Role of Staircase Hair Bundle Morphology in Auditory Mechanotransduction

描述（由申请人提供）：虽然已经确定了负责内耳毛细胞机械感觉束结构完整性的蛋白质数量，但这些蛋白质中的大多数如何参与机电转导仍然未知。同样未知的是MET的缺乏或修饰是否有助于耳聋和/或由毛束蛋白突变引起的前庭疾病的发展。当前项目的目标是确定肌球蛋白xva基立体纤毛延伸复合物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运动结构域的隐性点突变阻止了该蛋白正常定位到立纤毛尖端，导致立纤毛异常短。根据我们的初步数据，出生后幼龄Myo15sh2/sh2小鼠的耳蜗外毛细胞具有许多倾斜定向的“尖端连接”，并且机械转导明显正常。相比之下，Myo15sh2/sh2内毛细胞具有同样短的立体纤毛，没有任何斜向的尖端连接，但有许多垂直于立体纤毛核心的“顶对顶”连接。尽管形态异常，但Myo15sh2/sh2内毛细胞具有明显的“野生型”纳安培级振幅的转导电流，但方向敏感性异常，没有快速的Ca2+依赖性失活，称为“快速适应”。该建议的中心假设是基于肌球蛋白XVa的立体纤毛延伸复合体不是毛细胞机械敏感性所必需的，但可能影响定向敏感性和机械转导的适应性。本研究将确定：1)肌凝蛋白XVa在毛束定向敏感性中的作用；2)肌球蛋白xva缺陷毛束中纤毛连接已知分子成分的定位；3) myosin XVa及其分子伙伴whirlin在转导电流快速适应中的作用。我的长期目标是了解毛细胞如何在正常和病理条件下获得和维持机械敏感性，这项研究代表了我向这一目标迈出的一步。除了对理解毛细胞机械转导的基本机制具有重要意义外，本研究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/或尖端连接恢复过程中的机械转导。最后，我们的研究将为人类遗传性耳聋DFNB3和DFNB31的动物模型——摇鼠2和旋鼠的毛细胞功能提供丰富的数据。这项研究与公共卫生有关，因为它确切地调查了内耳感觉细胞在正常和病理条件下如何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理解、预防和开发内耳感觉细胞发育异常的治疗方法，这种发育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.