The role of tomosyn in synaptic transmission.

Tomosyn 在突触传递中的作用。

• 批准号: 7814143
• 负责人: Janet E Richmond
• 金额: $ 45.43万
• 依托单位: UNIVERSITY OF ILLINOIS AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-30 至 2011-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7814143
• 关键词: Address; Adopted; Adult; Affect; Affinity; Animal Model; Antibodies; Area; Behavioral; Binding; Binding Proteins; Biochemical; Biochemical Pathway; Biological Assay; Boxing; Brain; C-terminal; Caenorhabditis elegans; Calcium; Cell membrane; Cells; Charge; Classification; Collaborations; Complement; Complex; Cyclic AMP; Cyclin-Dependent Kinase 5; Cytosol; Data; Defect; Deletion Mutation; Dense Core Vesicle; Disease; Distal; Docking; Doctor of Philosophy; Drosophila genus; Drosophila melanogaster; Electrodes; Employment; Ensure; Event; Exhibits; Exocytosis; Figs - dietary; Foundations; Freeze Substitution; Freezing; Funding; Generations; Genes; Genetic; Genetic Models; Genetic Screening; Grant; Graph; Growth Cones; Human Resources; Hybrids; Immunoelectron Microscopy; In Vitro; Kinetics; Laboratories; Lead; Learning; Learning Disorders; Length; Link; Membrane; Messenger RNA; Modeling; Molecular; Molecular Conformation; Monomeric GTP-Binding Proteins; Muscle; Mutation; N-terminal; Nerve; Nervous system structure; Neurites; Neurologic; Neurons; Organism; Pattern; Phenotype; Phosphorylation; Phosphorylation Site; Phosphotransferases; Pilot Projects; Play; Process; Protein Isoforms; Proteins; Publishing; Qi; Qualifying; RNA Splicing; Rattus; Reagent; Regulation; Regulation of Exocytosis; Research; Research Design; Research Project Grants; Residual state; Role; SNAP receptor; Site; Site-Directed Mutagenesis; Structure; Synapses; Synaptic Transmission; Synaptic Vesicles; Synaptic plasticity; Tertiary Protein Structure; Testing; Tissues; Transgenic Organisms; Vacuolar Protein Sorting; Vesicle; WD Repeat; Wages; Work; Yeasts; base; cost; density; genetic analysis; graduate student; in vivo; insight; loss of function; mutant; nervous system disorder; neurotransmission; neurotransmitter release; novel; overexpression; phosphoric diester hydrolase; pressure; presynaptic; prevent; professor; promoter; protein function; protein kinase A kinase; public health relevance; research study; response; sample fixation; spatiotemporal; synaptic function; syntaxin; trafficking; vesicle-associated membrane protein; voltage clamp; yeast protein

DESCRIPTION (provided by applicant): Synaptic vesicle exocytosis is a highly specialized vesicle trafficking process in which calcium triggers fusion of synaptic vesicles with the plasma membrane, resulting in neurotransmitter release. SNARE complex assembly between synaptobrevin, SNAP-25 and syntaxin is a critical requirement preceding this vesicle fusion event. Several SNARE-interacting proteins have been shown to profoundly influence the strength of synaptic transmission, through their regulatory effects on the SNARE complex. Recently, a new SNARE binding partner, tomosyn was isolated from rat brain cytosol. Tomosyn has a SNARE binding domain that can compete with synaptobrevin for assembly into a tomosyn SNARE complex with syntaxin and SNAP-25. Based on these biochemical observations as well as tomosyn overexpression data, tomosyn is proposed to regulate vesicle release through an undefined mechanism. There are presently no loss-of-functions mutants available in any organism other than C. elegans. Therefore, we intend to examine the mechanism of tomosyn action at synapses in this powerful genetic model organism. Aim 1) Characterize the synaptic phenotype of tom-1 deletion mutants. We have obtained two tom-1 deletion mutants that have phenotypes consistent with increased synaptic transmission. We will conduct a detailed characterization of these tom-1 mutants including behavioral, cytoarchitectural, pharmacological, electrophysiological and ultrastructural analyses. Aim 2) Determine which TOM-1 isoforms regulate synaptic transmission. C. elegans tom-1 encodes three isoforms. The isoform expression patterns will be ascertained and mosaic analysis and tissue specific rescue experiments will be performed. Aim 3) Genetic analysis of TOM-1 function. We hypothesize that tomosyn regulates the priming step of exocytosis. To test this model we will generate and characterize double mutants between tom-1 and several mutants known to affect the vesicle primed pool (unc-13, unc-10, open-syntaxin and unc-18). Aim 4) Identify TOM-1 domains required for the regulation of synaptic transmission. TOM-1 protein domains essential for the regulation of exocytosis will be identified using a genetic screen for mutants that fail to complement the tom-1 mutation. These experiments are likely to further our understanding of neurotransmission, a foundation that may contribute to our understanding of neurological diseases and vesicle trafficking disorders. PUBLIC HEALTH RELEVANCE: Information flow within nervous systems occurs via specialized cell-cell contacts called 'synapses'. The mechanisms controlling information flow through synapses are incompletely understood. Here we propose supplemental work to study a protein called 'tomosyn', which our previous work, as well as work from other labs, has shown is an important regulator of synapse function. Specifically, our supplemental work will extend our previously funded work from worms into another genetic model organism, the fruitfly (Drosophila melanogaster), where we can probe the mechanism by which tomosyn works in more detail, and explore whether tomosyn plays a role in learning. A molecular understanding of tomosyn could contribute to treatments for a variety of neurological and learning disorders.

描述（由申请人提供）： 突触囊泡胞吐作用是一种高度特化的囊泡运输过程，其中钙触发突触囊泡与质膜融合，导致神经递质释放。SNARE复合物组装之间的突触泡蛋白，SNAP-25和syntaxin是一个关键的要求之前，这种囊泡融合事件。一些SNARE相互作用蛋白已被证明通过它们对SNARE复合物的调节作用而深刻地影响突触传递的强度。最近，一种新的SNARE结合配偶体，tomosyn分离自大鼠脑细胞质。Tomosyn具有SNARE结合结构域，其可以与小突触泡蛋白竞争组装成具有突触融合蛋白和SNAP-25的tomosyn SNARE复合物。基于这些生化观察以及tomosyn过表达数据，提出tomosyn通过未定义的机制调节囊泡释放。目前在除C外的任何生物体中没有可用的功能丧失突变体。优雅的因此，我们打算在这个强大的遗传模型生物体中研究tomosyn在突触中的作用机制。目的1）研究tom-1缺失突变体的突触表型。我们已经获得了两个tom-1缺失突变体，其表型与突触传递增加一致。我们将对这些tom-1突变体进行详细的表征，包括行为、细胞结构、药理学、电生理和超微结构分析。目的2）确定哪些TOM-1亚型调节突触传递。C.秀丽线虫TOM-1编码三种同种型。将确定同种型表达模式，并进行嵌合体分析和组织特异性拯救实验。目的3）TOM-1基因功能的遗传学分析。我们假设tomosyn调节胞吐的启动步骤。为了测试该模型，我们将产生和表征tom-1和已知影响囊泡引发池的几种突变体（unc-13、unc-10、开放突触融合蛋白和unc-18）之间的双突变体。目的4）鉴定突触传递调控所需的TOM-1结构域。TOM-1蛋白质结构域必不可少的调节胞吐作用将确定使用基因筛选突变体，不能补充tom-1突变。这些实验可能会进一步加深我们对神经传递的理解，这一基础可能有助于我们理解神经系统疾病和囊泡运输障碍。公共卫生相关性：神经系统内的信息流通过称为“突触”的专门细胞间接触发生。控制信息流经突触的机制还不完全清楚。在这里，我们提出了补充工作，研究一种名为“tomosyn”的蛋白质，我们以前的工作以及其他实验室的工作已经表明，它是突触功能的重要调节器。具体来说，我们的补充工作将把我们以前资助的工作从蠕虫扩展到另一种遗传模式生物果蝇（果蝇），在那里我们可以更详细地探索tomosyn的工作机制，并探索tomosyn是否在学习中发挥作用。对tomosyn的分子理解可能有助于治疗各种神经和学习障碍。