Spatial and molecular determinants of fusion probability and timing

融合概率和时间的空间和分子决定因素

• 批准号: 10226174
• 负责人: Shigeki Watanabe
• 金额: $ 35.82万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-09-30 至 2023-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10226174
• 关键词: Action Potentials; Address; Affinity; Back; Binding Sites; Biochemical; Bungarotoxins; Calcium; Calcium Channel; Calcium Channel Blockers; Cell membrane; Chemical Synapse; Cleaved cell; Communication; Consumption; Coupled; Couples; Data; Defect; Disease; Docking; Egtazic Acid; Electric Stimulation; Electron Microscopy; Electrons; Event; Exocytosis; Ferritin; Freezing; Genetic; Goals; Gold; Hippocampus (Brain); Image; Individual; Kinetics; Label; Liquid substance; Location; Maps; Mediating; Membrane; Methods; Modeling; Molecular; Molecular Biology; Mus; Neurons; Neurotransmitters; Nickel; Pathogenesis; Phase; Play; Presynaptic Terminals; Probability; Process; Proteins; Psyche structure; Recycling; Research; Resolution; Role; SNAP receptor; Site; Synapses; Synaptic Transmission; Synaptic Vesicles; Techniques; Testing; Time; VAMP-2; Vesicle; antibody test; chemical release; experimental study; millisecond; molecular modeling; nanomagnetic; nanoparticle; nanoscale; nervous system disorder; neurotransmission; neurotransmitter release; novel; particle; predictive modeling; pressure; recruit; sensor; synaptotagmin I; trafficking; vesicular SNARE proteins

At chemical synapses, neurons communicate each other through release of neurotransmitters. Synaptic transmission occurs in two kinetically distinct phases. Within milliseconds of an action potential, synaptic vesicles fuse with the plasma membrane synchronously across multiple synapses. Following synchronous fusion, synaptic vesicles continue to fuse with the plasma membrane over tens to hundreds of milliseconds. This phase of neurotransmission is called asynchronous release, and it becomes more apparent in pathophysiological conditions. Despite extensive research over the last 50 years, the nano-scale organization of synaptic vesicle fusion sites is poorly understood. Where do synchronous and asynchronous fusions take place within an active zone? Are fusion sites determined by the locations of calcium channels? Which calcium sensors are responsible for asynchronous fusion? Is there a separate pool of vesicles for asynchronous fusion? Or are the same pool of vesicles consumed for both phases? To address these questions, we have developed zap-and-freeze electron microscopy to follow membrane dynamics millisecond- by-millisecond. This technique couples electrical stimulation of neurons with high-pressure freezing to capture rapid membrane trafficking events at mammalian central synapses with unprecedented spatial (1 nm) and temporal (1 ms) resolution. Using this approach in combinations with advanced genetics, molecular biology, and biochemical techniques, we will determine how fusion sites are organized at mammalian central synapses. Defects in synaptic transmission play a causal role in neurological disorders. The proposed research aims to understand the molecular mechanisms underlying synaptic transmission with the ultimate goal of understanding the pathogenesis of these diseases.

在化学突触时，神经元通过释放神经递质相互交流。 突触传播发生在两个动力学不同的阶段。在行动潜力的毫秒内， 突触囊泡与质膜融合在多个突触中。下列的 同步融合，突触囊泡继续与质膜融合，超过数十至数百个 毫秒。这个神经传递的阶段称为异步释放，它变得更加明显 在病理生理状况下。尽管在过去的50年中进行了广泛的研究，但纳米级 突触囊泡融合位点的组织知之甚少。同步和异步在哪里 融合发生在活动区域​​内？融合位点是否由钙通道的位置确定？ 哪些钙传感器负责异步融合？有没有单独的囊泡池 异步融合？还是在两个阶段都消耗了同一囊泡？解决这些 问题，我们已经开发了ZAP和Freeze电子显微镜，以遵循膜动力学毫秒毫秒。 毫无疑问。该技术将神经元的电刺激与高压冻结捕获以捕获 哺乳动物中央突触的快速膜运输事件，其空间空间（1 nm）和 时间（1 ms）分辨率。使用这种方法与晚期遗传学，分子生物学的结合， 和生化技术，我们将确定如何在哺乳动物中央突触中组织融合位点。 突触传播中的缺陷在神经系统疾病中起因果作用。拟议的研究旨在 了解突触传播的分子机制，其最终目标是 了解这些疾病的发病机理。