Nucleation and dynamics of exocytotic fusion pores

描述（由申请人提供）：胞吐是神经递质和激素释放的基础。在神经元中，包裹着神经递质的突触囊泡（SV）与质膜融合，释放其内容物，并通过突触间隙被感知。这一过程受到严格调控：动作电位到达后，局部游离钙浓度的增加刺激释放。激素以类似的方式释放，使用一些相同的蛋白质机制，通过含有分泌颗粒（SG）的激素与质膜融合。SV或SG与质膜之间的初始连接是一个小孔（约1nm宽），可以连续打开和关闭，然后永久关闭（短暂的，或接吻和逃跑融合）或完全扩张。不同细胞类型之间的行为差异很大(孔隙打开时间跨度~100 ？S到10s的S)和在同一细胞内（一些毛孔闪烁，一些突然扩张）。毛孔闪烁受到刺激强度等生理输入的调节，对释放的物质（只有少量货物可以通过小毛孔逃逸）、时间过程以及胞吐作用如何与内吞作用耦合产生重要影响。尽管融合孔在调节神经递质和激素释放方面具有根本的重要性，但对孔成核和动力学的控制机制知之甚少。这主要是由于很难在具有明确定义的蛋白质和膜组分的重构系统中研究融合孔，从而分离出每种组分的作用。胞外SNARE蛋白及其调控因子介导的融合在过去的15年里得到了重构和研究。然而，现有的方法无法解析单个重构的融合孔隙，也无法以足够的时间分辨率跟踪孔隙动力学。我们的目标是(1)设计新的实验方法来探测胞外融合孔的成核和闪烁机制。结合电生理方法，单粒子荧光，微制造装置，和人工

DESCRIPTION (provided by applicant): Exocytosis underlies neurotransmitter and hormone release. In neurons, synaptic vesicles (SV) packaged with neurotransmitter fuse with the plasma membrane to release their content that is sensed across the synaptic cleft. This process is tightly regulated: release is stimulated by a local increase in the free calcium concentration following the arrival of an action potential. Hormones are released in a similar fashion using some of the same protein machinery, via fusion of hormone containing secretory granules (SG) with the plasma membrane. The initial connection between a SV or SG and the plasma membrane is a small pore (~1 nm wide) that can open and close in succession before either closing permanently (transient, or kiss-and-run fusion) or dilating fully. There is large variabiliy in behavior between cell types (pore open times span ~100 ?s to 10s of s) and within the same cell (some pores flicker, some dilate abruptly). Pore flickering is modulated by physiological inputs such as stimulation strength, with important consequences about what is released (only small cargo can escape through a small pore), on what time course, and how exocytosis is coupled to endocytosis. Despite the fundamental importance of fusion pores in regulating neurotransmitter and hormone release, very little is understood regarding mechanisms controlling pore nucleation and dynamics. This is mainly due to difficulties in studying fusion pores in reconstituted systems with well-defined protein and membrane components that would allow isolating the role of each component. Fusion mediated by exocytotic SNARE proteins and their regulators has been reconstituted and studied for the past 15 years. However, existing methods are not able to resolve single reconstituted fusion pores and follow pore dynamics with sufficient time resolution. We aim (1) to engineer novel experimental approaches to enable probing mechanisms of nucleation and flickering of exocytotic fusion pores. Combining electrophysiological methods, single-particle fluorescence, microfabricated devices, and artificial bilayer technologies we will develop in vitro assays that allow direct, simultaneous monitoring of single pore flickering and lipid mixing; counting protein numbers and/or probing protein-protein interactions; and controlling membrane curvature and tension. Using these assays, we will then (2) determine factors that govern nucleation and dynamics of SNARE- mediated fusion pores. We will resolve how membrane mechanics and the dynamics of fusion proteins together determine the number of SNARE complexes required for fusion. Further, we will quantify the roles of membrane tension, constraints mimicking the cytoskeleton, curvature, and mutations on pore flickering and expansion. These fundamental studies will advance our understanding of how neurotransmitter and hormone release are regulated, with potential impact on human health in the long term.