Summary Exocytosis of neurosecretory vesicles is mediated bythe SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins syntaxin-1, synaptobrevin, and SNAP-25, with synaptotagmin functioning as the major Ca2+-sensor for triggering membrane fusion. Here we show that bovine chromaffin granules readily fuse with large unilamellar liposomes in a SNARE-dependent manner. Fusion is enhanced by Ca2+ but only if the target liposomes contain PI(4,5)P2 and if polyphosphate anions such as nucleotides or pyrophosphate are present. Ca2+-dependent enhancement is mediated by endogenous synaptotagmin-1. Polyphosphates operate by an electrostatic mechanism that reverses an inactivating cis-association of synaptotagmin-1 with its own membrane whereas trans-binding is not affected. Hence, balancing trans- and cis-membrane interactions of synaptotagmin may be a crucial element in the pathway of Ca2+-dependent exocytosis.

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...

Mover, a member of the exquisitely small group of vertebrate-specific presynaptic proteins, has been discovered as an interaction partner of the scaffolding protein Bassoon, yet its function has not been elucidated. We used adeno-associated virus (AAV)-mediated shRNA expression to knock down Mover in the calyx of Held in vivo. Although spontaneous synaptic transmission remained unaffected, we found a strong increase of the evoked EPSC amplitude. The size of the readily releasable pool was unaltered, but short-term depression was accelerated and enhanced, consistent with an increase in release probability after Mover knockdown. This increase in release probability was not caused by alterations in Ca(2+) influx but rather by a higher Ca(2+) sensitivity of the release machinery, as demonstrated by presynaptic Ca(2+) uncaging. We therefore conclude that Mover expression in certain subsets of synapses negatively regulates synaptic release probability, constituting a novel mechanism to tune synaptic transmission.

Synaptic vesicles (SVs) are essential organelles that participate in the release of neurotransmitters from a neuron. Biochemical analysis of purified SVs was instrumental in the identification of proteins involved in exocytotic membrane fusion and neurotransmitter uptake. Although numerous protocols have been published detailing the isolation of SVs from the brain, those that give the highest-purity vesicles often have low yields. Here we describe a protocol for the small-scale isolation of SVs from mouse and rat brain. The procedure relies on standard fractionation techniques, including differential centrifugation, rate-zonal centrifugation and size-exclusion chromatography, but it has been optimized for minimal vesicle loss while maintaining a high degree of purity. The protocol can be completed in less than 1 d and allows the recovery of ∼150 μg of vesicle protein from a single mouse brain, thus allowing vesicle isolation from transgenic mice.

Synaptotagmin-1 regulates synaptic vesicle fusion, but little is known about the remaining syt isoforms. Syt-pHluorin reporters are used to show that only two syts are on synaptic vesicles, whereas other isoforms are selectively targeted to dendrites, axons, or both axons and dendrites, where they undergo exocytosis and endocytosis with distinct kinetics.