Proteomic characterization of the mechanotransduction complex in hair cells

 描述（由申请人提供）：声音和平衡的感知取决于机械转导，由此毛细胞静纤毛尖端的分子复合物将外部机械刺激转化为电信号，该电信号可以传播到中枢神经系统。听觉神经科学中一个关键的未知数是构成该复合物内离子传导转导通道的分子的身份。通过研究人类和小鼠的耳聋相关基因，研究人员认为TMC 1和TMC 2是强有力的机械传导通道候选者。不幸的是，具有足够的灵敏度和选择性来稳健地检测和定量机械转导复合物内的TMC或其他膜蛋白的生物化学方法尚未开发。基于抗体的方法，如蛋白质印迹和免疫细胞化学，经常遭受不良的特异性，低复用能力，和不精确的定量，从而防止它们提供准确的定量信息的组成的机械转导复合物。此外，高成本和有限的可用性的质量抗体，特别是膜蛋白，限制了筛选新的转导通道的候选人确定的其他手段。该提案的目标是表征毛细胞中机械转导复合物的组成。要做到这一点，我们将开发有针对性的蛋白质组学检测，以识别和准确定量转导蛋白，并测量它们的静纤毛位置和机械转导复合物的关联如何在耳聋和前庭功能障碍的小鼠模型中改变。我们将设计两套检测方法，这将使我们能够同时监测已知的机械转导复合物蛋白的浓度，以及检测在RNA水平上在毛细胞中鉴定的新的候选转导通道蛋白。这些测定与其他生物化学方法相结合，将使我们能够1）确定TMC 1，TMC 2或其他通道样膜蛋白是否定位于毛束，并能够与其他转导复合物蛋白相互作用，以及2）确定缺乏TMC 1和TMC 2的小鼠中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，这些测定将继续成为评估机械转导复合物的组成如何在各种小鼠模型中以及在毛细胞修复和再生的背景下在发育过程中发生变化的有价值的工具。

 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.