Characterization of the mechanotransduction complex in hair cells

毛细胞中机械转导复合物的表征

基本信息

批准号：
8885946
负责人：
Teresa A Nicolson
金额：
$ 32.73万
依托单位：
OREGON HEALTH & SCIENCE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-04-01 至 2020-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8885946
关键词：
Accounting Affect Animal Model Apical Auditory Binding Biochemical Birds Cadherins Cell physiology Co-Immunoprecipitations Collection Complex Coupled Dominant-Negative Mutation Family Family member Filament Fishes Functional disorder Genes Genetic Goals Hair Hair Cells Hearing Human Immunoblotting Individual Investigation Knowledge Labyrinth Lead Lesion Link Mammals Mass Spectrum Analysis Mediating Membrane Membrane Proteins Methods Modeling Molecular Mouse Protein Mus Mutation PCDH15 gene Phenotype Play Preparation Protein Binding Protein Isoforms Proteins Sensory Hair Sensory Receptors Signal Transduction Specificity Speed Stereocilium Structure Transgenic Organisms Two-Hybrid System Techniques Ubiquitin Usher Syndrome Vertebrates Vestibular Hair Cells Work Yeasts Zebrafish base deafness design extracellular follow-up gain of function hearing impairment human CDH23 protein in vivo in vivo imaging insight lateral line link protein loss of function member mouse Fish protein mutant novel overexpression protein function public health relevance research study sound yeast two hybrid system

项目摘要

DESCRIPTION (provided by applicant): Mutations in Protocadherin 15 (PCDH15) cause deafness in fish, mice, and humans. As a central and conserved component of the mechanotransduction complex in sensory hair cells, this unusual cadherin forms part of the extracellular filaments at the tips of stereocilia. These so-called `tip links' are thought to gate mechanically sensitive channels. To gain a better understanding of how PCDH15 is coupled to the mechanotransduction machinery, we performed an unbiased molecular screen using zebrafish Pcdh15a as bait in a membrane-based yeast two-hybrid screen. We identified a positive interaction with Tmc2a, an orthologue of mammalian TMC2. Tmc2 was recently implicated in deafness and vestibular dysfunction in mice, and its closely related gene, Tmc1, is associated with both recessive and dominant forms of hearing loss in mice and humans (DFNA36 and DFNB7/11). Our preliminary results recapitulate the protein interactions found in the screen among both the zebrafish and mouse TMC and PCDH15 orthologues. In addition, we have discovered both loss-of-function and gain-of-function effects on hair-cell mechanosensitivity upon overexpression of fragments of Tmc2a in wild-type fish. Together, the link to Pcdh15 and the dominant negative or activating effects in hair cells provide compelling evidence that Tmc1/2 proteins are central players of the mechanotransduction complex in hair cells. We will characterize the interaction of Pcdh15 with Tmc1/2 proteins with genetic and biochemical methods, and study structure/function aspects of the complex in vivo. This work will increase our knowledge of the molecular basis of mechanotransduction and the understanding of how lesion of Tmc1 leads to hearing loss.

描述（由申请人提供）：原钙粘蛋白 15 (PCDH15) 的突变会导致鱼类、小鼠和人类耳聋。作为感觉毛细胞中机械转导复合物的核心和保守成分，这种不寻常的钙粘蛋白形成了细胞外细丝的一部分。这些所谓的“尖端链接”被认为是门控。为了更好地了解 PCDH15 如何与机械传导机制耦合，我们使用斑马鱼 Pcdh15a 作为基于膜的酵母双杂交筛选中的诱饵进行了无偏分子筛选，我们发现了与 Tmc2a 的正相互作用。 Tmc2 是哺乳动物 TMC2 的直系同源基因，最近与小鼠耳聋和前庭功能障碍有关，其相关基因 Tmc1 是与小鼠和人类的隐性和显性听力损失相关（DFNA36 和 DFNB7/11）。我们的初步结果概括了在斑马鱼和小鼠 TMC 和 PCDH15 直系同源物之间的筛选中发现的蛋白质相互作用。在野生型鱼类中过度表达 Tmc2a 片段后，功能丧失和功能获得都会对毛细胞机械敏感性产生影响。 Pcdh15 和毛细胞中的显性负效应或激活效应提供了令人信服的证据，证明 Tmc1/2 蛋白是毛细胞中机械转导复合物的核心参与者，我们将通过遗传和生化方法表征 Pcdh15 与 Tmc1/2 蛋白的相互作用，并进行研究。这项工作将增加我们对机械转导分子基础的了解以及对 Tmc1 损伤如何导致听力损失的理解。