Defining the 'late' role for UNC-18 in synaptic vesicle exocytosis

定义 UNC-18 在突触小泡胞吐作用中的“晚期”作用

• 批准号: 8119072
• 负责人: Eric Gordon Bend
• 金额: $ 2.9万
• 依托单位: UNIVERSITY OF UTAH
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-08-15 至 2012-08-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8119072
• 关键词: Aldicarb; Behavior; Behavioral; Binding; Biological Assay; Caenorhabditis elegans; Calcium; Cell membrane; Cells; Cholinesterase Inhibitors; Complex; Conflict (Psychology); Data; Defect; Docking; Electron Microscopy; Electrophysiology (science); Event; Exocytosis; Frequencies; In Vitro; Individual; Kinetics; Locomotion; Measures; Mediating; Membrane Fusion; Modeling; Mutation; N-terminal; Neuromuscular Junction; Neurosecretion; Orthologous Gene; Peptides; Phase; Phenotype; Presynaptic Terminals; Process; Protein Binding; Proteins; Role; SNAP receptor; Specificity; Staging; Sucrose; Synapses; Synaptic Vesicles; Testing; Transgenic Animals; Transgenic Organisms; Vesicle; Work; in vivo; mutant; neurotransmission; research study; syntaxin; syntaxin 18; transmission process; vesicle-associated membrane protein

DESCRIPTION (provided by applicant): SM proteins are required for all membrane fusion steps in a cell, yet the mechanism of their action is not known. For example, studies of the SM protein at the synapse - the Unc18 proteins - suggest that these proteins can either promote or inhibit synaptic vesicle fusion. These conflicting results may be due to the multiple binding modes of Unc18. Unc18 can bind syntaxin in a closed form or it can bind to the N-terminus of the open form of syntaxin. Our preliminary data indicate that binding of the C. elegans Unc18 to the closed form of syntaxin is not essential for its function in fusion. The current working model is that Unc18 binding to the N-terminal peptide of syntaxin mediating the role of Unc18 in fusion via SNARE complex interaction. This model predicts, first, that the binding of UNC-18 to syntaxin in C. elegans will be conserved, and that mutations in the binding regions of UNC-18 and syntaxin will disrupt the N-peptide and SNARE interactions. I will assay the specificity of these mutations by using in vivo pull down experiments. Second, I will determine if these mutations disrupt fusion in vivo. Specifically, I will use electron microscopy to characterize vesicle docking at presynaptic terminals; I will measure vesicle priming by quantifying the size of the readily releasable pool; And, I will use in vivo electrophysiology to measure the kinetics of single vesicle fusion events (miniature EPSCs) and calcium evoked events. This analysis will determine the functional significance of Unc18's interaction with the assembled core complex and may elucidate the fusion-promoting role of Unc18 in synaptic vesicle exocytosis.

描述(由申请人提供)：SM蛋白是细胞中所有膜融合步骤所必需的，但其作用机制尚不清楚。例如，对突触上的SM蛋白--Unc18蛋白--的研究表明，这些蛋白可以促进或抑制突触小泡的融合。这些相互矛盾的结果可能是由于UNC18的多种绑定模式造成的。Unc18能以封闭的形式结合突触蛋白，也能与突触蛋白的开放形式的N末端结合。我们的初步数据表明，线虫Unc18与封闭形式的Synaxin的结合对其融合功能并不是必需的。目前的工作模式是通过SNARE复合体相互作用将Unc18结合到Synaxin的N端肽上，介导Unc18在融合中的作用。该模型预测，首先，在线虫中，UNC-18与Synaxin的结合将是保守的，UNC-18和Synaxin结合区的突变将破坏N-肽和SNARE的相互作用。我将通过活体下拉实验来分析这些突变的特异性。其次，我将确定这些突变是否会破坏体内的融合。具体地说，我将使用电子显微镜来描述突触前终末的小泡停靠；我将通过量化容易释放的池的大小来测量小泡的启动；我将使用体内电生理学来测量单个小泡融合事件(微型EPSCs)和钙诱发事件的动力学。这一分析将确定Unc18‘S与组装的核心复合体相互作用的功能意义，并可能阐明Unc18在突触小泡吐出中的促进融合作用。