Characterization of the Mechanotransduction complex in hair cells

 描述（由申请方提供）：原钙粘蛋白15（PCDH 15）突变导致鱼类、小鼠和人类耳聋。作为感觉毛细胞中机械传导复合体的中心和保守组分，这种不寻常的钙粘蛋白形成静纤毛尖端的细胞外丝的一部分。这些所谓的“提示链接”被认为是 机械敏感通道。为了更好地了解PCDH 15是如何耦合到机械转导机制，我们进行了一个公正的分子筛使用斑马鱼Pcdh 15 a作为诱饵，在基于膜的酵母双杂交筛选。我们确定了与哺乳动物TMC 2的直系同源物Tmc 2a的正相互作用。Tmc 2最近与小鼠的耳聋和前庭功能障碍有关，其密切相关的基因Tmc 1与小鼠和人类的隐性和显性听力损失有关（DFNA 36和DFNB 7/11）。我们的初步结果概括了在斑马鱼和小鼠TMC和PCDH 15直向同源物之间的筛选中发现的蛋白质相互作用。此外，我们已经发现，在野生型鱼中过度表达Tmc 2a片段后，毛细胞机械敏感性的功能丧失和功能获得效应。总之，与Pcdh 15的联系以及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Tmc 1/2蛋白是毛细胞中机械转导复合体的核心参与者。我们将用遗传学和生物化学方法表征Pcdh 15与Tmc 1/2蛋白的相互作用，并在体内研究复合物的结构/功能方面。这一工作将增加我们对机械传导的分子基础的了解，并了解Tmc 1的损伤如何导致听力损失。

 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.