Membrane fusion in eukaryotic cells is thought to be mediated by a highly conserved family of proteins called SNAREs (soluble N-ethyl maleimide sensitive-factor attachment protein receptors). The vesicle-associated v-SNARE engages with its partner t-SNAREs on the target membrane to form a coiled coil that bridges two membranes and facilitates fusion. As demonstrated by recent findings on the hemifusion state, identifying intermediates of membrane fusion can help unveil the underlying fusion mechanism. Observation of SNARE-driven fusion at the single-liposome level has the potential to dissect and characterize fusion intermediates most directly. Here, we report on the real-time observation of lipid-mixing dynamics in a single fusion event between a pair of SNARE-reconstituted liposomes. The assay reveals multiple intermediate states characterized by discrete values of FRET between membrane-bound fluorophores. Hemifusion, flickering of fusion pores, and kinetic transitions between intermediates, which would be very difficult to detect in ensemble assays, are now identified. The ability to monitor the time course of fusion events between two proteoliposomes should be useful for addressing many important issues in SNARE-mediated membrane fusion.FRET ͉ single-molecule spectroscopy ͉ lipid mixing

Ca2+-triggered, synchronized synaptic vesicle fusion underlies interneuronal communication. Complexin is a major binding partner of the SNARE complex, the core fusion machinery at the presynapse. The physiological data on complexin, however, have been at odds with each other, making delineation of its molecular function difficult. Here we report direct observation of two-faceted functions of complexin using the single-vesicle fluorescence fusion assay and EPR. We show that complexin I has two opposing effects on trans-SNARE assembly: inhibition of SNARE complex formation and stabilization of assembled SNARE complexes. Of note, SNARE-mediated fusion is markedly stimulated by complexin, and it is further accelerated by two orders of magnitude in response to an externally applied Ca2+ wave. We suggest that SNARE complexes, complexins and phospholipids collectively form a complex substrate for Ca2+ and Ca2+-sensing fusion effectors in neurotransmitter release.

Fusion pore formation and expansion, crucial steps for neurotransmitter release and vesicle recycling in soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)-dependent vesicle fusion, have not been well studied in vitro due to the lack of a reliable content-mixing fusion assay. Using methods detecting the intervesicular mixing of small and large cargoes at a single-vesicle level, we found that the neuronal SNARE complexes have the capacity to drive membrane hemifusion. However, efficient fusion pore formation and expansion require synaptotagmin 1 and Ca 2+ . Real-time measurements show that pore expansion detected by content mixing of large DNA cargoes occurs much slower than initial pore formation that transmits small cargoes. Slow pore expansion perhaps provides a time window for vesicles to escape the full collapse fusion pathway via alternative mechanisms such as kissand-run. The results also show that complexin 1 stimulates pore expansion significantly, which could put bias between two pathways of vesicle recycling. S oluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) mediate intracellular vesicle fusion in a wide variety of cellular activities such as neurotransmitter release. The fast synaptic vesicle fusion for neurotransmitter release is regulated with precision by various proteins including synaptotagmins, complexins, and SM proteins (1, 2). During this process, an initial fusion pore between two membranes can either close back or expand to a larger pore. Fusion pore expansion to the point where the vesicle membrane flattens on the plasma membrane surface, leading to the complete luminal contents release, is thought to be the final step in the fusion process (3, 4). SNAREs and accessary proteins may then be recycled to make fresh vesicles through endocytosis. Without pore expansion, however, the vesicles may be used again through the mechanism known as "kiss-and-run" (5). Therefore, pore expansion is an important event that determines how synaptic vesicles are regenerated.To dissect the SNARE-mediated membrane fusion process, we and others developed in vitro single-vesicle assays based on lipid mixing of proteoliposomes reconstituted with SNARE proteins and content mixing of small cargoes (6-10). However, these assays are blind to the expansion of the fusion pore and therefore unable to tell how the regulatory proteins are involved in this final step of the full-collapse fusion pathway, in which the small opening of the pore continues to expand to a large pore.To monitor fusion pore expansion, we developed a singlemolecule/vesicle content-mixing assay based on vesicle-encapsulated DNA molecules (11,12). This assay can detect expansion of the fusion pore that is large enough to pass ∼11-kDa DNA probes between two apposed proteoliposomes. With this method, we showed that yeast SNAREs alone can efficiently drive expansion of the fusion pore (12). In this work, we systematically dissect lipid mixing, fusion pore opening, and fusion pore expansion steps i...

SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins are a highly regulated class of membrane proteins that drive the efficient merger of two distinct lipid bilayers into one interconnected structure. This protocol describes our fluorescence resonance energy transfer (FRET)-based single vesicle-vesicle fusion assays for SNAREs and accessory proteins. Both lipid-mixing (with FRET pairs acting as lipophilic dyes in the membranes) and content-mixing assays (with FRET pairs present on a DNA hairpin that becomes linear via hybridization to a complementary DNA) are described. These assays can be used to detect substages such as docking, hemifusion, and pore expansion and full fusion. The details of flow cell preparation, protein-reconstituted vesicle preparation, data acquisition and analysis are described. These assays can be used to study the roles of various SNARE proteins, accessory proteins and effects of different lipid compositions on specific fusion steps. The total time required to finish one round of this protocol is 3–6 d.