The role of tomosyn in synaptic transmission.

描述（由申请人提供）：突触囊泡胞吐是一个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。synaptobrevin， SNAP-25和syntaxin之间的SNARE复合物组装是囊泡融合事件发生之前的关键条件。一些SNARE相互作用蛋白已被证明通过它们对SNARE复合体的调节作用深刻地影响突触传递的强度。近年来，从大鼠脑细胞质中分离到一种新的SNARE结合伙伴——tomosyn。Tomosyn有一个SNARE结合域，可以与synaptobrevin竞争，与syntaxin和SNAP-25组装成Tomosyn SNARE复合体。基于这些生化观察以及tomosyn过表达数据，我们提出tomosyn通过一种未定义的机制调节囊泡释放。目前，除了秀丽隐杆线虫之外，在任何生物体中都没有功能丧失突变体。因此，我们打算研究这种强大的遗传模式生物突触上的tomosyn作用机制。目的1)表征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，其表型与增加的突触传递一致。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。秀丽隐杆线虫tom-1编码三种同工异构体。将确定同种异构体的表达模式，并进行镶嵌分析和组织特异性拯救实验。目的3)TOM-1基因功能的遗传分析。我们假设tomosyn调节胞吐的启动步骤。为了测试这个模型，我们将在tom-1和几个已知影响囊泡引物池的突变体（unc-13, unc-10， open-syntaxin和unc-18）之间生成并表征双突变体。目的4)确定调节突触传递所需的TOM-1结构域。对胞吐调节至关重要的TOM-1蛋白结构域将通过基因筛选来确定不能补充TOM-1突变的突变体。这些实验可能会进一步加深我们对神经传递的理解，为我们理解神经系统疾病和囊泡运输障碍奠定基础。公共卫生相关性：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。控制信息通过突触流动的机制尚不完全清楚。在这里，我们提出补充工作来研究一种叫做“tomosyn”的蛋白质，我们之前的工作，以及其他实验室的工作，都表明它是突触功能的重要调节因子。具体来说，我们的补充工作将把我们之前资助的研究从蠕虫扩展到另一种遗传模式生物，果蝇（Drosophila melanogaster），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.