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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：
9901377
负责人：
Teresa A Nicolson
金额：
$ 33.26万
依托单位：
STANFORD 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 2021-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9901377
关键词：
Affect Animal Model Apical Auditory Binding Binding Proteins Biochemical Birds CDH23 gene Cadherins Cell physiology Co-Immunoprecipitations Collection Complex Coupled Dominant-Negative Mutation Family Family member Filament Fish Proteins Fishes Functional disorder Genes Genetic Goals Hair Hair Cells Hearing Human Immunoblotting Individual Investigation Knowledge Labyrinth Lead Lesion Link Mammals Mass Spectrum Analysis Mechanics Mediating Membrane Membrane Proteins Methods Modeling Molecular Mouse Protein Mus Mutation PCDH15 gene Phenotype Play Preparation Protein Isoforms Proteins Sensory Hair Sensory Receptors Signal Transduction Specificity Speed Structure Transgenic Organisms Two-Hybrid System Techniques Ubiquitin Usher Syndrome Vertebrates Vestibular Hair Cells Work Yeasts Zebrafish base deafness design experimental study extracellular follow-up gain of function hearing impairment in vivo in vivo imaging insight lateral line link protein loss of function mechanotransduction member mutant novel overexpression protein function public health relevance 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.