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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：
10013225
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 2022-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10013225
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.

项目总结/文摘