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: 10443756
• 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/10443756
• 关键词: Action Potentials; Address; Affinity; Antibodies; Back; Binding Sites; Biochemical; Bungarotoxins; Calcium; Calcium Channel; Calcium Channel Blockers; Cell membrane; Chemical Synapse; 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; 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.

在化学突触处，神经元通过释放神经递质相互交流。 突触传递发生在两个动力学上不同的阶段。在动作电位的几毫秒内， 突触小泡跨多个突触与质膜同步融合。下列的 同步融合，突触小泡持续与质膜融合数十至数百个 毫秒。这个神经传递阶段称为异步释放，它变得更加明显 在病理生理条件下。尽管过去 50 年进行了广泛的研究，但纳米级 对突触小泡融合位点的组织知之甚少。同步和异步在哪里 融合发生在活跃区域内？融合位点是由钙通道的位置决定的吗？ 哪些钙传感器负责异步融合？是否有单独的囊泡池 异步融合？或者两个阶段消耗的囊泡池是相同的吗？为了解决这些 问题，我们开发了快速冷冻电子显微镜来跟踪膜动力学毫秒- 以毫秒为单位。该技术将神经元的电刺激与高压冷冻结合起来以捕获 哺乳动物中央突触的快速膜运输事件具有前所未有的空间（1 nm）和 时间（1 毫秒）分辨率。将这种方法与先进的遗传学、分子生物学相结合， 和生化技术，我们将确定哺乳动物中央突触的融合位点是如何组织的。 突触传递缺陷在神经系统疾病中起着因果作用。拟议的研究旨在 了解突触传递的分子机制，最终目标是 了解这些疾病的发病机制。

项目成果

Transient docking of synaptic vesicles: Implications and mechanisms.

• DOI: 10.1016/j.conb.2022.102535
• 发表时间: 2022-06
• 期刊: Current opinion in neurobiology
• 影响因子: 5.7

Dynamin 1xA interacts with Endophilin A1 via its spliced long C-terminus for ultrafast endocytosis.

Dynamin 1xA 通过其剪接的长 C 末端与 Endophilin A1 相互作用，实现超快内吞作用。

• DOI: 10.1101/2023.09.21.558797
• 发表时间: 2023
• 期刊: bioRxiv : the preprint server for biology
• 作者: Imoto,Yuuta;Xue,Jing;Luo,Lin;Raychaudhuri,Sumana;Itoh,Kie;Ma,Ye;Craft,GeorgeE;Kwan,AnnH;Mackay,JoelP;Ha,Taekjip;Watanabe,Shigeki;Robinson,PhillipJ
• 通讯作者: Robinson,PhillipJ