Two synaptobrevin molecules are sufficient for vesicle fusion in central nervous system synapses

Abstract

Exocytosis of synaptic vesicles (SVs) during fast synaptic transmission is mediated by soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex assembly formed by the coil-coiling of three members of this protein family: vesicle SNARE protein, synaptobrevin 2 (syb2), and the presynaptic membrane SNAREs syntaxin-1A and SNAP-25. However, it is controversially debated how many SNARE complexes are minimally needed for SV priming and fusion. To quantify this effective number, we measured the fluorescence responses from single fusing vesicles expressing pHluorin (pHl), a pH-sensitive variant of GFP, fused to the luminal domain of the vesicular SNARE syb2 (spH) in cultured hippocampal neurons lacking endogenous syb2. Fluorescence responses were quantal, with the unitary signals precisely corresponding to single pHluorin molecules. Using this approach we found that two copies of spH per SV fully rescued evoked fusion whereas SVs expressing only one spH were unable to rapidly fuse upon stimulation. Thus, two syb2 molecules and likely two SNARE complexes are necessary and sufficient for SV fusion during fast synaptic transmission.synaptopHluorin | membrane fusion | single molecule bleaching | SNARE density I n conventional neuronal synapses, fast synaptic transmission is mediated by release of neurotransmitter upon Ca 2+ -triggered synaptic vesicle (SV) exocytosis. This process is exquisitely regulated both spatially and temporally. The core of the SV fusion machinery is formed by three members of the soluble N-ethylmaleimidesensitive factor attachment protein receptor (SNARE) protein family, which is characterized by conserved sequences of 60-70 amino acids called SNARE motifs: vesicle SNARE protein, synaptobrevin-2 (syb2), and the presynaptic membrane SNAREs syntaxin-1A and SNAP-25 (1-3).Zipper-like assembly of the SNARE motifs from their N-terminal ends toward their membrane-proximal C termini results in the formation of a highly stable heterotrimeric "trans-SNARE complex" (also called "SNAREpin"), consisting of four parallel α-helices, which brings the two membranes into close apposition for fusion (4-7). Previous studies have suggested that several of these SNARE complexes might assemble in rosette-shaped multivalent supercomplexes, forming a ring, around the fusion pore; however, there is no direct evidence in support of this model (5,8,9). Therefore, the precise number of SNARE complexes minimally required to drive membrane fusion is highly debated and current estimates range between 1 and 15 (10-18). Some of these results are based on single-molecule fluorescence measurements in artificially reconstituted liposomes, whereas others are based on theoretical models, kinetic analysis, and extrapolations from dose-response relationships. Therefore, it is essential to apply a more direct method capable of visualizing single SNARE complexes in real time in a physiological setting.In the present study we have used a unique direct approach to count the number of syb2 molecules required for fast...

Variable cooperativity in SNARE-mediated membrane fusion

“…In the first model, we suggest that although the threshold occurs at 23-30 sybs on the entire surface of the liposome this is the total amount required to locally enrich SNARE complexes, a process that implies stronger coupling and time synchronization in the cooperative assembly of the SNARE complexes. We have modeled a possible scenario and found that two to four SNARE complexes assembled in synchrony would fit in between two large liposomes as contact between membranes is initiated, in agreement with in vivo studies (12,15,33). However, we caution that this is a lower bound estimate because we cannot rule out lateral diffusion of neighboring SNAREs.…”

“…Whether and how SNAREs cooperate to mediate fusion has been a subject of intense study, with estimates ranging from a single SNARE complex to 15. Here we show that there is no universally conserved number of SNARE complexes involved as revealed by our observation that this varies greatly depending on membrane curvature.…”

“…Because we observe this high variability with neuronal SNAREs at constant density but different curvature, a corollary of this conclusion is that we would expect that cooperativity of SNAREs involved in other trafficking pathways will also be highly variable, not because of their inherent biochemical properties, but rather owing to the particular biophysical characteristics of the trafficking vesicle. We therefore suggest that the reported estimates of numbers of involved SNARE complexes likely reflect the particular type of fusion studied and that there is no universally conserved number, as has been often assumed (13,(15)(16)(17)(18)33).…”

“…Although some studies have left open the possibility that the number of SNARE complexes that cooperate during fusion is variable (11,12), much attention has been given to the notion of a preferred number of SNARE complexes, with estimates varying from a single SNARE complex (13) to 15 (14), although more recent estimates vary between two and eight (12,(15)(16)(17)(18). Unfortunately, this large disparity in results has not been appropriately explained, and it remains unclear whether the differences are a result of inherent properties of the particular set of SNAREs involved or rather originate from the biophysical characteristics of the fusing vesicles.…”

VAMP2 is implicated in the secretion of antibodies by human plasma cells and can be replaced by other synaptobrevins

“…In summary, the possible explanations for the partial effect observed with siRNA assays might be (a) molecular redundancy, that is, the protein levels remaining after knockdown are high enough to maintain their normal functionality; 27,55,56 (b) functional redundancy, that is, other VAMP members can substitute for the knockdown; 47 or (c) a combination of the two previous hypotheses. However, the results obtained with TeNT-LC and with VAMP2-ΔTMD lead us to prefer the functional redundancy hypothesis, if we assume that almost all detectable VAMP2 (and VAMP3) proteins were proteolyzed, as was observed by WB.…”