Nucleation and dynamics of exocytotic fusion pores

胞吐融合孔的成核和动力学

• 批准号: 10376228
• 负责人: ERDEM KARATEKIN
• 金额: $ 36.64万
• 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 2024-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10376228
• 关键词: Address; Affect; Affinity; Artificial Membranes; Binding; Biochemical; Biological Assay; Calcium; Cell fusion; Cell membrane; Cells; Charge; Communication; Complex; Coupled; Dependence; Docking; Electrophysiology (science); Endocrine; Endocytosis; Engineering; Event; Evolution; Exocytosis; Fluorescence Microscopy; Health; Hormones; Human; Individual; Ions; Kinetics; Lipids; Mediating; Membrane; Membrane Fusion; Membrane Proteins; Methods; Molecular; Monitor; Neuroendocrine Cell; Neurons; Neurotransmitters; Phase; Physiological; Probability; Process; Property; Protein Isoforms; Proteins; Recycling; Regulation; Resolution; Role; SNAP receptor; Secretory Vesicles; Shapes; Speed; Synaptic Cleft; Synaptic Vesicles; System; Technology; Testing; Time; Vesicle; cell type; experience; experimental study; flash photolysis; microdevice; nanodisk; neurotransmitter release; particle; reconstitution; response; sensor; synaptotagmin I; target SNARE proteins; ultraviolet; vesicular SNARE proteins

PROJECT SUMMARY: In neurons, synaptic vesicles (SV) packaged with neurotransmitter fuse with the plasma membrane to release their content that is sensed across the synaptic cleft. Release is triggered by a local increase in the calcium concentration following depolarization. Release kinetics comprise a synchronous phase (0.1-5 ms after calcium elevation), and a much slower asynchronous phase (~100 ms). How membrane fusion can be triggered so rapidly and how the kinetics are regulated are not well understood. Hormones are released in a similar fashion, with multiple kinetic phases, using some of the same protein machinery, via fusion of hormone containing secretory granules (SG) with the plasma membrane. The initial ~1-3 nm wide connection between the fusing compartments, called the fusion pore, can flicker open-closed in succession before either closing permanently (transient fusion) or dilating fully. There is large variability between cell types (pore open times span ~100 µs 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 importance of fusion pores in regulating 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. Fusion mediated by exocytic SNARE proteins and their regulators has been reconstituted and studied for the past 20 years. However, methods that can monitor single reconstituted fusion pores with sub- ms resolution have been lacking. During the last cycle, we developed such methods for the first time, and explored mechanisms regulating fusion pores induced by SNAREs alone. In the next cycle, we propose to use those methods to (1) define the role of SNARE-interacting proteins in nucleation and dynamics of fusion pores and the selectivity of small pores. To characterize how the calcium sensors for exocytosis and other essential components of the release machinery contribute to fusion pore properties, we will use electrophysiology, nanodiscs, engineered cells, single-particle fluorescence microscopy, microfabricated devices, and artificial bilayers. We will also characterize selectivity of small fusion pores for ions, which is highly relevant for determining what is released during transient fusion events. We will then (2) dissect mechanisms contributing to kinetics of calcium-triggered exocytosis. The approaches will be augmented to allow rapid (~1 ms) [Ca2+] elevation using microperfusion or ultraviolet flash photolysis. These will enable defining how different sensors and release complexes regulate release kinetics and what determines the high calcium-cooperativity of release. 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.

项目概要： 在神经元中，包裹着神经递质的突触小泡 (SV) 与质膜融合并释放 通过突触间隙感知到的它们的含量是由局部钙的增加触发的。 去极化后的浓度。释放动力学包括同步阶段（钙后 0.1-5 毫秒）。 海拔），以及慢得多的异步阶段（~100 ms）如何触发膜融合。 激素以类似的方式释放，但动力学如何调节尚不清楚。 具有多个动力学阶段，使用一些相同的蛋白质机制，通过含有激素的融合 分泌颗粒 (SG) 与质膜之间最初约 1-3 nm 宽的连接。 称为融合孔的隔室可以连续闪烁打开或关闭，然后永久关闭 （瞬时融合）或完全扩张 细胞类型之间存在很大差异（孔开放时间跨度约为 100 µs）。 到 10 秒）并且在同一细胞内（一些孔闪烁，一些突然扩张）。 生理输入，例如刺激强度，对释放的内容产生重要影响（仅 小货物可以通过小孔逸出），在什么时间过程中，以及胞吐作用如何与 尽管融合孔在调节释放方面很重要，但人们对内吞作用知之甚少。 控制孔成核和动力学的机制这主要是由于研究融合的困难。 重构系统中的孔具有明确的蛋白质和膜成分，可以分离 由胞吐 SNARE 蛋白及其调节因子介导的融合的作用已被重建并 然而，在过去的 20 年里，人们一直在研究能够监测单个重构融合孔的方法。 在上一个周期中，我们首次开发了此类方法，并且缺乏毫秒分辨率。 探索了仅由 SNARE 诱导的融合孔调节机制。在下一个周期中，我们建议使用。 这些方法 (1) 定义 SNARE 相互作用蛋白在成核和动力学中的作用 融合孔和小孔的选择性来表征钙传感器如何进行胞吐作用。 和释放机械的其他重要部件有助于融合孔特性，我们将使用 电生理学、纳米圆盘、工程细胞、单颗粒荧光显微镜、微加工 我们还将表征小融合孔对离子的选择性，即 与确定瞬态融合事件期间释放的内容高度相关，然后我们将 (2) 进行剖析。 有助于钙触发胞吐作用动力学的机制。 使用微灌注或紫外线闪光光解增强以允许快速（~1 ms）[Ca2+]升高。 将能够定义不同的传感器和释放复合物如何调节释放动力学以及决定因素 这些基础研究将增进我们对释放的高钙协同性的理解。 神经递质和激素释放受到调节，从长远来看对人类健康具有潜在影响。