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: 10528722
• 负责人: Manfred LINDAU
• 金额: $ 36.52万
• 依托单位: UNIVERSITY OF MIAMI SCHOOL OF MEDICINE
• 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 2024-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10528722
• 关键词: Development; resolution; assay; characterize; exocytotic

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.

抽象的 递质释放是由神经分泌囊泡与质膜的融合介导的。虽然它是 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 机器中，导致融合孔形成的精确分子重排仍然未知。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原理 了解与神经分泌囊泡融合相关的结构分子重排 质膜。该方法将电化学检测器 (ECD) 阵列与重构的 支持膜通过电流分析法和总和同时研究分离的嗜铬颗粒的融合 内反射荧光 (TIRF) 成像。我们之前曾联合进行过 ECD 和 TIRF 使用完整嗜铬细胞进行实验，发现 SNAP25 相关的快速构象变化 与融合事件。然而，这些测量是使用 FRET 结构进行的，并结合了 CFP/金星和结构变化的实际性质仍然未知。继续重组 系统将使得在 SNARE 蛋白或其他蛋白的任意位点掺入小标签成为可能 辅助蛋白，这项技术将能够精确识别 SNARE 中的氨基酸 复杂蛋白和辅助蛋白在融合过程中的特定时间移动并改变距离。这 电流记录可以以毫秒或更短的时间分辨率进行，并通过平均 多次融合事件导致的荧光变化，这种荧光变化的时间相对于融合 可以以非常高的精度确定事件，不受荧光中使用的曝光时间的限制 图像采集。通过名为“事件相关性”的时间超分辨率方法，这已成为可能 显微镜 (ECOM)，由 Lindau 实验室开发。我们建议开发的技术具有高风险，因为 我们需要建立一个协议来在 ECD 阵列之上形成受支持的双层，并探索如何 在给定的 ECD 阵列上可以实现足够高的融合事件率，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）。众所周知，当支持的双层结合 SNARE 蛋白在石英或氧化硅表面形成，致密核心囊泡确实发生融合。这 该项目将由开发了 ECD 和 ECOM 方法的 Lindau 博士合作执行 Kiessling 博士是支持双层系统中 SNARE 蛋白构象研究的先驱。 如果成功，该技术将能够通过实验鉴定囊泡中的详细分子步骤 融合并测试结构工作和分子动力学模拟的预测。