Nucleation and dynamics of exocytotic fusion pores

胞吐融合孔的成核和动力学

• 批准号: 8615066
• 负责人: ERDEM KARATEKIN
• 金额: $ 31.64万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-02-15 至 2019-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8615066
• 关键词: Action Potentials; Aspirate substance; Behavior; Biochemistry; Biological Assay; Calcium; Cell membrane; Cells; Chimeric Proteins; Complex; Coupled; Cytoskeleton; Detection; Devices; Docking; Endocrine; Endocytosis; Engineering; Evolution; Exocytosis; Fluorescence; Fluorescence Microscopy; Goals; Health; Hormones; Human; In Vitro; Lipid Bilayers; Lipids; Liposomes; Measurement; Measures; Mechanics; Mediating; Membrane; Membrane Fusion; Membrane Proteins; Methods; Microelectrodes; Microfabrication; Molecular; Monitor; Mutation; Neurons; Neurotransmitters; Physiological; Process; Property; Proteins; Resolution; Role; Running; SNAP receptor; Secretory Vesicles; Speed; Structure; Surface; Synaptic Cleft; Synaptic Vesicles; System; Technology; Time; Transmembrane Domain; Vesicle; Work; cell type; electrical measurement; in vitro Assay; in vivo; innovation; millisecond; new technology; novel; particle; protein protein interaction; proteoliposomes; public health relevance; reconstitution; single molecule; target SNARE proteins; vesicular SNARE proteins

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.

描述(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小泡(SV)与质膜融合，释放出通过突触间隙感受到的内容。这一过程受到严格的调控：在动作电位到达后，局部游离钙浓度的增加会刺激释放。通过含有分泌颗粒(SG)的激素与质膜融合，使用一些相同的蛋白质机制，激素以类似的方式释放。SV或SG与质膜之间的初始连接是一个小孔(~1 nm宽)，在永久关闭(瞬时或亲吻并运行融合)或完全扩张之前可以连续打开和关闭。不同细胞类型(张孔时间约100s？S的S到10s)和同一细胞内(有些毛孔闪烁，有些突然扩张)的行为有很大的差异。毛孔闪烁受到刺激强度等生理输入的调节，对释放什么(只有微小的货物才能通过小孔逃逸)、时间进程以及胞吐作用与内吞作用如何耦合产生重要影响。尽管融合孔在调节神经递质和激素释放方面具有重要意义，但对控制孔成核和动力学的机制知之甚少。这主要是因为在含有明确定义的蛋白质和膜成分的重组系统中很难研究融合孔，这些蛋白质和膜成分可以分离出每个成分的作用。在过去的15年里，由胞吐SNARE蛋白及其调控因子介导的融合已经被重组和研究。然而，现有的方法不能以足够的时间分辨率来分辨单个重组融合孔并遵循孔动力学。我们的目标是(1)设计新的实验方法，以实现胞吐融合毛孔成核和闪烁的探测机制。结合电生理方法、单粒子荧光、微细加工设备和人工 双层技术我们将开发体外检测，允许直接、同时监测单孔闪烁和脂质混合；计数蛋白质数量和/或探测蛋白质-蛋白质相互作用；以及控制膜的曲率和张力。使用这些分析，我们将(2)确定控制SNARE介导的融合孔的成核和动力学的因素。我们将解决膜机制和融合蛋白的动力学如何共同决定融合所需的SNARE复合体的数量。此外，我们将量化膜张力、模拟细胞骨架的约束、曲率和突变对孔闪烁和扩张的作用。这些基础研究将促进我们对神经递质和激素释放如何调节的理解，并对人类健康产生潜在的长期影响。