Characterization of the Mechanotransduction complex in hair cells

 描述(由申请人提供)：原钙粘附素15(PCDH15)的突变会导致鱼类、老鼠和人类耳聋。作为感觉毛细胞机械转导复合体的中心和保守成分，这种不同寻常的钙粘附素形成了立体纤毛顶端的细胞外细丝的一部分。这些所谓的“尖端链接”被认为是 机械敏感通道。为了更好地了解PCDH15是如何与机械转导机制耦合的，我们在基于膜的酵母双杂交筛选中以斑马鱼Pcdh15a为诱饵进行了无偏分子筛选。我们发现Tmc2a与哺乳动物TMC2的同源基因Tmc2a存在正相互作用。TMC2最近被认为与小鼠耳聋和前庭功能障碍有关，其密切相关的基因TMC1与小鼠和人类的隐性和显性听力损失有关(DFNA36和DFNB7/11)。我们的初步结果概括了斑马鱼与小鼠TMC和PCDH15同源物之间的蛋白质相互作用。此外，我们还发现，在野生型鱼类中过表达Tmc2a片段会对毛细胞的机械敏感性产生功能丧失和功能获得的影响。总之，与PCDH15的联系以及毛细胞中主要的负或激活效应提供了令人信服的证据，表明TMC1/2蛋白是毛细胞机械转导复合体的中心参与者。我们将用遗传学和生物化学方法表征PCDH15与TMC1/2蛋白的相互作用，并在体内研究该复合体的结构和功能。这项工作将增加我们对机械转导的分子基础的了解，以及对TMC1损伤如何导致听力损失的理解。

 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.