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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/摘要几乎相同的诱捕机器如何根据环境以显著不同的速度运行，通常比单个SNAREpin快得多？这是当今推动该领域的核心问题之一，而它所体现的问题在神经元突触上表现得最大胆，所以我们就在这里关注关键蛋白质机械的结构、生物物理和生理特性。我们的总体假设是同时发布的多个SNAREPin，每个都已经接近触发融合的点，合作实现核聚变比单独实现任何一个都要快得多。在当前的支持期间，我们发现钙感受器突触素(通常固定在突触小泡中)在体外可以自我组装成钙离子。敏感的环状低聚物直径约30 nm，并表明这种环在突触小泡(或胰岛素分泌小泡)和质膜将阻止释放，直到它们被钙离子干扰。我们的具体假设是，这种突触素的环状寡聚体(SYT)是一个中心胞吐组织原理，可夹紧并快速同步释放 SNAREPINS。最近的实验有力地支持了这一假设，在这些实验中，靶向突变(F349A) 破坏Syt1环的稳定显著增加了自发和诱发的释放，并在海马区并显著降低释放与动作电位的同步性。我们建议1) 检验突触素环状寡聚体调节胞吐作用的假说；2)检验以下假说 Syt1和Syt7在从 3)阐明了Munc13及其寡聚体的动力学和拓扑结构以及定位为囊泡锚链和外环伴侣的双重作用模板SNAREpins；4)通过单一颗粒低温电子显微镜和低温电子显微镜断层扫描功能释放部位的高分辨结构原位被困在定义的功能状态中。类似的机制调节神经内分泌分泌生理学，包括胰腺胰岛素分泌，所以我们预计答案将与 NIDDK。此外，在后瘦素时代，神经系统在新陈代谢中的关键作用几乎是毫无疑问的。平衡和疾病。

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）

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

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

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.

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.

Binding of an N-ethylmaleimide-sensitive fusion protein to Golgi membranes requires both a soluble protein(s) and an integral membrane receptor.