Planar Pore-Spanning Membranes: A Platform to Study Snare-Mediated Fusion Processes

Abstract

In most physiological fusion processes, a strongly to moderately curved membrane (e.g. synaptic vesicle, virion) fuses with a planar membrane (e.g. (neuronal) plasma membrane). Despite these geometric considerations of the fusing membranes in nature, most in vitro results on fusion have been obtained from bulk vesicle assays, where two curved membranes fuse with each other resulting in an increase in vesicle size, i.e. the membrane's curvature changes during the fusion process.To establish a fusion assay that resembles the in vivo situation more closely, we aimed at a planar membrane architecture that is long-term stable and provides laterally mobile membrane components. Moreover, this membrane system should allow for the detection of single vesicle fusion events, should be accessible from both sides and should provide enough space for the incoming lipid material during the fusion process. One membrane system that suffice these requirements are pore-spanning membranes.We demonstrate that planar pore-spanning membranes harboring neuronal SNARE-proteins can be successfully generated on highly ordered and functionalized micrometer-sized pore arrays in silicon nitride by fusion of giant unilamellar vesicles. Full mobility of the membrane components was demonstrated by fluorescence correlation spectroscopy. Individual fusion events of unilamellar vesicles were analyzed by confocal laser scanning fluorescence microscopy in a time-resolved manner allowing to readily distinguish between vesicle docking, intermediate states such as hemifusion and full fusion. With this assay in hand, the impact of individual components, such as the PIP2 concentration in the planar membrane, on the fusion process can be analyzed in depth.