Molecular basis for the regulation of SNARE assembly in neuronal exocytosis

神经元胞吐作用中 SNARE 组装调节的分子基础

• 批准号: 10202630
• 负责人: DAVID S CAFISO
• 金额: $ 37.26万
• 依托单位: UNIVERSITY OF VIRGINIA
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-04-01 至 2024-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10202630
• 关键词: Automobile Driving; Binding; Biochemical; Biochemistry; Biophysics; C2 Domain; Calcium; Cell membrane; Collaborations; Complex; Data; Docking; Electron Spin Resonance Spectroscopy; Electrons; Electrostatics; Environment; Event; Exocytosis; Goals; Heterogeneity; Length; Lipids; Magnetic Resonance; Measures; Membrane; Membrane Fusion; Membrane Proteins; Methods; Microscopy; Modeling; Molecular; Molecular Conformation; Molecular Structure; NMR Spectroscopy; Nerve; Neuraxis; Neurobiology; Neurons; Physiologic pulse; Physiological; Positioning Attribute; Procedures; Process; Proteins; Regulation; Resolution; Role; SNAP receptor; Site; Structure; Synaptic Vesicles; System; Testing; Time; Total Internal Reflection Fluorescent; Vesicle; base; controlled release; design; genetic regulatory protein; imaging modality; innovation; millisecond; nervous system disorder; neurotransmitter release; phosphatidylinositol phosphate, PtdIns(4,5)P2; presynaptic; presynaptic neurons; programs; protein complex; reconstitution; restraint; sensor; spectroscopic imaging; stoichiometry; structural biology; synaptotagmin; synaptotagmin I; syntaxin; vesicle-associated membrane protein

PROJECT SUMMARY The overall goal of this program project is to elucidate the precise molecular mechanism and regulation of the fusion machine that drives exocytosis for the controlled release of neurotransmitter at nerve terminals. The assembly of SNARE molecules residing in the synaptic vesicle and presynaptic plasma membrane takes center stage and provides the driving energy for this process. Even though we know the structure of the fully assembled cis-SNARE complex after fusion in atomic detail and have detailed conformational models for several of the SNAREs before fusion, we do not precisely know how (i) they are conditioned with regulatory proteins such as Munc18 and Munc13 to form an active acceptor complex on the plasma membrane, (ii) how this acceptor SNARE complex engages with the synaptic vesicle SNARE upon encounter, and (iii) how this high-energy trans-SNARE complex is ultimately triggered by the synaptic vesicle protein synaptotagmin and calcium to proceed to full assembly and fusion. Three projects led by three expert leaders in the biochemistry, structural biology, and biophysics of neuronal exocytotic membrane fusion are designed to jointly unravel the precise molecular interactions that drive the neuronal fusion machine through the vesicle docking, priming, and fusion steps with the highest possible structural and time resolution. The team will seek to define the structures and configurations of the active presynaptic acceptor SNARE complex and the fusion-restricted trans-SNARE complex between two membranes, and the team will strive to uncover the molecular mechanism, by which calcium-synaptotagmin engages with the membranes and/or complex to release their fusion-restriction. To achieve this goal the team will use a unique combination of approaches ranging from highly innovative biochemical procedures to reconstitute the relevant proteins, EPR, DEER, and NMR spectroscopy to characterize the pertinent structures in membrane environments, and FLIC and single vesicle TIRF microscopy to measure membrane topology and read out fusion on the millisecond timescale.

项目摘要 该计划项目的总体目标是阐明精确的分子机制和调节 融合机驱动胞吐作用，以控制神经递质在神经末端的释放。这 驻留在突触囊泡和突触前质膜中的圈套分子的组装 中心舞台，并为此过程提供驱动能量。即使我们知道完全的结构 聚集在原子细节后融合后组装顺式螺旋络合物，并具有详细的构象模型 融合前的几只网罗 蛋白（例如Munc18和Munc13）在质膜上形成活性受体复合物，（ii）如何 这种受体圈套复合物在相遇时与突触小囊泡互动，（iii）如何 高能式透射式复合物最终是由突触囊泡蛋白突触蛋白和 钙要进行完全组装和融合。 三个由生物化学，结构生物学和神经元生物物理学领导的三个项目领导 胞吐膜融合旨在共同揭示驱动驱动的精确分子相互作用 神经元融合机穿过囊泡对接，启动和融合步骤，最高 结构和时间分辨率。团队将寻求定义活动的结构和配置 突触前受体圈圈复合物和两个之间的融合限制性跨效复合物 膜，团队将努力揭示分子机制，钙珠蛋白质机制 与膜和/或复合物互动以释放其融合限制。 为了实现这一目标，团队将使用独特的方法组合，包括高度创新性 重建相关蛋白质，EPR，鹿和NMR光谱的生化程序 表征膜环境中相关结构，以及flic和单囊TIRF显微镜 测量膜拓扑并读取毫秒时刻表上的融合。