Regulation of Membrane Fusion in Exocytosis

胞吐作用中膜融合的调节

基本信息

批准号：
10246265
负责人：
SHYAM S KRISHNAKUMAR
金额：
$ 67.23万
依托单位：
YALE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
1991
资助国家：
美国
起止时间：
1991-09-30 至 2023-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10246265
关键词：
Action Potentials Automobile Driving Binding Biochemical Biophysics Calcium Caliber Cell membrane Closure by clamp Complex Cryo-electron tomography Cryoelectron Microscopy Diglycerides Disease Docking Equilibrium Exocytosis Fluorescence Resonance Energy Transfer Geometry Goals Hippocampus (Brain)In Situ In Vitro Insulin Leptin Ligands Light Lipids Measures Mediating Membrane Fusion Metabolic Metabolic Diseases Mission Molecular Chaperones Mutation National Institute of Diabetes and Digestive and Kidney Diseases Negative Staining Nervous system structure Neurites Neuronal Differentiation Neurons Neurosecretory Systems PC12 Cells Pancreas Pharmacology Phosphatidylinositol 4,5-Diphosphate Physiological Physiology Play Point Mutation Polymers Process Property Proteins Regulation Regulation of Exocytosis Resolution Role SNAP receptor Secretory Vesicles Shapes Site Speed Structure Synapses Synaptic Vesicles System Testing Uncertainty Vesicle base experimental study imaging approach improved insulin secretion monolayer mutant nanocluster nanodisk neurotransmitter release particle prevent reconstitution sensor single molecule synaptotagmin three dimensional structure trigger point virtual

项目摘要

Project Summary/Abstract How can virtually the same SNARE machine operate at dramatically different speeds depending on context, often far faster than a single SNAREpin? This is one of the central questions driving the field today, and the problem it embodies stands in boldest relief at the neuronal synapse, so it is here that we focus on the structures, biophysics, and physiological properties of the key protein machinery. Our overall hypothesis is that multiple SNAREpins released synchronously, each already close to the point of triggering fusion, co-operate to achieve fusion dramatically faster than any one alone. During the current period of support we discovered that the calcium sensor Synaptotagmin (normally anchored in synaptic vesicle) can self-assemble in vitro into Ca2+- sensitive, ring-like oligomers ~30 nm in diameter and have suggested that such rings forming between the synaptic vesicle (or insulin secretory vesicle) and the plasma membrane would prevent release until they are disrupted by Ca2+. Our specific hypothesis is that such ring oligomers of Synaptotagmin (Syt) are a central organizing principle for exocytosis, enabling the clamping and rapid synchronous release of multiple SNAREpins. This hypothesis is strongly supported by recent experiments in which a targeted mutation (F349A) that de-stabilizes Syt1 rings dramatically increases spontaneous and evoked release and in hippocampal neurons, and dramatically reduces the synchronicity of release with the action potential. We propose to 1) Test the hypothesis that ring-like oligomers of Synaptotagmins regulate exocytosis; 2) Test the hypothesis that Syt1 and Syt7 play distinct structural and functional roles in synchronous and asynchronous release from the same docked vesicles; 3) Elucidate the dynamics and topology of Munc13 and its proposed oligomers and the posited dual roles as vesicle tether and outer ring chaperone templating SNAREpins; and 4) Obtain by single particle cryo-EM and cryo EM tomography high resolution structures of functional release sites in vitro and in situ trapped in defined functional states. Similar machinery mediates neuroendocrine secretory physiology, including pancreatic insulin secretion, so we expect the answers will be highly relevant to the mission of NIDDK. Further, there is little doubt in the post-leptin era of the key role of the nervous system in metabolic balance and diseases.

项目摘要/摘要几乎可以根据上下文的不同速度以不同的速度运行相同的弹力机器通常比单个Snarepin快得多？这是今天推动该领域的核心问题之一，它体现的问题在神经元突触时最大地浮出水面，因此我们在这里专注于关键蛋白质机械的结构，生物物理学和生理特性。我们的总体假设是多个snarepins同步释放，每个sn蛋白已经接近触发融合点，合作到比单独的任何一个人都要快地实现融合的速度。在当前的支持期间，我们发现钙传感器突触抗体（通常以突触囊泡为锚定）可以在体外自组装到Ca2+ - - 直径敏感，类似环状的低聚物约30 nm，并提出这种环在突触囊泡（或胰岛素分泌囊泡）和质膜将阻止释放，直到它们为止被CA2+破坏。我们的具体假设是，突触量（SYT）的这种环寡聚物是中央组织胞吐作用的原理，使多个夹紧和快速同步释放 Snarepins。最近的实验得到了强烈支持，其中有针对性的突变（F349a）使SYT1环的稳定幅度大大增加了自发和诱发的释放，并在海马中神经元，并大大降低了与动作电位的释放同步性。我们建议1）检验以下假设，即突触毒素的环状低聚物调节胞吐作用； 2）检验以下假设 SYT1和SYT7在同步和异步释放中起独特的结构和功能作用相同的对接囊泡； 3）阐明Munc13及其提议的低聚物的动力学和拓扑结构及将双重作用作为囊泡系绳和外环伴侣模板snarepins； 4）单一获得粒子冷冻EM和冷冻EM断层扫描高分辨率释放位点的高分辨率结构，并在体外和IN 被困在定义的功能状态中的原位。类似的机械介导神经内分泌分泌生理学，包括胰腺胰岛素分泌，因此我们希望答案将与 niddk。此外，毫无疑问，在神经系统中的关键作用在代谢中的关键作用时，毫无疑问平衡与疾病。

项目成果

期刊论文数量（55）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Diacylglycerol-dependent hexamers of the SNARE-assembling chaperone Munc13-1 cooperatively bind vesicles.

DOI：
10.1073/pnas.2306086120
发表时间：
2023-10-31
期刊：
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
影响因子：
11.1
作者：
Li, Feng;Grushin, Kirill;Coleman, Jeff;Pincet, Frederic;Rothman, James E.
通讯作者：
Rothman, James E.

Chromosome Territorial Organization Drives Efficient Protein Complex Formation: A Hypothesis.

染色体区域组织驱动有效的蛋白质复合物形成：一个假设。

DOI：
发表时间：
2019
期刊：
The Yale journal of biology and medicine
影响因子：
0
作者：
Bera,Manindra;KalyanaSundaram,RamalingamVenkat
通讯作者：
KalyanaSundaram,RamalingamVenkat

Purification of three related peripheral membrane proteins needed for vesicular transport.

纯化囊泡运输所需的三种相关外周膜蛋白。

DOI：
发表时间：
1990
期刊：
The Journal of biological chemistry
影响因子：
0
作者：
Clary,DO;Rothman,JE
通讯作者：
Rothman,JE

Snapshot of sequential SNARE assembling states between membranes shows that N-terminal transient assembly initializes fusion.

膜之间顺序 SNARE 组装状态的快照显示 N 端瞬时组装初始化融合。

DOI：
10.1073/pnas.1518935113
发表时间：
2016
期刊：
Proceedings of the National Academy of Sciences of the United States of America
影响因子：
11.1
作者：
Wang,YongJian;Li,Feng;Rodriguez,Nicolas;Lafosse,Xavier;Gourier,Christine;Perez,Eric;Pincet,Frederic
通讯作者：
Pincet,Frederic

Molecular determinants of complexin clamping and activation function.

DOI：
10.7554/elife.71938
发表时间：
2022-04-20
期刊：
ELIFE
影响因子：
7.7
作者：
Bera, Manindra;Ramakrishnan, Sathish;Coleman, Jeff;Krishnakumar, Shyam S.;Rothman, James E.
通讯作者：
Rothman, James E.