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The Role of Wrb in Vertebrate Ribbon Synapse Formation

Wrb 在脊椎动物带状突触形成中的作用

基本信息

批准号：
8489300
负责人：
Brian D Perkins
金额：
$ 18.64万
依托单位：
CLEVELAND CLINIC LERNER COM-CWRU
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-07-01 至 2015-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8489300
关键词：
Affect Afferent Neurons Affinity Chromatography Amino Acids Anatomy Antibodies Architecture Auditory Binding Biochemistry Biological Assay Blindness Candidate Disease Gene Cells Coiled-Coil Domain Communication Complex Data Defect Development Disease Docking Ear Electron Microscopy Endocytosis Exhibits Functional disorder Gene Expression Genes Hair Cells Hearing Human Immunohistochemistry In Situ Hybridization Investigation Knowledge Labyrinth Lead Learning Mass Spectrum Analysis Membrane Membrane Potentials Modeling Molecular Molecular Probes Morphology Mutate Mutation Neurons Perception Phenotype Photoreceptors Property Protein Binding Proteins Reagent Research Retina Role Sensory Sensory Hair Stimulus Structure Synapses Synaptic Cleft Synaptic Transmission Synaptic Vesicles System Testing Transgenes Transgenic Animals Transgenic Organisms Transmembrane Domain Vertebrate Photoreceptors Vesicle Vision Visual Zebrafish abstracting base deafness hearing impairment in vivo insight interdisciplinary approach mutant neurotransmitter release novel photoreceptor degeneration postsynaptic presynaptic protein protein interaction research study response ribbon synapse synaptic function synaptogenesis

项目摘要

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.

项目总结/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进行分析。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在体内系统中表征BCLB将为突触的研究开辟新的途径，结构，并可能提供对盲/聋疾病的候选基因的洞察。