SNARE-Mediated Single-Vesicle Fusion Events with Supported and Freestanding Lipid Membranes

Abstract

In vitro single-vesicle fusion assays are important tools to analyze the details of SNARE-mediated fusion processes. In this study, we employed planar pore-spanning membranes (PSMs) prepared on porous silicon substrates with large pore diameters of 5 μm, allowing us to compare the process of vesicle docking and fusion on the supported parts of the PSMs (s-PSMs) with that on the freestanding membrane parts (f-PSM) under the exact same experimental conditions. The PSMs harbor the t-SNARE ΔN49-complex to investigate the dynamics and fusogenicity of single large unilamellar vesicles doped with the v-SNARE synaptobrevin 2 by means of spinning-disc confocal microscopy with a time resolution of 10 ms. Our results demonstrate that vesicles docked to the s-PSM were fully immobile, whereas those docked to the f-PSM were mobile with a mean diffusion coefficient of 0.42 μm2/s. Despite the different dynamics of the vesicles on the two membrane types, similar fusion kinetics were observed, giving rise to a common fusion mechanism. Further investigations of individual lipid mixing events on the s-PSMs revealed semi-stable post-fusion structures.