The Role of Wrb in Vertebrate Ribbon Synapse Formation

项目摘要/摘要 脊椎动物感光细胞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。在插入突变的筛查中 为了影响斑马鱼的视觉功能，我们鉴定了斑马鱼WRB突变体，该突变体表现出 光感受器退化和听力损失。这种突变会扰乱Wrb基因，而Wrb基因 编码一个新的170个氨基酸的蛋白质，功能未知。在初步研究中，我们 发现Wrb突变体表现出条带突触组件的错误定位，减少了 光感受器突触处停靠的条带数量，并显著降低ERG 回应。这些数据有力地表明WRB可能完整地参与了带状突触 装配和/或功能。为了验证这一假设，我们将研究WRB在斑马鱼中的作用 通过组织学和分子学相结合的方法实现突触功能。在目标1中，我们 将使用转基因方法确定WRB的亚细胞定位，我们将确定是否 WRB影响发育过程中的带状组装，WRB的丢失如何影响树枝晶形态 突触后细胞，以及WRB是否阻止突触囊泡释放或膜 内吞作用。在目标2中，我们将使用转基因方法确定WRB的结合伙伴 提纯标记的WRB络合物，并进行质谱分析。这些研究 将深入了解WRB的作用，WRB是一种新的蛋白质，可能是 功能区突触的结构和功能。WRB绑定伙伴的身份以及随后的 WRB在体内系统的表征将为突触的研究开辟新的途径 结构，并可能提供对失明/耳聋疾病候选基因的洞察。

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.