Observing isolated synaptic vesicle association and fusion ex vivo.

Here, we present a protocol for isolating functionally intact glutamatergic synaptic vesicles from whole-mouse brain tissue and using them in a single-vesicle assay to examine their association and fusion with plasma membrane mimic vesicles. This is a Protocol Extension, building on our previous protocol, which used a purely synthetic system comprised of reconstituted proteins in liposomes. We also describe the generation of a peptide based on the vesicular glutamate transporter, which is essential in the isolation process of glutamatergic synaptic vesicles. This method uses easily accessible reagents to generate fusion-competent glutamatergic synaptic vesicles through immunoisolation. The generation of the vGlut peptide can be accomplished in 6 d, while the isolation of the synaptic vesicles by using the peptide can be accomplished in 2 d, with an additional day to fluorescently label the synaptic vesicles for use in a single-vesicle hybrid fusion assay. The single-vesicle fusion assay can be accomplished in 1 d and can unambiguously delineate synaptic vesicle association, dissociation, Ca2+-independent and Ca2+-dependent fusion modalities. This assay grants control of the synaptic vesicle environment while retaining the complexity of the synaptic vesicles themselves. This protocol can be adapted to studies of other types of synaptic vesicles or, more generally, different secretory or transport vesicles. The workflow described here requires expertise in biochemistry techniques, in particular, protein purification and fluorescence imaging. We assume that the laboratory has protein-purification equipment, including chromatography systems.

Beyond the MUN domain, Munc13 controls priming and depriming of synaptic vesicles.

Synaptic vesicle docking and priming are dynamic processes. At the molecular level, SNAREs (soluble NSF attachment protein receptors), synaptotagmins, and other factors are critical for Ca2+-triggered vesicle exocytosis, while disassembly factors, including NSF (N-ethylmaleimide-sensitive factor) and α-SNAP (soluble NSF attachment protein), disassemble and recycle SNAREs and antagonize fusion under some conditions. Here, we introduce a hybrid fusion assay that uses synaptic vesicles isolated from mouse brains and synthetic plasma membrane mimics. We included Munc18, Munc13, complexin, NSF, α-SNAP, and an ATP-regeneration system and maintained them continuously-as in the neuron-to investigate how these opposing processes yield fusogenic synaptic vesicles. In this setting, synaptic vesicle association is reversible, and the ATP-regeneration system produces the most synchronous Ca2+-triggered fusion, suggesting that disassembly factors perform quality control at the early stages of synaptic vesicle association to establish a highly fusogenic state. We uncovered a functional role for Munc13 ancillary to the MUN domain that alleviates an α-SNAP-dependent inhibition of Ca2+-triggered fusion.