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Development of a high resolution assay to characterize exocytotic vesicle fusion

开发高分辨率测定法来表征胞吐囊泡融合

基本信息

批准号：
10041876
负责人：
Manfred LINDAU
金额：
$ 9.55万
依托单位：
CORNELL UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-06-01 至 2021-10-01
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10041876
关键词：
Alzheimer&apos s Disease Amino Acids Biological Assay C-terminal Cattle Cell membrane Cells Chromaffin Cells Chromaffin granule Collaborations Complex Dementia Dense Core Vesicle Deposition Development Docking Drug Targeting Drug usage Ensure Environment Event Fluorescence Fluorescence Recovery After Photobleaching Fluorescence Resonance Energy Transfer Hormones Image Individual Label Length Lipids Measurement Measures Mediating Membrane Membrane Fluidity Methods Microscopy Molecular Molecular Conformation Molecular Machines Molecular Structure Names Nature Neurodegenerative Disorders Neurons Oxides Parkinson Disease Pattern Pharmaceutical Preparations Process Protein Conformation Proteins Protocols documentation Quartz Recombinants Regulation Resolution SNAP receptor Secretory Vesicles Silicon Site Structure Surface System Technology Testing Time Transmembrane Domain Universities VAMP-2 Validation Venus Vesicle Virginia Work detector experimental study fluorescence imaging fluorophore high risk innovative technologies millisecond molecular dynamics molecular rearrangement monolayer neurotransmitter release novel protein function receptor reconstitution single molecule syntaxin 1 technology development time use

项目摘要

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 at Cornell 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.

递质释放是由神经分泌囊泡与质膜融合介导的。当它是