Development of a high resolution assay to characterize exocytotic vesicle fusion.

点击翻译按钮获取中文摘要

Abstract Transmitter release is mediated by fusion of neurosecretory vesicles with the plasma membrane. While it is known that Soluble NSF Attachment REceptor (SNARE) proteins form the core complex of the molecular fusion machine, the precise molecular rearrangements leading to fusion pore formation are still unknown. We will develop a highly innovative technology that will enable experiments to achieve a precise mechanistic understanding of structural molecular rearrangements associated with the fusion of neurosecretory vesicles at the plasma membrane. The approach combines electrochemical detector (ECD) arrays, with reconstituted supported membranes to study fusion of isolated chromaffin granules simultaneously by amperometry and total internal reflection fluorescence (TIRF) imaging. We have previously performed combined ECD and TIRF experiments using intact chromaffin cells and discovered a rapid conformational change in SNAP25 associated with fusion events. However, these measurements were performed using a FRET construct incorporating CFP/Venus and the actual nature of the structural change remains unknown. Proceeding to the reconstituted system will make it possible to incorporate small labels at arbitrary sites in the SNARE proteins or other accessory proteins, a technology that will make it possible to identify precisely which amino acids in the SNARE complex and accessory proteins move and change distance at specific times during the fusion process. The amperometric recordings can be performed with a time resolution of a millisecond or less and by averaging fluorescence changes from multiple fusion events, the time of such fluorescence changes relative to the fusion event can be determined with very high precision, not limited by the exposure time used in the fluorescence image acquisition. This has become possible with the time super-resolution approach named Event Correlation Microscopy (ECOM), developed in the Lindau Lab. The technology we propose to develop is high risk because we need to establish a protocol to form the supported bilayers on top of the ECD arrays and explore how a sufficiently high rate of fusion events at a given ECD array can be achieved to perform the required averaging of large numbers of fusion events (as we did in the cells). It is known, that when supported bilayers incorporating SNARE proteins are formed on a quartz or silicon oxide surface, fusion of dense core vesicles does occur. The project will be performed in collaboration between Dr. Lindau who has developed the ECD and ECOM methods and Dr. Kiessling who has pioneered the study of SNARE protein conformations in the supported bilayer system. If successful, this technology will enable the experimental identification of the detailed molecular steps in vesicle fusion and to test the predictions from structural work as well as molecular dynamics simulations.