Visualizing Release of Single Fluorophores at Membrane Fusion Sites

Abstract

SNARE proteins play a central role in nearly all intracellular fusion reactions; fusion is driven by formation of trans-SNARE complexes (SNAREpins) through pairing of vesicle-associated v-SNAREs with complementary t-SNAREs on target membranes. We recently reported a docking and fusion assay in which single small unilamellar vesicles containing the synaptic/exocytic v-SNAREs VAMP/synaptobrevin (v-SUVs) fuse rapidly with planar, supported bilayers containing the synaptic/exocytic t-SNARE syntaxin-SNAP25 (t-SBLs), with single fusion events occurring in ∼130 ms after docking. We optimized acquisition conditions such that now the release of single fluorescently labeled lipids can be visualized. As the fluorophores diffuse away from the release site, individual spots become discernible and can be tracked with ∼16 ms resolution. More than 90% of the tracked spots bleach in a single step, strongly suggesting they represent single fluorescent lipid molecules. Intensity-drops as spots disappear follow a normal distribution whose mean defines the intensity of a single fluorophore. This allows us to estimate the total number of lipids in a vesicle prior to its fusion with the t-SBL, given the label density. An independent estimate of vesicle size is based on extrapolating the number of surviving spots as a function of time to just before fusion.We studied the effects of lipid composition. In bilayers containing nearly physiological amounts of cholesterol, the mean squared displacement of single fluorescent lipids that are released into the t-SBL increases linearly in time indicating a diffusive process and yields a diffusion coefficient of ∼0.3 µm2/s, a value that is similar to those found for lipid diffusivities in the plasma membrane of live cells. In addition, the majority of the vesicles that dock end up fusing. In contrast, in bilayers devoid of cholesterol, lipid diffusion is much faster, but only 40-50% of docked vesicles fuse.