Isolation of Recycling Synaptic Vesicles for Proteomic Analyses

用于蛋白质组分析的回收突触小泡的分离

• 批准号: 8458913
• 负责人: Sandra M Bajjalieh
• 金额: $ 23.18万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-12-01 至 2014-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8458913
• 关键词: Adaptor Signaling Protein; Address; Affect; Attention; Australia; Brain; Cell Fractionation; Cell membrane; Cell physiology; Cerebral cortex; Child; Clathrin; Clathrin Adaptors; Clathrin-Coated Vesicles; Complex; Development; Dynamin; Endocytosis; Endosomes; Event; Goals; Guanosine Triphosphate Phosphohydrolases; Half-Life; Intervention; Investigation; Knowledge; Mediating; Medical Research; Modeling; Molecular; Mus; Neurosciences; Personal Communication; Pharmaceutical Preparations; Population; Preparation; Procedures; Process; Proteins; Proteome; Proteomics; Protocols documentation; Recycling; Regulation; Research Institute; Scheme; Scientist; Site; Sorting - Cell Movement; Source; Synapses; Synaptic Vesicles; Testing; Vesicle; Wild Type Mouse; Work; genetic manipulation; inhibitor/antagonist; neurotransmission; neurotransmitter release; presynaptic; prevent; protein transport; public health relevance; response; synaptotagmin; tool development; trafficking

DESCRIPTION (provided by applicant): In response to RFA-MH-12-140, "Development of Tools to Explore the Synaptome", we propose to develop a procedure to trap and isolate recycling synaptic vesicles as an essential first step in analyzing how vesicle protein composition varies with changes in synaptic activity, drug treatment, and under pathological conditions. The ability of synaptic vesicles to undergo multiple rounds of fusion is crucial to maintaining fast, efficient neurotransmission. Synaptic vesicles are formed and recycle via endocytosis. At many synapses, a small proportion of the total vesicle population mediates the majority of neurotransmission while the larger portion remains untapped in a reserve pool. Therefore, proteomic analyses using traditional vesicle purification schemes cannot track changes in the protein composition of the recycling pool. To address this we will develop a scalable, readily applied purification protocol for recycling synaptic vesicles that relies on transiently trapping recycling vesicles as a means of separating them from the total vesicle pool. This approach will open a new field of investigation into the complex endocytotic machinery that regulates vesicle recycling. It will provide a means to test the effects of drugs, genetic manipulations, and pathological conditions on this very important process.

描述（由申请人提供）：响应RFA-MH-12-140，“开发探索突触器的工具”，我们建议开发一种程序来捕获和分离回收突触囊泡作为分析囊泡蛋白组成如何随突触活动，药物治疗，药物治疗和病理条件的变化而变化的第一步。突触囊泡进行多轮融合的能力对于维持快速，有效的神经传递至关重要。形成突触囊泡并通过内吞作用回收。在许多突触中，一小部分的总囊泡种群介导了大多数神经传递，而较大的部分仍未在储备库中均未开发。因此，使用传统囊泡纯化方案进行蛋白质组学分析无法跟踪回收库蛋白质组成的变化。为了解决这个问题，我们将开发一种可扩展的，易于应用的纯化协议，用于回收突触囊泡，该协议依赖于瞬时捕获回收囊泡，作为将它们与总囊泡池分开的一种手段。这种方法将为调节囊泡回收的复杂内吞机制开设一个新的调查领域。它将提供一种测试药物，遗传操纵和病理条件对这个非常重要过程的影响的方法。