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Botulinum neurotoxins (BoNTs) are valuable tools to unveil molecular mechanisms of exocytosis in neuronal and non-neuronal cells due to their peptidase activity on exocytic isoforms of SNARE proteins. They are produced by Clostridia as single-chain polypeptides that are proteolytically cleaved into light, catalytic domains covalently linked via disulfide bonds to heavy, targeting domains. This format of two subunits linked by disulfide bonds is required for the full neurotoxicity of BoNTs. We have generated a recombinant version of BoNT/B that consists of the light chain of the toxin fused to the protein transduction domain of the human immunodeficiency virus-1 (TAT peptide) and a hexahistidine tag. His6-TAT-BoNT/B-LC, expressed in Escherichia coli and purified by affinity chromatography, penetrated membranes and exhibited strong enzymatic activity, as evidenced by cleavage of the SNARE synaptobrevin from rat brain synaptosomes and human sperm cells. Proteolytic attack of synaptobrevin hindered exocytosis triggered by a calcium ionophore in the latter. The novel tool reported herein disrupts the function of a SNARE protein within minutes in cells that may or may not express the receptors for the BoNT/B heavy chain, and without the need for transient transfection or permeabilization., (© 2024 The Author(s).)

The soluble N -ethylmaleimide-sensitive factor attachment receptor (SNARE) proteins synaptobrevin (Syb), syntaxin, and SNAP-25 function in Ca 2+ -triggered exocytosis in both endocrine cells and neurons. The transmembrane domains (TMDs) of Syb and syntaxin span the vesicle and plasma membrane, respectively, and influence flux through fusion pores in endocrine cells as well as fusion pores formed during SNARE-mediated fusion of reconstituted membranes. These results support a model for exocytosis in which SNARE TMDs form the initial fusion pore. The present study sought to test this model in synaptic terminals. Patch-clamp recordings of miniature EPSCs (mEPSCs) were used to probe fusion pore properties in cultured hippocampal neurons from mice of both sexes. Mutants harboring tryptophan at four different sites in the Syb TMD reduced the rate-of-rise of mEPSCs. A computer model that simulates glutamate diffusion and receptor activation kinetics could account for this reduction in mEPSC rise rate by slowing the flux of glutamate through synaptic fusion pores. TMD mutations introducing positive charge also reduced the mEPSC rise rate, but negatively charged residues and glycine, which should have done the opposite, had no effect. The sensitivity of mEPSCs to pharmacological blockade of receptor desensitization was enhanced by a mutation that slowed the mEPSC rate-of-rise, suggesting that the mutation prolonged the residence of glutamate in the synaptic cleft. The same four Syb TMD residues found here to influence synaptic release were found previously to influence endocrine release, leading us to propose that a similar TMD-lined fusion pore functions widely in Ca 2+ -triggered exocytosis in mammalian cells. SIGNIFICANCE STATEMENT SNARE proteins function broadly in biological membrane fusion. Evidence from non-neuronal systems suggests that SNARE proteins initiate fusion by forming a fusion pore lined by transmembrane domains, but this model has not yet been tested in synapses. The present study addressed this question by testing mutations in the synaptic vesicle SNARE synaptobrevin for an influence on the rise rate of miniature synaptic currents. These results indicate that synaptobrevin's transmembrane domain interacts with glutamate as it passes through the fusion pore. The sites in synaptobrevin that influence this flux are identical to those shown previously to influence flux through endocrine fusion pores. Thus, SNARE transmembrane domains may function in the fusion pores of Ca 2+ -triggered exocytosis of both neurotransmitters and hormones., (Copyright © 2018 the authors 0270-6474/18/387179-13$15.00/0.)

Secretory vesicle exocytosis is a fundamental biological event and the process by which hormones (like insulin) are released into the blood. Considerable progress has been made in understanding this precisely orchestrated sequence of events from secretory vesicle docked at the cell membrane, hemifusion, to the opening of a membrane fusion pore. The exact biophysical and physiological regulation of these events implies a close interaction between membrane proteins and lipids in a confined space and constrained geometry to ensure appropriate delivery of cargo. We consider some of the still open questions such as the nature of the initiation of the fusion pore, the structure and the role of the Soluble N-ethylmaleimide-sensitive-factor Attachment protein REceptor (SNARE) transmembrane domains and their influence on the dynamics and regulation of exocytosis. We discuss how the membrane composition and protein-lipid interactions influence the likelihood of the nascent fusion pore forming. We relate these factors to the hypothesis that fusion pore expansion could be affected in type-2 diabetes via changes in disease-related gene transcription and alterations in the circulating lipid profile. Detailed characterisation of the dynamics of the fusion pore in vitro will contribute to understanding the larger issue of insulin secretory defects in diabetes., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Vesicle fusion is mediated by an assembly of SNARE proteins between opposing membranes, but it is unknown whether transmembrane domains (TMDs) of SNARE proteins serve mechanistic functions that go beyond passive anchoring of the force-generating SNAREpin to the fusing membranes. Here, we show that conformational flexibility of synaptobrevin-2 TMD is essential for efficient Ca(2+)-triggered exocytosis and actively promotes membrane fusion as well as fusion pore expansion. Specifically, the introduction of helix-stabilizing leucine residues within the TMD region spanning the vesicle's outer leaflet strongly impairs exocytosis and decelerates fusion pore dilation. In contrast, increasing the number of helix-destabilizing, ß-branched valine or isoleucine residues within the TMD restores normal secretion but accelerates fusion pore expansion beyond the rate found for the wildtype protein. These observations provide evidence that the synaptobrevin-2 TMD catalyzes the fusion process by its structural flexibility, actively setting the pace of fusion pore expansion.

Ribbon synapses of the vertebrate retina are specialized synapses that release neurotransmitter by synaptic vesicle exocytosis in a manner that is proportional to the level of depolarization of the cell. This release property is different from conventional neurons, in which the release of neurotransmitter occurs as a short-lived burst triggered by an action potential. Synaptic vesicle exocytosis is a calcium regulated process that is dependent on a set of interacting synaptic proteins that form the so-called SNARE (soluble N-ethylmaleimide sensitive factor attachment protein receptor) complex. Syntaxin 3B has been identified as a specialized SNARE molecule in ribbon synapses of the rodent retina. However, the best physiologically-characterized neuron that forms ribbon-style synapses is the rod-dominant or Mb1 bipolar cell of the goldfish retina. We report here the molecular characterization of syntaxin 3B from the goldfish retina. Using a combination of reverse transcription (RT) polymerase chain reaction (PCR) and immunostaining with a specific antibody, we show that syntaxin 3B is highly enriched in the plasma membrane of bipolar cell synaptic terminals of the goldfish retina. Using membrane capacitance measurements we demonstrate that a peptide derived from goldfish syntaxin 3B inhibits synaptic vesicle exocytosis. These experiments demonstrate that syntaxin 3B is an important factor for synaptic vesicle exocytosis in ribbon synapses of the vertebrate retina., (Copyright 2010 IBRO. Published by Elsevier Ltd. All rights reserved.)

Abstract Neurotransmitter and hormone exocytosis depends on SNARE protein transmembrane domains and membrane lipids but their interplay is poorly understood. We investigated the interaction of the structure of VAMP2, a vesicular transmembrane SNARE protein, and membrane lipid composition by infrared spectroscopy using either the wild-type transmembrane domain (TMD), VAMP2 TM22 , or a peptide mutated at the central residues G 100 /C 103 (VAMP2 TM22 VV) previously identified by us as being critical for exocytosis. Our data show that the structure of VAMP2 TM22 , in terms of α-helices and β-sheets is strongly influenced by peptide/lipid ratios, by lipid species including cholesterol and by membrane surface charges. Differences observed in acyl chain alignments further underscore the role of the two central small amino acid residues G 100 /C 103 within the transmembrane domain during lipid rearrangements in membrane fusion. Graphical abstract Unlabelled Image Highlights • The transmembrane domain (TMD) of VAMP2 and lipids influences membrane fusion. • VAMP2-TMD undergoes concentration dependent reversible structural changes in bilayers. • VAMP2 TMD structures are highly sensitive to the composition of the lipid environment. • Mutating 2 central residues, crucial for exocytosis, increases β-sheet content. • The same mutation increases acyl chain perturbation. [ABSTRACT FROM AUTHOR]

The soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) proteins synaptobrevin (Syb), syntaxin, and SNAP-25 function in Ca2+ -triggered exocytosis in both endocrine cells and neurons. The transmembrane domains (TMDs) of Syb and syntaxin span the vesicle and plasma membrane, respectively, and influence flux through fusion pores in endocrine cells as well as fusion pores formed during SNARE-mediated fusion of reconstituted membranes. These results support a model for exocytosis in which SNARE TMDs form the initial fusion pore. The present study sought to test this model in synaptic terminals. Patch-damp recordings of miniature EPSCs (mEPSCs) were used to probe fusion pore properties in cultured hippocampal neurons from mice of both sexes. Mutants harboring tryptophan at four different sites in the Syb TMD reduced the rate-of-rise of mEPSCs. A computer model that simulates glutamate diffusion and receptor activation kinetics could account for this reduction in mEPSC rise rate by slowing the flux of glutamate through synaptic fusion pores. TMD mutations introducing positive charge also reduced the mEPSC rise rate, but negatively charged residues and glycine, which should have done the opposite, had no effect. The sensitivity of mEPSCs to pharmacological blockade of receptor desensitization was enhanced by a mutation that slowed the mEPSC rate-of-rise, suggesting that the mutation prolonged the residence of glutamate in the synaptic cleft. The same four Syb TMD residues found here to influence synaptic release were found previously to influence endocrine release, leading us to propose that a similar TMD-lined fusion pore functions widely in Ca2+ -triggered exocytosis in mammalian cells. [ABSTRACT FROM AUTHOR]

As a SNARE binding protein, tomosyn has been reported to negatively regulate synaptic exocytosis via arresting syntaxin‐1 and SNAP‐25 into a nonfusogenic product that precludes synaptobrevin‐2 entry, raising the question how the assembly of the SNARE complex is achieved. Here, we have investigated new functions of tomosyn in SNARE complex formation and SNARE‐mediated vesicle fusion. Assisted by NSF/α‐SNAP, syntaxin‐1 escapes tomosyn arrest and assembles into the Munc18‐1/syntaxin‐1 complex. Munc13‐1 then catalyzes the transit of syntaxin‐1 from the Munc18‐1/syntaxin‐1 complex to the SNARE complex in a manner specific to synaptobrevin‐2 but resistant to tomosyn. Our data suggest that tomosyn ensures SNARE assembly in a way amenable to tight regulation by Munc18‐1 and Munc13‐1. [ABSTRACT FROM AUTHOR]

Parallel zippering of the SNARE domains of syntaxin 1A/B, SNAP-25, and VAMP/synaptobrevin 2 is widely regarded as supplying the driving force for exocytotic events at nerve terminals and elsewhere. However, in spite of intensive research, no consensus has been reached concerning the molecular mechanism by which these SNARE proteins catalyze membrane fusion. As an alternative to SNARE-based models, a scenario was developed in which synaptotagmin 1 (or, 2) can serve as a template to guide lipid movements that underlie fast, synchronous exocytosis at nerve terminals. This "dyad model" advanced a novel proposal concerning the membrane disposition of the palmitoylated, cysteine-rich region of these synaptotagmins. Unexpectedly, it now emerges that a similar principle can be exploited to reveal how the hydrophobic, carboxyl-terminal domains of syntaxin 1A and synaptobrevin 2 can perturb membrane structure at the interface between a docked synaptic vesicle and the plasma membrane. These "β-to-α transition" models will be compared and contrasted with other proposals for how macromolecules are thought to intervene to drive membrane fusion. [ABSTRACT FROM AUTHOR]

Assembly of the SNARE proteins syntaxin1, SNAP25, and synaptobrevin into a SNARE complex is essential for exocytosis in neurons. For efficient assembly, SNAREs interact with additional proteins but neither the nature of the intermediates nor the sequence of protein assembly is known. Here, we have characterized a ternary complex between syntaxin1, SNAP25, and the SM protein Munc18-1 as a possible acceptor complex for the R- SNARE synaptobrevin. The ternary complex binds synaptobrevin with fast kinetics, resulting in the rapid formation of a fully zippered SNARE complex to which Munc18-1 remains tethered by the N-terminal domain of syntaxin1. Intriguingly, only one of the synaptobrevin truncation mutants (Syb1-65) was able to bind to the syntaxin1: SNAP25:Munc18-1 complex, suggesting either a cooperative zippering mechanism that proceeds bidirectionally or the progressive R- SNARE binding via an SM template. Moreover, the complex is resistant to disassembly by NSF. Based on these findings, we consider the ternary complex as a strong candidate for a physiological intermediate in SNARE assembly. [ABSTRACT FROM AUTHOR]

The soluble N-ethylmaleimide-sensitive factor attachment protein receptor ( SNARE) protein syntaxin-1 adopts a closed conformation when bound to Munc18-1, preventing binding to synaptobrevin-2 and SNAP-25 to form the ternary SNARE complex. Although it is known that the MUN domain of Munc13-1 catalyzes the transition from the Munc18-1/syntaxin-1 complex to the SNARE complex, the molecular mechanism is unclear. Here, we identified two conserved residues (R151, I155) in the syntaxin-1 linker region as key sites for the MUN domain interaction. This interaction is essential for SNARE complex formation in vitro and synaptic vesicle priming in neuronal cultures. Moreover, this interaction is important for a tripartite Munc18-1/syntaxin-1/ MUN complex, in which syntaxin-1 still adopts a closed conformation tightly bound to Munc18-1, whereas the syntaxin-1 linker region changes its conformation, similar to that of the LE mutant of syntaxin-1 when bound to Munc18-1. We suggest that the conformational change of the syntaxin-1 linker region induced by Munc13-1 initiates ternary SNARE complex formation in the neuronal system. [ABSTRACT FROM AUTHOR]

Objective To identify the molecular basis and elucidate the pathogenesis of a fatal congenital myasthenic syndrome. Methods We performed clinical electrophysiology studies, exome and Sanger sequencing, and analyzed functional consequences of the identified mutation. Results Clinical electrophysiology studies of the patient revealed several-fold potentiation of the evoked muscle action potential by high frequency nerve stimulation pointing to a presynaptic defect. Exome sequencing identified a homozygous c.340delA frameshift mutation in synaptobrevin 1 (SYB1), one of the three SNARE proteins essential for synaptic vesicle exocytosis. Analysis of both human spinal cord gray matter and normal human muscle revealed expression of the SYB1A and SYB1D isoforms, predicting expression of one or both isoforms in the motor nerve terminal. The identified mutation elongates the intravesicular C-terminus of the A isoform from 5 to 71, and of the D isoform from 4 to 31 residues. Transfection of either mutant isoform into bovine chromaffin cells markedly reduces depolarization-evoked exocytosis, and transfection of either mutant isoform into HEK cells significantly decreases expression of either mutant compared to wild type. Interpretation The mutation is pathogenic because elongation of the intravesicular C-terminus of the A and D isoforms increases the energy required to move their C-terminus into the synaptic vesicle membrane, a key step for fusion of the synaptic vesicle with the presynaptic membrane, and because it is predicted to reduce expression of either isoform in the nerve terminal. [ABSTRACT FROM AUTHOR]

Membrane fusion is a key process in all living organisms that contributes to a variety of biological processes including viral infection, cell fertilization, as well as intracellular transport, and neurotransmitter release. In particular, the various membrane-enclosed compartments in eukaryotic cells need to exchange their contents and communicate across membranes. Efficient and controllable fusion of biological membranes is known to be driven by cooperative action of SNARE proteins, which constitute the central components of the eukaryotic fusion machinery responsible for fusion of synaptic vesicles with the plasma membrane. During exocytosis, vesicle-associated v-SNARE (synaptobrevin) and target cell-associated t-SNAREs (syntaxin and SNAP-25) assemble into a core trans-SNARE complex. This complex plays a versatile role at various stages of exocytosis ranging from the priming to fusion pore formation and expansion, finally resulting in the release or exchange of the vesicle content. This review summarizes current knowledge on the intricate molecular mechanisms underlying exocytosis triggered and catalyzed by SNARE proteins. Particular attention is given to the function of the peptidic SNARE membrane anchors and the role of SNARE-lipid interactions in fusion. Moreover, the regulatory mechanisms by synaptic auxiliary proteins in SNAREdriven membrane fusion are briefly outlined. [ABSTRACT FROM AUTHOR]

Vesicle fusion is mediated by an assembly of SNARE proteins between opposing membranes, but it is unknown whether transmembrane domains (TMDs) of SNARE proteins serve mechanistic functions that go beyond passive anchoring of the force-generating SNAREpin to the fusing membranes. Here, we show that conformational flexibility of synaptobrevin-2 TMD is essential for efficient Ca2+-triggered exocytosis and actively promotes membrane fusion as well as fusion pore expansion. Specifically, the introduction of helix-stabilizing leucine residues within the TMD region spanning the vesicle’s outer leaflet strongly impairs exocytosis and decelerates fusion pore dilation. In contrast, increasing the number of helix-destabilizing, ß-branched valine or isoleucine residues within the TMD restores normal secretion but accelerates fusion pore expansion beyond the rate found for the wildtype protein. These observations provide evidence that the synaptobrevin-2 TMD catalyzes the fusion process by its structural flexibility, actively setting the pace of fusion pore expansion.

Neurotransmitters released at synapses activate neighboring astrocytes, which in turn, modulate neuronal activity by the release of diverse neuroactive substances that include classical neurotransmitters such as glutamate, GABA or ATP. Neuroactive substances are released from astrocytes through several distinct molecular mechanisms, for example, by diffusion through membrane channels, by translocation via plasmalemmal transporters or by vesicular exocytosis. Vesicular release regulated by a stimulus-mediated increase in cytosolic calcium involves soluble N-ethyl maleimide-sensitive fusion protein attachment protein receptor (SNARE)-dependent merger of the vesicle membrane with the plasmalemma. Up to 25 molecules of synaptobrevin 2 (Sb2), a SNARE complex protein, reside at a single astroglial vesicle; an individual neuronal, i.e. synaptic, vesicle contains ∼70 Sb2 molecules. It is proposed that this paucity of Sb2 molecules in astrocytic vesicles may determine the slow secretion. In the present essay we shall overview multiple aspects of vesicular architecture and types of vesicles based on their cargo and dynamics in astroglial cells. [ABSTRACT FROM AUTHOR]

The vesicular protein synaptobrevin II (sybII) constitutes a central component of the SNARE complex, which mediates vesicle fusion in neuronal exocytosis. Previous studies revealed that the transmembrane domain (TMD) of sybII is playing a critical role in the fusion process and is involved in all distinct fusion stages from priming to fusion pore opening. Here, we analyzed sequence-dependent effects of sybII and of mutants of sybII on both structure and flexibility of the protein and the interactions with a phospholipid bilayer by means of microsecond atomistic simulations. The sybII TMD was found to direct the folding of both the juxtamembrane helix and of the connecting linker and thus to influence both the intrinsic helicity and flexibility. Fusion active peptides revealed two helical segments, one for the juxtamembrane region and one for the TMD, connected by a flexible linker. In contrast, a fusion-inactive poly-leucine TMD mutant assumes a structure with a comparably rigid linker that is suggested to hinder the formation of the trans -SNARE complex during fusion. Kinking of the TMD at the central glycine together with anchoring of the TMD via conserved tryptophans and a lysine in position 94 likely yields an enhanced flexibility of sybII for different membrane thickness. All studied peptides were found to deform the outer membrane layer by altering the lipid head group orientation, causing partial membrane dehydration and enhancing lipid protrusions. These effects weaken the integrity of the outer membrane layer and are attributed mainly to the highly charged linker and JM regions of sybII. [ABSTRACT FROM AUTHOR]

SNARE proteins catalyze many forms of biological membrane fusion, including Ca2+-triggered exocytosis. Although fusion mediated by SNAREs generally involves proteins anchored to each fusing membrane by a transmembrane domain (TMD), the role of TMDs remains unclear, and previous studies diverge on whether SNAREs can drive fusion without a TMD. This issue is important because it relates to the question of the structure and composition of the initial fusion pore, as well as the question of whether SNAREs mediate fusion solely by creating close proximity between two membranes versus a more active role in transmitting force to the membrane to deform and reorganize lipid bilayer structure. To test the role of membrane attachment, we generated four variants of the synaptic v-SNARE synaptobrevin-2 (syb2) anchored to the membrane by lipid instead of protein. These constructs were tested for functional efficacy in three different systems as follows: Ca2+-triggered dense core vesicle exocytosis, spontaneous synaptic vesicle exocytosis, and Ca2+-synaptotagmin-enhanced SNARE-mediated liposome fusion. Lipid-anchoring motifs harboring one or two lipid acylation sites completely failed to support fusion in any of these assays. Only the lipid-anchoring motif from cysteine string protein-α, which harbors many lipid acylation sites, provided support for fusion but at levels well below that achieved with wild type syb2. Thus, lipid-anchored syb2 provides little or no support for exocytosis, and anchoring syb2 to a membrane by a TMD greatly improves its function. The low activity seen with syb2-cysteine string protein-α may reflect a slower alternative mode of SNARE-mediated membrane fusion. [ABSTRACT FROM AUTHOR]

Key points Mouse cortical astrocytes express VAMP3 but not VAMP2., VAMP3 vesicles undergo Ca2+-independent exo- and endocytotic cycling at the plasma membrane., VAMP3 vesicle traffic regulates the recycling of plasma membrane glutamate transporters., cAMP modulates VAMP3 vesicle cycling and glutamate uptake., Abstract Previous studies suggest that small synaptic-like vesicles in astrocytes carry vesicle-associated vSNARE proteins, VAMP3 (cellubrevin) and VAMP2 (synaptobrevin 2), both contributing to the Ca2+-regulated exocytosis of gliotransmitters, thereby modulating brain information processing. Here, using cortical astrocytes taken from VAMP2 and VAMP3 knock-out mice, we find that astrocytes express only VAMP3. The morphology and function of VAMP3 vesicles were studied in cultured astrocytes at single vesicle level with stimulated emission depletion (STED) and total internal reflection fluorescence (TIRF) microscopies. We show that VAMP3 antibodies label small diameter (∼80 nm) vesicles and that VAMP3 vesicles undergo Ca2+-independent exo-endocytosis. We also show that this pathway modulates the surface expression of plasma membrane glutamate transporters and the glutamate uptake by astrocytes. Finally, using pharmacological and optogenetic tools, we provide evidence suggesting that the cytosolic cAMP level influences astrocytic VAMP3 vesicle trafficking and glutamate transport. Our results suggest a new role for VAMP3 vesicles in astrocytes. [ABSTRACT FROM AUTHOR]

Ca2+-triggered release of neurotransmitters and hormones depends on soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) to drive the fusion of the vesicle and plasma membranes. The formation of the SNARE complex by the vesicle SNARE synaptobrevin 2 (syb2) and the two plasma membrane SNAREs syntaxin (syx) and SNAP-25 draws the two membranes together, but the events that follow membrane juxtaposition, and the ways that SNAREs remodel lipid membranes remain poorly understood. The SNAREs syx and syb2 have transmembrane domains (TMDs) that can exert force directly on the lipid bilayers. The TMD of syx influences fusion pore flux in a manner that suggests it lines the nascent fusion pore through the plasma membrane. The TMD of syb2 traverses the vesicle membrane and is the most likely partner to syx in completing a proteinaceous fusion pore through the vesicle membrane, but the role of this vesicle SNARE in fusion pores has yet to be tested. Here amperometry and conductance measurements were performed to probe the function of the syb2 TMD in fusion pores formed during catecholamine exocytosis in mouse chromaffin cells. Fusion pore flux was sensitive to the size and charge of TMD residues near the N terminus; fusion pore conductance was altered by substitutions at these sites. Unlike syx, the syb2 residues that influence fusion pore permeation fell along two a-helical faces of its TMD, rather than one. These results indicate a role for the syb2 TMD in nascent fusion pores, but in a very different structural arrangement from that of the syx TMD. [ABSTRACT FROM AUTHOR]

How clathrin-mediated endocytosis ( CME) retrieves vesicle proteins into newly formed synaptic vesicles ( SVs) remains a major puzzle. Besides its roles in stimulating clathrin-coated vesicle formation and regulating SV size, the clathrin assembly protein AP180 has been identified as a key player in retrieving SV proteins. The mechanisms by which AP180 recruits SV proteins are not fully understood. Here, we show that following acute inactivation of AP180 in Drosophila, SV recycling is severely impaired at the larval neuromuscular synapse based on analyses of FM 1-43 uptake and synaptic ultrastructure. More dramatically, AP180 activity is important to maintain the integrity of SV protein complexes at the plasma membrane during endocytosis. These observations suggest that AP180 normally clusters SV proteins together during recycling. Consistent with this notion, SV protein composition and distribution are altered in AP180 mutant flies. Finally, AP180 co-immunoprecipitates with SV proteins, including the vesicular glutamate transporter and neuronal synaptobrevin. These results reveal a new mode by which AP180 couples protein retrieval to CME of SVs. AP180 is also genetically linked to Alzheimer's disease. Hence, the findings of this study may provide new mechanistic insight into the role of AP180 dysfunction in Alzheimer's disease. [ABSTRACT FROM AUTHOR]

SNAP25, an essential component of the soluble NSF (N-ethylmaleimide-sensitive factor) attachment protein receptor (SNARE) complex that mediates exocytosis, is not considered to play a role in endocytosis, which couples to exocytosis by retrieving a similar amount of exocytosed vesicles. By knocking down SNAP25 and imaging slow endocytosis at a conventional synapse, the rat cultured hippocampal synapse, we found that SNAP25 is involved in slow, clathrin-dependent endocytosis. With similar techniques, we found that not only SNAP25, but also synaptobrevin is involved in slow endocytosis. These results provide the first evidence showing the dual role of SNAP25 and synaptobrevin in both exocytosis and slow endocytosis at conventional synapses. Such a dual role may contribute to mediate the coupling between exocytosis and clathrin-dependent endocytosis at conventional synapses, a mechanism critical for the maintenance of synaptic transmission and the normal structure of nerve terminals. [ABSTRACT FROM AUTHOR]

Parkinson disease and dementia with Lewy bodies are featured with the formation of Lewy bodies composed mostly of α-synuclein (α-Syn) in the brain. Although evidence indicates that the large oligomeric or protofibril forms of α-Syn are neurotoxic agents, the detailed mechanisms of the toxic functions of the oligomers remain unclear. Here, we show that large α-Syn oligomers efficiently inhibit neuronal SNARE-mediated vesicle lipid mixing. Large α-Syn oligomers preferentially bind to the N-terminal domain of a vesicular SNARE protein, synaptobrevin-2, which blocks SNARE-mediated lipid mixing by preventing SNARE complex formation. In sharp contrast, the α-Syn monomer has a negligible effect on lipid mixing even with a 30-fold excess compared with the case of large α-Syn oligomers. Thus, the results suggest that large α-Syn oligomers function as inhibitors of dopamine release, which thus provides a clue, at the molecular level, to their neurotoxicity. [ABSTRACT FROM AUTHOR]

Four evolutionarily conserved proteins are required for mammalian regulated exocytosis: three SNARE proteins, syntaxin, SNAP-25, and synaptobrevin, and the SM protein, Munc18-1. Here, using single-molecule imaging, we measured the spatial distribution of large cohorts of single Munc18-1 molecules correlated with the positions of single secretory vesicles in a functionally rescued Munc18-1-null cellular model. Munc18-1 molecules were nonrandomly distributed across the plasma membrane in a manner not directed by mode of interaction with syntaxin1, with a small mean number of molecules observed to reside under membrane resident vesicles. Surprisingly, we found that the majority of vesicles in fully secretion-competent cells had no Munc18-1 associated within distances relevant to plasma membrane-vesicle SNARE interactions. Live cell imaging of Munc18-1 molecule dynamics revealed that the density of Munc18-1 molecules at the plasma membrane anticorrelated with molecular speed, with single Munc18-1 molecules displaying directed motion between membrane hotspots enriched in syntaxin1a. Our findings demonstrate that Munc18-1 molecules move between membrane depots distinct from vesicle morphological docking sites. [ABSTRACT FROM AUTHOR]

Abstract: Synaptobrevin 2 (Syb2), syntaxin (Sx1A), and SNAP-25, generate a force to induce fusion pore formation. The v-SNARE, Syb2, is anchored to the vesicle membrane by a single transmembrane domain. Here we show that 2 tryptophans (W89/W90) located in the juxtamembrane domain of Syb2, which stabilize the transmembrane (TM) domain position, control the ratio of spontaneous vs. stimulated membrane fusion events in chromaffin cells. Changing the 2 hydrophobic tryptophans to neutral alanines promotes spontaneous membrane fusion, faster transmitter release kinetics and complete release from individual vesicles. The results indicate that the two tryptophans act as a fusion clamp making fusion stimulus-dependent. [Copyright &y& Elsevier]

Soluble NSF attachment protein receptors (SNAREs) are the core proteins in membrane fusion. The Sneuron-speciic synaptic v-SNARE n-syb (neuronal Synaptobrevin) plays a key role during synaptic vesicle exocytosis. In this paper, we report that loss of n-syb caused slow neurodegeneration independent of its role in neurotransmitter release in adult Drosophila melanogaster photoreceptor neurons. In addition to synaptic vesicles, n-Syb localized to endosomal vesicles. Loss of n-syb lead to endosomal accumulations, transmembrane protein degradation defects, and a secondary increase in autophagy. Our evidence suggests a primary defect of impaired delivery of vesicles that contain degradation proteins, including the acidification-activated Cathepsin proteases and the neuron-specific proton pump and V0 adenosine triphosphatase component V100. Overexpressing V100 partially rescued n-syb-dependent degeneration through an acidification-independent endosomal sorting mechanism. Collectively, these findings reveal a role for n-Syb in a neuron-specific sort and-degrade mechanism that protects neurons from degeneration. Our findings further shed light on which intraneuronal compartments exhibit increased or decreased neurotoxicity. [ABSTRACT FROM AUTHOR]

Abstract: Although synaptotagmin I, which is a calcium (Ca2+)-binding synaptic vesicle protein, may trigger soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)-mediated synaptic vesicle exocytosis, the mechanisms underlying the interaction between these proteins remain controversial, especially with respect to the identity of the protein(s) in the SNARE complex that bind(s) to synaptotagmin and whether Ca2+ is required for their highly effective binding. To address these questions, native proteins were solubilized, immunoprecipitated from rat brain extracts, and analyzed by immunoblotting. SNARE complexes comprising syntaxin 1, 25-kDa synaptosomal-associated protein (SNAP-25), and synaptobrevin 2 were coprecipitated with synaptotagmin I in the presence of ethylene glycol tetraacetic acid. The amount of coprecipitated proteins was significantly unaltered by the addition of Ca2+ to the brain extract. To identify the component of the SNARE complex that bound to synaptotagmin, SNARE was coexpressed with synaptotagmin in HEK293 cells and immunoprecipitated. Syntaxin, but not SNAP-25 and synaptobrevin, bound to synaptotagmin in a Ca2+-independent manner, and the binding was abolished in the presence of 1M NaCl. Synaptotagmin contains 2 Ca2+-binding domains (C2A, C2B). Mutating the positively charged lysine residues in the putative effector-binding region of the C2B domain, which are critical for transmitter release, markedly inhibited synaptotagmin–syntaxin binding, while similar mutations in the C2A domain had no effect on binding. Synaptotagmin–syntaxin binding was reduced by mutating multiple negatively charged glutamate residues in the amino-terminal half of the syntaxin SNARE motif. These results indicate that synaptotagmin I binds to syntaxin 1 electrostatically through its C2B domain effector region in a Ca2+-independent fashion, providing biochemical evidence that synaptotagmin I binds SNARE complexes before Ca2+ influx into presynaptic nerve terminals. [Copyright &y& Elsevier]

Abstract: Exocytosis is one of the most crucial and ubiquitous processes in all of biology. This event is mediated by the formation of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) complexes, ternary assemblies of syntaxin, SNAP23/SNAP25 (synaptosomal-associated protein of 23 or 25kDa), and synaptobrevin. The exocytotic process can be further regulated by complexin, which interacts with the SNARE complex. Complexin is involved in a Ca2+-triggered exocytotic process. In eukaryotic cells, multiple isoforms of SNARE proteins are expressed and are involved in distinct types of exocytosis. To understand the underlying biochemical mechanism of various exocytotic processes mediated by different SNARE protein isoforms, we systematically analyzed the interactions among syntaxin, SNAP23/SNAP25, synaptobrevin, and complexin by employing a newly developed yeast four-hybrid interaction assay. The efficiency of SNARE complex formation and the specificity of complexin binding are regulated by the different SNARE protein isoforms. Therefore, various types of exocytosis, occurring on different time scales with different efficiencies, can be explained by the involved SNARE complexes composed of different combinations of SNARE protein isoforms. [Copyright &y& Elsevier]

Astrocytes possess much of the same exocytotic protein machinery as neurons do and can release various gliotransmitters stored in their secretory vesicles. An essential component of this exocytotic machinery is the vesicle-associated membrane protein synaptobrevin 2 (Sb2). In order to assess whether vesicular age plays a role in determining the intracellular location of vesicles in astrocytes, we generated a fluorescent chimeric form of Sb2. We appended the Sb2 cytosolic N-terminus with the fluorescent ‘timer’ protein DsRedE5, which changes its fluorescence emission from green to red as it ages. We found that Sb2-containing vesicles in astrocytes segregate and localize intracellularly in an age dependent manner. Younger vesicles predominately localize at the periphery of cell somata and processes, while older vesicles predominately locate at the central portion of the cell body. These findings raise the notion that there might be differential astrocyte-neuron signaling at sites away or at the tripartite synapse that could be modulated by the age of vesicles and/or their cargo. [ABSTRACT FROM AUTHOR]

Lead (Pb2+) exposure is known to affect presynaptic neurotransmitter release in both in vivo and cell culture models. However, the precise mechanism by which Pb2+ impairs neurotransmitter release remains unknown. In the current study, we show that Pb2+ exposure during synaptogenesis in cultured hippocampal neurons produces the loss of synaptophysin (Syn) and synaptobrevin (Syb), two proteins involved in vesicular release. Pb2+ exposure also increased the number of presynaptic contact sites. However, many of these putative presynaptic contact sites lack Soluble NSF attachment protein receptor complex proteins involved in vesicular exocytosis. Analysis of vesicular release using FM 1-43 dye confirmed that Pb2+ exposure impaired vesicular release and reduced the number of fast-releasing sites. Because Pb2+ is a potent N-methyl-D-aspartate receptor (NMDAR) antagonist, we tested the hypothesis that NMDAR inhibition may be producing the presynaptic effects. We show that NMDAR inhibition by aminophosphonovaleric acid mimics the presynaptic effects of Pb2+ exposure. NMDAR activity has been linked to the signaling of the transsynaptic neurotrophin brain-derived neurotrophic factor (BDNF), and we observed that both the cellular expression of proBDNF and release of BDNF were decreased during the same period of Pb2+ exposure. Furthermore, exogenous addition of BDNF rescued the presynaptic effects of Pb2+. We suggest that the presynaptic deficits resulting from Pb2+ exposure during synaptogenesis are mediated by disruption of NMDAR-dependent BDNF signaling. [ABSTRACT FROM PUBLISHER]

Synaptic vesicle fusion is catalyzed by assembly of synaptic SNARE complexes, and is regulated by the synaptic vesicle GTP-binding protein Rab3 that binds to RIM and to rabphilin. RIM is a known physiological regulator of fusion, but the role of rabphilin remains obscure. We now show that rabphilin regulates recovery of synaptic vesicles from use-dependent depression, probably by a direct interaction with the SNARE protein SNAP-25. Deletion of rabphilin dramatically accelerates recovery of depressed synaptic responses; this phenotype is rescued by viral expression of wild-type rabphilin, but not of mutant rabphilin lacking the second rabphilin C2 domain that binds to SNAP-25. Moreover, deletion of rabphilin also increases the size of synaptic responses in synapses lacking the vesicular SNARE protein synaptobrevin in which synaptic responses are severely depressed. Our data suggest that binding of rabphilin to SNAP-25 regulates exocytosis of synaptic vesicles after the readily releasable pool has either been physiologically exhausted by use-dependent depression, or has been artificially depleted by deletion of synaptobrevin. [ABSTRACT FROM AUTHOR]

Exocytosis is initiated within a highly localized region of contact between two biological membranes. Small areas of these membranes draw close, molecules on the two surfaces interact, and structural transformations take place. Membrane fusion requires the action of proteins specialized for this task, and these proteins act as a fusion machine. At a critical point in this process, a fusion pore forms within the membrane contact site and then expands as the spherical vesicle merges with the flat target membrane. Hence, the operation of a fusion machine must be realized through the formation and expansion of a fusion pore. Delineating the relation between the fusion machine and the fusion pore thus emerges as a central goal in elucidating the mechanisms of membrane fusion. We summarize present knowledge of fusion machines and fusion pores studied in vitro, in neurons, and in neuroendocrine cells, and synthesize this knowledge into some specific and detailed hypotheses for exocytosis. [ABSTRACT FROM AUTHOR]

Abstract: In addition to being the conduit for pathogens into hosts, tick saliva contains a broad array of secretory products that facilitate prolonged tick attachment and blood feeding. Proteins found in tick saliva modulate host hemostasis and immune responses. However, it is not clear whether ticks manipulate the immune responses of their hosts by disrupting the antigen-processing pathways of the hosts. Protein secretion into tick saliva from the salivary glands is due to exocytosis of vesicular membrane-bound granular material regulated by SNARE complex proteins. Proteins associated with vesicles (v-SNAREs) are essential components of the exocytotic process. In this study, we assessed the functional significance of synaptobrevin, a SNARE protein, and cystatin, a cysteine protease inhibitor to blood feeding success, in the lone star tick, Amblyomma americanum, using in vivo RNA interference. In separate experiments, tick salivary cystatin and synpatobrevin genes were silenced by injecting adult ticks with 500ng of dsRNA complementing each gene sequence. Silencing was demonstrated by reduced transcript in midguts and salivary glands. Additionally, disrupting expression of cystatin and synaptobrevin by RNAi reduced the ability of ticks to feed successfully, as demonstrated by feeding inhibition and reduced engorgement weights. Moreover, normal ticks exposed to a rabbit previously exposed to cystatin-silenced ticks exhibited significant resistance to tick feeding. Based on these findings, ticks appear to skillfully evade the host immune system by secreting cystatin, which disrupts normal antigen processing in antigen-presenting cells of hosts. [Copyright &y& Elsevier]

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. [ABSTRACT FROM AUTHOR]