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

• 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
• 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' 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

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的融合介导的。虽然 已知可溶性NSF附着受体（SNARE）蛋白形成分子融合的核心复合物 然而，导致融合孔形成的精确分子重排仍然是未知的。我们将 开发一种高度创新的技术，使实验能够实现精确的机械 了解与神经分泌囊泡融合相关的结构分子重排. 质膜。该方法结合了电化学检测器（ECD）阵列， 支持膜研究融合的孤立嗜铬颗粒同时通过安培法和总 内反射荧光（TIRF）成像。我们以前进行过联合ECD和TIRF 实验使用完整的嗜铬细胞，并发现SNAP 25相关的快速构象变化， 融合事件。然而，这些测量是使用FRET构建体进行的，所述FRET构建体掺入了 CFP/金星和结构变化的实际性质仍然未知。前往重组的 该系统将使得有可能在SNARE蛋白或其他蛋白中的任意位点掺入小标记。 辅助蛋白，一项技术，将有可能准确地确定哪些氨基酸在陷阱 复合蛋白和辅助蛋白在融合过程中的特定时间移动和改变距离。的 可以以毫秒或更小的时间分辨率进行安培记录 当荧光从多个融合事件改变时，这种荧光的时间相对于融合事件改变， 可以非常高的精度确定事件，而不受荧光测量中使用的曝光时间的限制 图像采集这已经成为可能的时间超分辨率方法命名为事件相关 显微镜（ECOM），在林道实验室开发。我们建议开发的技术是高风险的，因为 我们需要建立一个在ECD阵列上形成支撑双层的方案，并探索如何在ECD阵列上形成支撑双层。 在给定的ECD阵列处，可以实现足够高的聚变事件速率，以执行所需的平均 大量的融合事件（就像我们在细胞中所做的那样）。已知当支撑双层时， SNARE蛋白在石英或氧化硅表面形成，致密核心囊泡的融合确实发生。的 该项目将与康奈尔大学的林道博士合作进行，他开发了ECD和ECOM 方法和Kiessling博士谁率先研究的陷阱蛋白构象的支持双层 系统如果成功的话，这项技术将使详细的分子步骤的实验鉴定成为可能。 在囊泡融合和测试的预测，从结构工作以及分子动力学模拟。