Development of a high resolution assay to characterize exocytotic vesicle fusion

递质的释放是通过神经分泌囊泡与质膜的融合来调节的。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体 在机器上，导致熔合孔形成的精确分子重排仍然未知。我们会 开发一种高度创新的技术，使实验能够实现精确的机械 与神经分泌囊泡融合相关的结构分子重排的理解 质膜。该方法将电化学检测器(ECD)阵列与重组 安培法和全电流法同时研究分离的嗜铬粒细胞的融合 内反射荧光(TIRF)成像。我们以前进行过ECD和TIRF的联合手术 使用完整的嗜铬细胞进行实验，发现SNAP25相关的构象快速变化 核聚变事件。然而，这些测量是使用结合了FRET结构的FRET进行的 CFP/金星和结构变化的实际性质仍不得而知。继续向重组的 该系统将使在SNARE蛋白质或其他蛋白质中的任意位置结合小标签成为可能 辅助蛋白质，这项技术将使准确识别圈套中的哪些氨基酸成为可能 在融合过程中，复杂蛋白和辅助蛋白在特定的时间移动和改变距离。这个 安培记录可以在毫秒或更短的时间分辨率下执行，并通过平均 来自多个融合事件的荧光变化，这种荧光相对于融合的时间发生变化 可以以非常高的精度确定事件，而不受荧光中使用的曝光时间的限制 图像采集。这通过名为事件关联的时间超分辨率方法成为可能 显微镜(ECOM)，由林道实验室开发。我们建议开发的技术风险很高，因为 我们需要建立一个协议来在ECD阵列上形成受支持的双层，并探索如何 可以在给定的ECD阵列上实现足够高的融合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)。已知的是，当结合有 SNARE蛋白在石英或硅氧化物表面形成，致密的核心小泡发生融合。这个 该项目将在康奈尔大学的Lindau博士之间进行，他开发了ECD和ECOM 方法和Kiessling博士，他是研究支撑双层中SNARE蛋白构象的先驱 系统。如果成功，这项技术将使详细的分子步骤的实验鉴定成为可能 在囊泡融合中，并测试来自结构工作以及分子动力学模拟的预测。

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.