The Role of Wrb in Vertebrate Ribbon Synapse Formation

项目概要/摘要 脊椎动物光感受器和感觉毛细胞的突触释放神经递质 通过被称为突触带的特殊结构。在筛选插入突变时， 我们鉴定了斑马鱼wrb突变体，它表现出 光感受器退化和听力损失。突变破坏了wrb基因， 编码一种新的170个氨基酸的蛋白质，其功能未知。在初步研究中，我们 发现wrb突变体表现出带状突触成分的错误定位，减少 光感受器突触处的停靠条带数量，并显著降低ERG 应答这些数据有力地表明，BMPb可能是完整地参与带状突触 组装和/或功能。为了验证这一假设，我们将研究斑马鱼中的BMPB的作用 通过组织学和分子方法的组合来研究突触功能。目标1： 将使用转基因方法确定BCLB的亚细胞定位，我们将确定是否 在发展过程中，BRB影响带状组装，BRB的损失如何影响树枝状形态 突触后细胞的，以及是否bcb阻断突触囊泡释放或膜 内吞作用在目标2中，我们将使用转基因方法鉴定BMPb的结合伴侣， 纯化标记的BisB复合物并随后通过质谱分析。这些研究 将提供对BMPb作用的深入了解，BMPb是一种新的蛋白质，可能是 带状突触的结构和功能。BCLb结合伴侣的身份和随后的 Wrb在体内系统中的表征将开辟突触研究的新途径 结构，并可能提供对盲/聋疾病的候选基因的洞察。

Project Summary/Abstract The synapses of vertebrate photoreceptors and sensory hair cells release neurotransmitter through specialized structures known as synaptic ribbons. In a screen for insertional mutations affecting visual function in zebrafish, we identified the zebrafish wrb mutant, which exhibited photoreceptor degeneration and hearing loss. The mutation disrupts the wrb gene, which encodes a novel 170 amino acid protein with no known function. In preliminary studies, we found that wrb mutants exhibited mislocalization of ribbon synapse components, reduced numbers of docked ribbons at photoreceptor synapses, and significantly reduced ERG responses. These data strongly suggest that Wrb could be integrally involved in ribbon synapse assembly and/or function. To validate this hypothesis, we will study the role of Wrb in zebrafish synaptic function through a combination of histological and molecular approaches. In Aim 1, we will identify the subcellular localization of Wrb using transgenic approaches, we will determine if Wrb affects ribbon assembly during development, how loss of Wrb affects dendritic morphology of postsynaptic cells, and whether Wrb blocks synaptic vesicle release or membrane endocytosis. In Aim 2, we will identify binding partners of Wrb using a transgenic approach to purify tagged Wrb complexes and subsequent analysis by mass spectroscopy. These studies will provide insights into the role of Wrb, a novel protein that may be a critical component to ribbon synapse structure and function. The identity of Wrb binding partners and the subsequent characterization of Wrb in an in vivo system will open new avenues of research on synaptic architecture and possibly provide insight into candidate genes for blindness/deafness disorders.