Synaptic vesicle membrane fusion complex: action of clostridial neurotoxins on assembly.

Clostridial neurotoxins inhibit neurotransmitter release by selective and specific intracellular proteolysis of synaptobrevin/VAMP, synaptosomal-associated protein of 25 kDa (SNAP-25) or syntaxin. Here we show that in binary reactions synaptobrevin binds weakly to both SNAP-25 and syntaxin, and SNAP-25 binds to syntaxin. In the presence of all three components, a dramatic increase in the interaction strengths occurs and a stable sodium dodecyl sulfate-resistant complex forms. Mapping of the interacting sequences reveals that complex formation correlates with the presence of predicted alpha-helical structures, suggesting that membrane fusion involves intermolecular interactions via coiled-coil structures. Most toxins only attack the free, and not the complexed, proteins, and proteolysis of the proteins by different clostridial neurotoxins has distinct inhibitory effects on the formation of synaptobrevin-syntaxin-SNAP-25 complexes. Our data suggest that synaptobrevin, syntaxin and SNAP-25 associate into a unique stable complex that functions in synaptic vesicle exocytosis.     

Dense-core secretory vesicle docking and exocytotic membrane fusion in Paramecium cells

Work with Paramecium has contributed to the actual understanding of certain aspects of exocytosis regulation, including membrane fusion. The system is faster and more synchronous than any other dense-core vesicle system described and its highly regular design facilitates correlation of functional and ultrastructural (freeze-fracture) features. From early times on, several crucial aspects of exocytosis regulation have been found in Paramecium cells, e.g. genetically controlled microdomains (with distinct ultrastructure) for organelle docking and membrane fusion, involvement of calmodulin in establishing such microdomains, priming by ATP, occurrence of focal fusion with active participation of integral and peripheral proteins, decay of a population of integral proteins ("rosettes", mandatory for fusion capacity) into subunits and their lateral dispersal during fusion, etc. The size of rosette particles and their dispersal upon focal fusion would be directly compatible with proteolipid V(0) subunits of a V-ATPase, much better than the size predicted for oligomeric SNARE pins (SCAMPs are unknown from Paramecium at this time). However, there are some restrictions for a straightforward interpretation of ultrastructural results. The rather pointed, nipple-like tip of the trichocyst membrane could accommodate only one (or very few) potential V(0) counterpart(s), while the overlaying domain of the cell membrane contains numerous rosette particles. Particle size is compatible with V(0), but larger than that assumed for the SNARE complexes. When membrane fusion is induced in the presence of antibodies against cell surface components, focal fusion is seen to occur with dispersing rosette particles but without dispersal of their subunits and without pore expansion. Clearly, this is required for completing fusion and pore expansion. After cloning SNARE and V(0) components in Paramecium (with increasing details becoming rapidly available), we may soon be able to address the question more directly, whether any of these components or some new ones to be detected, serve exocytotic and/or any other membrane fusions in Paramecium.     

Reconstituted synaptotagmin I mediates vesicle docking, priming, and fusion

The synaptic vesicle protein synaptotagmin I (syt) promotes exocytosis via its ability to penetrate membranes in response to binding Ca(2+) and through direct interactions with SNARE proteins. However, studies using full-length (FL) membrane-embedded syt in reconstituted fusion assays have yielded conflicting results, including a lack of effect, or even inhibition of fusion, by Ca(2+). In this paper, we show that reconstituted FL syt promoted rapid docking of vesicles (<1 min) followed by a priming step (3-9 min) that was required for subsequent Ca(2+)-triggered fusion between v- and t-SNARE liposomes. Moreover, fusion occurred only when phosphatidylinositol 4,5-bisphosphate was included in the target membrane. This system also recapitulates some of the effects of syt mutations that alter synaptic transmission in neurons. Finally, we demonstrate that the cytoplasmic domain of syt exhibited mixed agonist/antagonist activity during regulated membrane fusion in vitro and in cells. Together, these findings reveal further convergence of reconstituted and cell-based systems.     

Synaptic vesicle endocytosis

The functions of Ca2+ are many and varied within cells, but in the nerve terminals of neurons it has had a very defined role. That is, the influx of extracellular Ca2+ through voltage-dependent Ca2+ channels stimulates neurotransmitter release by exocytosis. For years this was assumed to be the main role for Ca2+ in this specialized subcellular region. However recent studies have shown that Ca2+ also has multiple roles in synaptic-vesicle endocytosis. This review will present evidence for three Ca2+-dependent and -independent steps; a high-affinity Ca2+-dependent triggering step, a Ca2+-independent maintenance phase, and a low-affinity Ca2+-dependent inhibition step. How the control of endocytosis by Ca2+ might impact on different neuronal functions such as synaptic transmission, the nucleation of SV endocytosis, and the repair of damaged membrane is then discussed.     

Synaptic vesicle pools

Communication between cells reaches its highest degree of specialization at chemical synapses. Some synapses talk in a 'whisper'; others 'shout'. The 'louder' the synapse, the more synaptic vesicles are needed to maintain effective transmission, ranging from a few hundred (whisperers) to nearly a million (shouters). These vesicles reside in different 'pools', which have been given a bewildering array of names. In this review, we focus on five tissue preparations in which synaptic vesicle pools have been identified and thoroughly characterized. We argue that, in each preparation, each vesicle can be assigned to one of three distinct pools.     

Synaptic vesicle pools and dynamics

Synaptic vesicles release neurotransmitter at chemical synapses, thus initiating the flow of information in neural networks. To achieve this, vesicles undergo a dynamic cycle of fusion and retrieval to maintain the structural and functional integrity of the presynaptic terminals in which they reside. Moreover, compelling evidence indicates these vesicles differ in their availability for release and mobilization in response to stimuli, prompting classification into at least three different functional pools. Ongoing studies of the molecular and cellular bases for this heterogeneity attempt to link structure to physiology and clarify how regulation of vesicle pools influences synaptic strength and presynaptic plasticity. We discuss prevailing perspectives on vesicle pools, the role they play in shaping synaptic transmission, and the open questions that challenge current understanding.     

TRAPP, a highly conserved novel complex on the cis-Golgi that mediates vesicle docking and fusion.

We previously identified BET3 by its genetic interactions with BET1, a gene whose SNARE-like product acts in endoplasmic reticulum (ER)-to-Golgi transport. To gain insight into the function of Bet3p, we added three c-myc tags to its C-terminus and immunopurified this protein from a clarified detergent extract. Here we report that Bet3p is a member of a large complex ( approximately 800 kDa) that we call TRAPP (transport protein particle). We propose that TRAPP plays a key role in the targeting and/or fusion of ER-to-Golgi transport vesicles with their acceptor compartment. The localization of Bet3p to the cis-Golgi complex, as well as biochemical studies showing that Bet3p functions on this compartment, support this hypothesis. TRAPP contains at least nine other constituents, five of which have been identified and shown to be highly conserved novel proteins.     

Synaptic vesicle recycling and Alzheimer's disease

Loss of synapses correlates well with cognitive decline in Alzheimer's disease (AD). However, the molecular mechanisms underlying the synaptic dysfunction and loss are not well understood. Synaptic vesicle (SV) recycling is a key process for synaptic transmission. A body of evidences suggested that malfunction or loss of the machinery for SV recycling occurred in AD, which could result in disruption of neuronal circuitry. In this article, we summarized the recent progress in the research of synaptic proteins for SV recycling and the pathological changes of some proteins in AD.     

Synaptic vesicle trafficking and Parkinson's disease

Presynaptic terminals maintain neurotransmitter release during repeated rounds of stimulation using local recycling of synaptic vesicles (SV). During each SV cycle, protein complex assembly and disassembly results in accumulation of inactive (unfolded) protein intermediates that may render synaptic terminals vulnerable to activity-dependent degeneration. SV trafficking is affected in many neurodegenerative conditions including Alzheimer' and Parkinson's disease (PD) suggesting that alteration of this process might be an important aspect of disease pathogenesis. This article reviews our current understanding for a role of causative PD genes in the SV cycle and speculates on the potential role of aberrant SV trafficking in the neurodegenerative cascade of PD.     

Single vesicle assaying of SNARE-synaptotagmin-driven fusion reveals fast and slow modes of both docking and fusion and intrasample heterogeneity

Lipid mixing between vesicles functionalized with SNAREs and the cytosolic C2AB domain of synaptotagmin-1 recapitulates the basic Ca²⁺ dependence of neuronal exocytosis. However, in the conventional ensemble lipid mixing assays it is not possible to discriminate whether Ca²⁺ accelerates the docking or the fusion of vesicles. Here we report a fluorescence microscopy-based assay to monitor SNARE-mediated docking and fusion of individual vesicle pairs. In situ measurement of the concentration of diffusing particles allowed us to quantify docking rates by a maximum-likelihood approach. This analysis showed that C2AB and Ca²⁺ accelerate vesicle-vesicle docking with more than two orders of magnitude. Comparison of the measured docking rates with ensemble lipid mixing kinetics, however, suggests that in most cases bilayer fusion remains the rate-limiting step. Our single vesicle results show that only ∼60% of the vesicles dock and only ∼6% of docked vesicles fuse. Lipid mixing on single vesicles was fast (t_mix < 1 s) while an ensemble assay revealed two slow mixing processes with t_mix ∼ 1 min and t_mix ∼ 20 min. The presence of several distinct docking and fusion pathways cannot be rationalized at this stage but may be related to intrasample heterogeneities, presumably in the form of lipid and/or protein composition.     

Synaptic vesicle dynamics sans dynamin

The neuron-specific guanosine triphosphatase dynamin 1 has been hypothesized to be critically required for pinching off synaptic vesicles during endocytosis. In a recent publication in Science, Ferguson et al. describe a series of experiments demonstrating an unexpectedly selective requirement of dynamin 1 in synaptic vesicle endocytosis only during high frequency (<10 Hz) stimulation, but not after cessation of the stimulus train.     

Synaptic vesicle recycling intermediates revealed

Neurotransmitter release takes place by the exocytosis of loaded synaptic vesicles. The vesicles then fuse to the presynaptic membrane and are recycled by an endocytotic mechanism. A quantitative optical assay that detects uptake and release of a fluorescent dye during presynaptic activity was recently developed and used on the frog neauromuscular junction. I discuss a report⁽¹⁾ that demonstrates the effective application of this method to a Drosophila preparation. The authors use the shibire mutation and a spider venom to identify two intermediates in vesicle recycling. Their report, along with other recent studies, demonstrates the power and promise of the genetic approach for the understanding of mechanisms of synapse function and development.     

Synaptic vesicle recycling: steps and principles

Synaptic vesicle recycling is one of the best‐studied cellular pathways. Many of the proteins involved are known, and their interactions are becoming increasingly clear. However, as for many other pathways, it is still difficult to understand synaptic vesicle recycling as a whole. While it is generally possible to point out how synaptic reactions take place, it is not always easy to understand what triggers or controls them. Also, it is often difficult to understand how the availability of the reaction partners is controlled: how the reaction partners manage to find each other in the right place, at the right time. I present here an overview of synaptic vesicle recycling, discussing the mechanisms that trigger different reactions, and those that ensure the availability of reaction partners. A central argument is that synaptic vesicles bind soluble cofactor proteins, with low affinity, and thus control their availability in the synapse, forming a buffer for cofactor proteins. The availability of cofactor proteins, in turn, regulates the different synaptic reactions. Similar mechanisms, in which one of the reaction partners buffers another, may apply to many other processes, from the biogenesis to the degradation of the synaptic vesicle.     

Synaptic vesicle distribution by conveyor belt

The equal distribution of synaptic vesicles among synapses along the axon is critical for robust neurotransmission. Wong et al. show that the continuous circulation of synaptic vesicles throughout the axon driven by molecular motors ultimately yields this even distribution.     

Class C Vps protein complex regulates vacuolar SNARE pairing and is required for vesicle docking/fusion

In yeast, the Class C Vps protein complex (C-Vps complex), composed of Vps11, Vps16, Vps18, and Vps33, functions in Golgi-to-vacuole protein transport. In this study, we characterized and purified this complex and identified its interaction with the syntaxin homolog Vam3. Vam3 pairs with the SNAP-25 homolog Vam7 and VAMP homolog Vti1 to form SNARE complexes during vesicle docking/fusion with the vacuole. The C-Vps complex does not bind to Vam3-Vti1-Vam7 paired SNARE complexes but instead binds to unpaired Vam3. Antibodies to a component of this complex inhibited in vitro vacuole-to-vacuole fusion. Furthermore, temperature-conditional mutations in the Class C VPS genes destabilized Vam3-Vti1-Vam7 pairing. Therefore, we propose that the C-Vps complex associates with unpaired (activated) Vam3 to mediate the assembly of trans-SNARE complexes during both vesicle docking/fusion and vacuole-to-vacuole fusion.     

Munc18-1 promotes large dense-core vesicle docking.

Secretory vesicles dock at the plasma membrane before Ca(2+) triggers their exocytosis. Exocytosis requires the assembly of SNARE complexes formed by the vesicle protein Synaptobrevin and the membrane proteins Syntaxin-1 and SNAP-25. We analyzed the role of Munc18-1, a cytosolic binding partner of Syntaxin-1, in large dense-core vesicle (LDCV) secretion. Calcium-dependent LDCV exocytosis was reduced 10-fold in mouse chromaffin cells lacking Munc18-1, but the kinetic properties of the remaining release, including single fusion events, were not different from controls. Concomitantly, mutant cells displayed a 10-fold reduction in morphologically docked LDCVs. Moreover, acute overexpression of Munc18-1 in bovine chromaffin cells increased the amount of releasable vesicles and accelerated vesicle supply. We conclude that Munc18-1 functions upstream of SNARE complex formation and promotes LDCV docking.     

Synaptic vesicle protein 2 binds adenine nucleotides

Synaptic vesicle protein 2 (SV2) is required for normal calcium-regulated secretion of hormones and neurotransmitters. Neurons lacking the two most widely expressed isoforms, SV2A and SV2B, have a reduced readily releasable pool of synaptic vesicles, indicating that SV2 contributes to vesicle priming. The presence of putative ATP-binding sites in SV2 suggested that SV2 might be an ATP-binding protein. To explore this, we examined the binding of the photoaffinity reagent 8-azido-ATP[gamma] biotin to purified, recombinant SV2 in the presence and absence of other nucleotides. Our results indicate that SV2A and SV2B bind nucleotides, with the highest affinity for adenine-containing nucleotides. SV2A contains two binding sites located in the cytoplasmic domains preceding the first and seventh transmembrane domains. These results suggest that SV2-mediated vesicle priming could be regulated by adenine nucleotides, which might provide a link between cellular energy levels and regulated secretion.     

The protein machinery of vesicle budding and fusion.

A general protein machinery that buds and fuses transport vesicles is harnessed to generate the complex web of intracellular transport pathways critical for such diverse processes as cell growth, endocytosis, hormone release, and neurotransmission. With this appreciation, the challenge of understanding the precise molecular mechanisms of these many facets of cell biology has been reduced to a series of problems in protein structure and chemistry.