Fusion pore formation and expansion induced by Ca ²⁺ and synaptotagmin 1

Abstract

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...

Variable cooperativity in SNARE-mediated membrane fusion

“…For instance, when using full-length SNAREs, only a very low percentage of large SNARE liposomes undergo fusion and instead remain either stably docked or hemifused even at very high l/p ratios of 50 and 200:1 (37,38), a finding we have also observed in our bulk assays. It is not clear why this is the case, but the observation that synaptotagmin 1 and Ca 2+ substantially enhances fusion by presumably lowering the activation energy of certain intermediates might suggest that the energy barriers in these systems are higher than in ours.…”

Synaptotagmin-1 Is an Antagonist for Munc18-1 in SNARE Zippering

“…Protein Expression and Purification-Protein expression and purification were described previously (41,43). Briefly, all recombinant proteins were expressed in Escherichia coli BL21 (DE3).…”

“…For v-vesicles, the mixture was diluted twice with dialysis buffer containing 1 mM EDTA and dialyzed in 2 liters of dialysis buffer with EDTA at 4°C overnight. Details for reconstitution were described in our previous work (39,43).…”

“…Remaining free SRB was removed using the PD-10 desalting column (GE Healthcare) after dialysis. Details for reconstitution were described in our previous work (39,43).…”

Local electrostatic interactions determine the diameter of fusion pores

“…10 Nevertheless, several other SNARE-binding proteins, be they individual or in complexes, influence the formation and the expansion of fusion pores; including synaptotagmins, 11 complexins, 12 and Sec1/Munc18 (SM) proteins. 13 Lipids also affect the fusibility of lipid bilayers 14 and can regulate the fusion process by manipulating the SNARE complex.…”