Role of Staircase Hair Bundle Morphology in Auditory Mechanotransduction

描述（由申请人提供）：虽然已经鉴定了负责内耳毛细胞的机械感觉束的结构完整性的蛋白质的数量，但这些蛋白质中的大多数如何有助于机械-电转导仍然是未知的。同样未知的是MET的缺乏或修饰是否有助于由毛束蛋白突变引起的耳聋和/或前庭疾病的发展。目前的项目的目标是确定肌球蛋白Xa为基础的静纤毛延长复合物在机械转导的作用。在纯合子shaker 2小鼠（Myo 15 sh 2/sh 2）中，肌球蛋白XVa运动域中的隐性点突变阻止了该蛋白质在静纤毛尖端的正常定位，导致静纤毛异常短。根据我们的初步数据，耳蜗外毛细胞的年轻出生后Myo 15 sh 2/sh 2小鼠具有许多倾斜定向的“尖端链接”和明显正常的机械转导。相比之下，Myo 15 sh 2/sh 2内毛细胞具有同样短的静纤毛，没有任何倾斜定向的尖端链接，但具有许多垂直于静纤毛核心的“顶到顶”链接。尽管它们的异常形态，Myo 15 sh 2/sh 2内毛细胞具有突出的转导电流，具有“野生型”纳安级幅度，但方向敏感性异常，并且没有快速的Ca 2+依赖性失活，称为“快速适应”。该提议的中心假设是基于肌球蛋白XVa的静纤毛延伸复合物不是毛细胞的机械敏感性所需的，但可能影响机械转导的方向敏感性和适应性。这项研究将确定：1）肌球蛋白XVa在毛束的方向敏感性中的作用; 2）静纤毛连接的已知分子组分在肌球蛋白XVa缺陷的毛束中的定位; 3）肌球蛋白XVa及其分子伴侣whirling在转导电流的快速适应中的作用。这项研究代表了我的长期目标，即了解毛细胞在正常和病理条件下如何获得和维持机械敏感性。除了理解毛细胞机械转导的基本机制是重要的，这项研究将建立如何发展异常的静纤毛生长可能会影响转导机制。该项目也将是第一个研究哺乳动物耳蜗毛细胞毛束形态和/或尖端链接恢复过程中的机械转导。最后，我们的研究将提供大量关于shaker 2和whirler小鼠毛细胞功能的数据，这些小鼠是人类DFNB 3和DFNB 31遗传性耳聋的动物模型。这项研究与公共卫生有关，因为它确切地研究了内耳的感觉细胞如何在正常和病理条件下获得和维持机械敏感性。我们的实验结果将帮助科学家更好地了解，预防和开发内耳感觉细胞发育异常的治疗方法，这些异常导致先天性耳聋。

DESCRIPTION (provided by applicant): While number of proteins responsible for structural integrity of the mechanosensory bundle of the inner ear hair cells has been identified, how most of these proteins contribute to mechano-electrical transduction is still unknown. Also unknown is whether the lack or modification of MET contributes to the development of deafness and/or vestibular disorders that result from mutations of hair bundle proteins. The goal of the current project is to determine the role of the myosin XVa-based stereocilia elongation complex in mechanotransduction. In homozygous shaker 2 mice (Myo15sh2/sh2), a recessive point mutation in the motor domain of myosin XVa prevents normal localization of this protein to the tips of stereocilia, resulting in abnormally short stereocilia. According to our preliminary data, cochlear outer hair cells of young postnatal Myo15sh2/sh2 mice possess numerous obliquely oriented "tip links" and apparently normal mechanotransduction. In contrast, Myo15sh2/sh2 inner hair cells have equally short stereocilia without any obliquely oriented tip links, but with numerous "top-to-top" links perpendicular to the core of stereocilia. In spite of their abnormal morphology, Myo15sh2/sh2 inner hair cells have a prominent transduction current with "wild type" nanoampere-scale amplitude but abnormal directional sensitivity and no rapid Ca2+dependent deactivation, known as "fast adaptation". The central hypothesis of the proposal is that the myosin XVa- based stereocilia elongation complex is not required for mechanosensitivity of hair cells but may affect directional sensitivity and adaptation of mechanotransduction. This study will determine: 1) the role of myosin XVa in directional sensitivity of the hair bundle; 2) localization of known molecular components of stereocilia links in the myosin XVa-deficient hair bundles; 3) the role of myosin XVa and its molecular partner, whirlin in the fast adaptation of the transduction current. This study represents a step toward my long-term goal to understand how the hair cells acquire and maintain mechanosensitivity in normal and pathological conditions. Apart from being important for understanding the basic mechanisms of hair cell mechanotransduction, this study will establish how developmental abnormalities of stereocilia growth may affect the transduction machinery. This project will also be the first to study mechanotransduction during restoration of the hair bundle morphology and/or tip links in mammalian cochlear hair cells. Finally, our study will provide a wealth of data on hair cell function in shaker 2 and whirler mice, the animal models for DFNB3 and DFNB31 hereditary deafness in humans. This research is relevant to public health because it investigates exactly how sensory cells of the inner ear acquire and maintain mechanosensitivity in normal and pathological conditions. Our experimental results will help scientists to better understand, prevent, and develop treatments for developmental abnormalities in the sensory cells of the inner ear, which lead to congenital deafness.