Role of Staircase Hair Bundle Morphology in Auditory Mechanotransduction

描述(申请人提供)：虽然许多负责内耳毛细胞机械感觉束结构完整性的蛋白质已经确定，但这些蛋白质中的大多数如何对机械-电转导起作用仍是未知的。同样未知的是，MET的缺失或修饰是否会导致耳聋和/或前庭疾病的发生，这些疾病是由毛束蛋白的突变引起的。本项目的目标是确定基于肌球蛋白XVA的立体纤毛延长复合体在机械转导中的作用。在纯合子Shaker 2小鼠(Myo15sh2/Sh2)中，肌球蛋白XVA运动域的隐性点突变阻止了该蛋白在立体纤毛尖端的正常定位，导致异常短的立体纤毛。根据我们的初步数据，出生后幼年的Myo15sh2/Sh2小鼠的耳蜗外毛细胞具有许多倾斜定向的“尖端连接”和明显正常的机械转导。相比之下，Myo15sh2/Sh2内毛细胞有同样短的立体纤毛，没有任何倾斜定向的尖端链接，但有许多垂直于立体纤毛核心的“自上而上”的链接。尽管其形态异常，但Myo15sh2/Sh2内毛细胞仍具有显著的传导电流，其幅度为纳安级，但方向敏感度异常，没有钙离子依赖的快速失活，被称为“快速适应”。该方案的中心假设是，基于肌球蛋白XVA的立体纤毛延长复合体不是毛细胞机械敏感性所必需的，但可能影响机械转导的方向敏感性和适应性。本研究将确定：1)肌球蛋白XVA在毛束方向敏感性中的作用；2)肌球蛋白XVA缺失毛束中已知的纤毛连接分子成分的定位；3)肌球蛋白XVA及其分子伴侣Whirlin在快速适应转导电流中的作用。这项研究代表着我朝着了解毛细胞在正常和病理条件下如何获得和保持机械敏感性的长期目标迈出了一步。这项研究除了对了解毛细胞机械转导的基本机制很重要外，还将确定立体纤毛生长的发育异常如何影响转导机制。该项目还将首次研究在恢复哺乳动物耳蜗毛细胞的毛束形态和/或尖端连接过程中的机械转导。最后，我们的研究将提供大量有关Shaker 2和Whirler小鼠毛细胞功能的数据，这两种动物模型是人类遗传性耳聋的动物模型。这项研究与公共健康有关，因为它准确地调查了内耳的感觉细胞在正常和病理条件下如何获得和保持机械敏感性。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，这种异常会导致先天性耳聋。

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.