The opening of a fusion pore during exocytosis creates the first aqueous connection between the lumen of a vesicle and the extracellular space. Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) mediate the formation of these dynamic structures, and their kinetic transitions are tightly regulated by accessory proteins at the synapse. Here, we utilize two single molecule approaches, nanodisc-based planar bilayer electrophysiology and single-molecule FRET, to address the relationship between SNARE complex assembly and rapid (micro-millisecond) fusion pore transitions, and to define the role of accessory proteins. Synaptotagmin (syt) 1, a major Ca 2+ -sensor for synaptic vesicle exocytosis, drove the formation of an intermediate: committed trans-SNARE complexes that form large, stable pores. Once open, these pores could only be closed by the action of the ATPase, NSF. Timeresolved measurements revealed that NSF-mediated pore closure occurred via a complex 'stuttering' mechanism. This simplified system thus reveals the dynamic formation and dissolution of fusion pores.

Current methods to isolate synaptic vesicles (SVs), the organellar quanta of synaptic transmission, require highly specialized materials and up to 24 hours. These technical obstacles have thus far limited the study of SVs in models of synaptic function and pathophysiology. Here, we describe techniques for the rapid isolation of SVs by immunoprecipitation with widely available antibodies conjugated to magnetic beads. We report that the inexpensive rho1D4 monoclonal antibody binds SVs and show that elution with the 1D4 peptide yields native vesicles that are ≥ 10-fold purer than those obtained with classical techniques. These methods substantially widen the accessibility of SVs, enabling their purification in 60-90 minutes for downstream analyses including mass spectrometry and cryoelectron microscopy. Immunopurified SV preparations from mouse brain contained apolipoprotein E (ApoE), the low-density lipoprotein (LDL) receptor Lrp1, and enzymes involved in lipid metabolism, suggesting that SVs may play direct roles in lipid homeostasis and lipoprotein trafficking at the nerve terminal.Significance Statement Synaptic vesicles (SVs) are small organelles that form and recycle at nerve terminals to enable synaptic transmission. Much remains unknown about the processes that enable the formation and function of SVs. Moreover, nerve terminals appear to be particularly vulnerable to pathophysiologic processes underlying neurodegenerative diseases and schizophrenia. While techniques to purify synaptic vesicles thus have the potential to yield significant insights into physiology and pathophysiology of nerve terminals, current methods rely on either esoteric materials or expression of transgenes. This paper addresses these problems by establishing robust, efficient methods for SV purification using widely available materials, and it highlights several promising areas of future study arising from proteomic analyses of immunopurified SVs.

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