Proteomic characterization of the mechanotransduction complex in hair cells

 描述(申请人提供)：对声音和平衡的感知依赖于机械转导，即毛细胞立体纤毛顶端的分子复合体将外部机械刺激转化为电信号，电信号可以传播到中枢神经系统。听觉神经科学中一个未知的关键是构成这个复合体中离子传导转导通道的分子的身份。通过在人类和小鼠中研究与耳聋相关的基因，研究人员认为TMC1和TMC2是强有力的机械转导通道候选基因。不幸的是，具有足够的灵敏度和选择性的生化方法还没有被开发出来，以强有力地检测和定量机械转导复合体中的TMCs或其他膜蛋白。以抗体为基础的方法，如免疫印迹和免疫细胞化学，通常存在特异性差、多重能力低和定量不准确的问题，因此无法提供有关机械转导复合体组成的准确定量信息。此外，高质量的抗体，特别是膜蛋白抗体的高成本和有限的可获得性，限制了通过其他手段确定的新的转导通道候选的筛选。这项建议的目标是表征毛细胞中机械转导复合体的组成。为此，我们将开发有针对性的蛋白质组分析，以识别和准确量化转导蛋白，并测量在耳聋和前庭功能障碍的小鼠模型中，它们的立体纤毛位置和机械转导复合体关联是如何改变的。我们将设计两套检测方法，使我们能够同时监测已知的机械转导复杂蛋白的浓度，以及在RNA水平上检测在毛细胞中发现的新的候选转导通道蛋白。这些分析结合其他生化方法，将使我们能够1)确定TMC1、TMC2或其他类似通道的膜蛋白是否定位于毛束并能够与其他转导复杂蛋白相互作用，以及2)确定缺乏TMC1和TMC2的小鼠机械转导蛋白的定位如何变化。总之，这些研究的结果将大大有助于我们理解听力和耳聋的分子基础。从长远来看，这些分析将继续是有价值的工具，用于评估机械转导复合体的组成在各种小鼠模型中的发育过程中以及在毛细胞修复和再生的背景下如何变化。

 DESCRIPTION (provided by applicant): Perception of sound and balance depends on mechanotransduction, whereby a molecular complex at the tips of hair cell stereocilia converts an external mechanical stimulus into an electrical signal, which can propagate to the central nervous system. A key unknown in auditory neuroscience is the identity of the molecules that constitute the ion-conducting transduction channel within this complex. By studying deafness- linked genes in humans and mice, investigators have suggested that TMC1 and TMC2 are strong mechanotransduction channel candidates. Unfortunately, biochemical methods with sufficient sensitivity and selectivity to robustly detect and quantify the TMCs or other membrane proteins within the mechanotransduction complex have yet to be developed. Antibody-based methods such as Western blotting and immunocytochemistry often suffer from poor specificity, low multiplexing capabilities, and imprecise quantitation, thus preventing them from providing accurate quantitative information about the composition of the mechanotransduction complex. In addition, the high cost and limited availability of quality antibodies, especially for membrane proteins, limits screening for new transduction-channel candidates identified by other means. The goal of this proposal is to characterize the composition of the mechanotransduction complex in hair cells. To do so, we will develop targeted proteomic assays to identify and accurately quantify transduction proteins and measure how their stereocilia location and mechanotransduction complex association are altered in mouse models of deafness and vestibular dysfunction. We will design two sets of assays, which will allow us to simultaneously monitor the concentrations of known mechanotransduction complex proteins, as well as detect new candidate transduction channel proteins identified in hair cells at the RNA level. These assays, combined with other biochemical approaches, will allow us to 1) determine whether TMC1, TMC2, or other channel-like membrane proteins are localized to the hair bundle and are able to interact with other transduction complex proteins, and 2) determine how the localization of mechanotransduction proteins change in mice lacking TMC1 and TMC2. Together, the results of these studies will significantly contribute to our understanding of the molecular basis of hearing and deafness. In the long term, these assays will continue to be valuable tools for assessing how the composition of the mechanotransduction complex changes across development in various mouse models, as well as in the context of hair cell repair and regeneration.