FLIC Microscopy Reveals Different Conformational States of Syntaxin 1a in Supported Lipid Bilayers

Abstract

The fusion of synaptic vesicles with the presynaptic membrane is a fast and highly regulated process that is catalyzed by the exocytotic SNAREs synaptobrevin-2 (VAMP-2), SNAP-25, and syntaxin-1a (Syx1a). In addition to the SNAREs a number of protein players that regulate the fusion process have been identified and characterized. Despite the tremendous progress in this field we are still missing a molecular timeline that leads from docking of synaptic vesicles to the plasma membrane, to priming of the fusion machinery and eventually to fusion of the two membranes once an action potential reaches the synaptic terminal. One of the difficulties in approaching these questions arises from the fact that the lipid environment plays an active role in regulating this protein driven process.Reconstituting SNAREs into supported lipid bilayers allows us to characterize the state of these proteins in different lipid environments. Using fluorescence interference contrast (FLIC) microscopy we measure distances of specific residues in Syx1a from the membrane surface. Here, we report how regulatory proteins like Munc18 and Munc13 modulate the state of Syx1a in the presence and absence of SNAP-25.