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 递质释放是通过神经分泌囊泡与质膜融合的介导的。虽然是 知道可溶性NSF附着受体（SNARE）蛋白形成分子融合的核心复合物 机器，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，该技术将使实验能够实现精确的机械性 了解与神经分泌囊泡融合有关的结构分子重排 质膜。该方法结合了电化学探测器（ECD）阵列，并重组 支撑的膜以通过安培法同时研究孤立的铬蛋白颗粒的融合 内部反射荧光（TIRF）成像。我们以前已经进行了ECD和TIRF的组合 实验使用完整的铬蛋白细胞，发现SNAP25相关的快速构象变化 与融合事件。但是，这些测量是使用合并的FRET结构进行的 CFP/Venus和结构变化的实际性质仍然未知。前往重构的 系统将有可能在军鼓蛋白或其他的任意位置合并小标签 辅助蛋白，这项技术将有可能精确识别网罗中哪些氨基酸 在融合过程中，复杂和附件蛋白在特定时间移动和更改距离。这 可以通过毫秒或更少的时间分辨率进行安培记录，并通过平均 荧光因多个融合事件而变化，这种荧光时间相对于融合而变化 事件可以用很高的精度确定，不受荧光中使用的暴露时间的限制 图像获取。随着时间的超分辨率方法名为事件相关，这已成为可能 显微镜（ECOM），在Lindau Lab中开发。我们建议开发的技术是高风险，因为 我们需要建立一个协议，以在ECD阵列之上形成受支持的双层，并探索如何 可以实现给定ECD阵列的融合事件的足够高率，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，当支持双层时 SNARE蛋白是在石英或氧化硅表面形成的，确实发生了致密核囊泡的融合。这 项目将与开发ECD和ECOM方法的Lindau博士合作进行 Kiessling博士率先研究了受支持的双层系统中的军鼓构象。 如果成功，该技术将实现囊泡中详细分子步骤的实验鉴定 融合并测试结构工作以及分子动力学模拟的预测。