The role of tomosyn in synaptic transmission.

描述(申请人提供)：突触小泡胞吐是一种高度专门化的囊泡运输过程，在这个过程中，钙离子触发突触小泡与质膜融合，导致神经递质释放。Synaptobrevin、SNAP-25和Synaxin之间的SNARE复杂组装是这次囊泡融合事件之前的一个关键要求。一些与SNARE相互作用的蛋白已经被证明通过它们对SNARE复合体的调节作用而深刻地影响突触传递的强度。最近，从大鼠脑胞浆中分离到一种新的诱捕结合蛋白Tomosyn。Tomosyn有一个SNARE结合域，它可以与Synaptobrevin竞争，与Synaxin和SNAP-25组装成TomosynSNARE复合体。基于这些生化观察和Tomosyn的过度表达数据，Tomosyn被认为通过一种未知的机制来调节囊泡的释放。目前，除线虫外，没有任何生物体中存在功能丧失的突变体。因此，我们打算在这个强大的遗传模式生物体中研究Tomosyns在突触上的作用机制。目的1)研究突触突触表型。我们已经获得了两个tom-1缺失突变体，它们的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TOM-1的哪些亚型调控突触传递。线虫Tom-1编码三种亚型。将确定亚型表达模式，并进行镶嵌分析和组织特异性抢救实验。目的3)TOM-1功能的遗传分析。我们假设Tomosyn调控胞吐作用的启动步骤。为了测试这一模型，我们将在tom-1和几个已知影响囊泡启动池的突变体(UNC-13、UNC-10、开放合成素和UNC-18)之间产生并表征双突变体。目的4)确定突触传递调控所需的TOM-1结构域。对于胞吐作用的调节至关重要的TOM-1蛋白结构域将通过对未能补充TOM-1突变的突变进行基因筛选来鉴定。这些实验可能会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疾病和囊泡运输障碍。与公共健康相关：神经系统内的信息流通过称为“突触”的特殊细胞-细胞接触发生。通过突触控制信息流的机制还不完全清楚。在这里，我们建议进行补充工作来研究一种名为Tomosyn的蛋白质，我们之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因子。具体地说，我们的补充工作将把我们之前资助的工作从蠕虫扩展到另一种遗传模式生物--果蝇(Drophila Blackogaster)，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。

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.