SNARE-Mediated Transient Fusion of Liposomes to Supported Bilayers Probed by Two-Color pTIRFM

Abstract

In membrane fusion opening of a fusion pore establishes the first connection between two compartments. During neurotransmitter or hormone release via exocytosis, fusion pores can transiently open and close repeatedly (flicker) before resealing (‘kiss-and-run’) or dilating irreversibly (full fusion). Pore dynamics regulate the amount and size of released cargo and determine the mode of vesicle recycling, yet what governs pore dynamics is poorly understood. We established an assay to monitor membrane fusion by polarized total internal reflection (pTIRF) microscopy with single molecule sensitivity and ∼15 ms time resolution in a biochemically well-defined system. In our assay, fusion of small unilamellar vesicles containing v-SNARE proteins (v-SUVs) fuse to a flat bilayer reconstituted with cognate t-SNAREs, supported on a soft polymer cushion (t-SBL). Exploiting the signal enhancement when a label transfers from the SUV to the SBL membrane in pTIRF, we could monitor the release kinetics of vesicular lipid labels into the SBL upon fusion. We found release times were much longer than would be expected for permanently open pores. Our sensitivity to single fluorescent lipids allowed us to calculate the lipid diffusivity and SUV size for each fusion event. This was used in a model we developed assuming the retardation was due to flickering pores, to estimate pore openness P0, the fraction of time a pore remains open during fusion. Increasing cholesterol levels increased P0. However, retardation of lipid release could also be due to steric hindrance of lipid diffusion through the walls of the fusion pore by SNARE proteins. To exclude this, we encapsulated soluble markers into the SUVs and simultaneously monitored lipid and contents release. Release of both lipid and soluble cargo started simultaneously. Interestingly, many pores resealed after partially releasing their contents.