

A Role for Synaptotagmin VII-Regulated Exocytosis of Lysosomes in Neurite Outgrowth from Primary Sympathetic Neurons

Neurite outgrowth is mediated by the exocytosis of intracellular vesicles at the tips of elongating neuronal processes. The lysosomal vesicle-associated soluble N-ethylmaleimide-sensitive factor attachment protein receptor tetanus neurotoxin insensitive vesicle-associated membrane protein (TI-VAMP)/VAMP7 was previously implicated in membrane fusion events mediating neurite outgrowth, but the participation of lysosomes in this exocytic process has remained unclear. Here, we show that VAMP7 and the lysosomal glycoprotein Lamp1 extensively colocalize in vesicles present throughout the soma and neurite outgrowths of primary sympathetic neurons. Synaptotagmin VII (Syt VII), a Ca(2+)-sensing synaptotagmin isoform previously shown to interact with VAMP7 during lysosomal exocytosis in fibroblasts, was detected on a subset of these lysosomal glycoprotein 1 (Lamp1)/VAMP7-positive neuronal vesicles. Ionophore-stimulated exocytosis triggered exposure of the luminal domains of both Lamp1 and Syt VII at overlapping sites on the neuronal surface, indicating that the Syt VII-containing lysosomal compartments fuse with the plasma membrane in response to [Ca2+]i elevation. To determine whether Syt VII was required for the exocytic events mediating neurite extension, we followed the development of superior cervical ganglion neurons explanted from Syt VII-deficient mice. The results revealed a marked defect in neurite outgrowth and arborization, suggesting that Ca(2+)-dependent, Syt VII-regulated exocytosis of late endosomes/lysosomes plays a role in the addition of new membrane to developing neurite extensions.

Cdc42 and Actin Control Polarized Expression of TI-VAMP Vesicles to Neuronal Growth Cones and Their Fusion with the Plasma Membrane

Tetanus neurotoxin-insensitive vesicle-associated membrane protein (TI-VAMP)-mediated fusion of intracellular vesicles with the plasma membrane is crucial for neurite outgrowth, a pathway not requiring synaptobrevin-dependent exocytosis. Yet, it is not known how the TI-VAMP membrane trafficking pathway is regulated or how it is coordinated with cytoskeletal dynamics within the growth cone that guide neurite outgrowth. Here, we demonstrate that TI-VAMP, but not synaptobrevin 2, concentrates in the peripheral, F-actin-rich region of the growth cones of hippocampal neurons in primary culture. Its accumulation correlates with and depends upon the presence of F-actin. Moreover, acute stimulation of actin remodeling by homophilic activation of the adhesion molecule L1 induces a site-directed, actin-dependent recruitment of the TI-VAMP compartment. Expression of a dominant-positive mutant of Cdc42, a key regulator of cell polarity, stimulates formation of F-actin- and TI-VAMP-rich filopodia outside the growth cone. Furthermore, we report that Cdc42 activates exocytosis of pHLuorin tagged TI-VAMP in an actin-dependent manner. Collectively, our data suggest that Cdc42 and regulated assembly of the F-actin network control the accumulation and exocytosis of TI-VAMP-containing membrane vesicles in growth cones to coordinate membrane trafficking and actin remodeling during neurite outgrowth.

The Tetanus Neurotoxin‐Sensitive and Insensitive Routes to and from the Plasma Membrane: Fast and Slow Pathways?

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.

A molecular basis underlying differences in the toxicity of botulinum serotypes A and E.

Botulinum neurotoxins (BoNTs) block neurotransmitter release through their specific proteolysis of the proteins responsible for vesicle exocytosis. Paradoxically, two serotypes of BoNTs, A and E, cleave the same molecule, synaptosome-associated protein with relative molecular mass 25K (SNAP-25), and yet they cause synaptic blockade with very different properties. Here we compared the action of BoNTs A and E on the plasma membrane fusion machinery composed of syntaxin and SNAP-25. We now show that the BoNT/A-cleaved SNAP-25 maintains its association with two syntaxin isoforms in vitro, which is mirrored by retention of SNAP-25 on the plasma membrane in vivo. In contrast, BoNT/E severely compromises the ability of SNAP-25 to bind the plasma membrane syntaxin isoforms, leading to dissociation of SNAP-25. The distinct properties of botulinum intoxication, therefore, can result from the ability of shortened SNAP-25 to maintain its association with syntaxins-in the case of BoNT/A poisoning resulting in unproductive syntaxin/SNAP-25 complexes that impede vesicle exocytosis.

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

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.

Tetanus neurotoxin-insensitive vesicle-associated membrane protein localizes to a presynaptic membrane compartment in selected terminal subsets of the rat brain

Tetanus neurotoxin-insensitive vesicle-associated membrane protein (TI-VAMP) is a vesicular soluble N-ethyl maleimide-sensitive fusion protein attachment protein receptor (SNARE) that has been implicated in neurite outgrowth. It has previously been reported that TI-VAMP is localised in the somatodendritic compartment of neurons indicating a role in membrane fusion events within dendrites. Using a newly produced monoclonal antibody to TI-VAMP that improves signal/noise immunodetection, we report that TI-VAMP is also present in subsets of axon terminals of the adult rat brain. Four distinctive populations of labelled axon terminals were identified: 1) the hippocampal mossy fibres of the dentate gyrus and of CA3, 2) the striatal peridendritic terminal plexuses in the globus pallidus (GP), substantia nigra pars reticulata (SNr), 3) peridendritic plexuses in the central nucleus of the amygdala, and 4) the primary sensory afferents in the dorsal horn of the spinal cord. The presynaptic localisation of TI-VAMP in these locations was demonstrated by co-localisation with synaptophysin. Ultrastructural studies showed TI-VAMP labelling over synaptic vesicles in the mossy fibres, whereas it was localised in tubulo-vesicular structures and multivesicular bodies in the pyramidal cell dendrites. The presynaptic localisation of TI-VAMP occurred by P15, so relatively late during development. In contrast, dendritic labelling was most prominent during the early post-natal period. Co-localisation with markers of neurotransmitters showed that TI-VAMP-positive terminals are GABAergic in the GP and SNr and glutamatergic in the mossy fibre system and in the dorsal root afferents. Most of these terminals are known to co-localise with neuropeptides. We found met-enkephalin-immunoreactivity in a sizeable fraction of the TI-VAMP positive terminals in the GP, amygdala, and dorsal horn, as well as in a few mossy fibre terminals. The function of TI-VAMP in subsets of mature axon terminals remains to be elucidated; it could participate in the exocytotic molecular machinery and/or be implicated in particular growth properties of the mature axon terminals. Thus, the presence of TI-VAMP in the mossy fibres may correspond to the high degree of plasticity that characterises this pathway throughout adult life.

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.