Structural Basis of Outer Hair Cell Function

描述(由申请人提供)：在过去的二十年里，我们对外毛细胞(OHC)的结构和功能的了解有了很大的提高。例如，我们知道耳蜗的放大与OHC的快速运动有关，OHC的快速运动是由质膜中由数千个完整的膜蛋白prestin拷贝的构象变化产生的力驱动的。我们知道，这些力量被皮质细胞骨架利用并平行于OHC的长轴定向，皮质细胞骨架是一个由肌动蛋白细丝组成的二维各向异性网络，它位于整个OHC横向质膜的下方，我们知道这是可能的，因为质膜通过数千个结构性“支柱”与细胞骨架肌动蛋白细丝相连。然而，我们仍然不知道这些支柱的性质。我们不知道它们是如何工作的，也不知道它们是否在调节由预制素产生的力量方面起到任何作用。我们也不知道通过调节细胞骨架组织及其与质膜的支柱介导的连接来控制OHC长度和运动性的分子机制。这项建议的目的是通过解决以下具体目标来填补我们对OHC结构和功能的这些知识空白，这是我们寻求了解耳蜗功能、涉及OHC形状变化的故障以及对过度刺激做出反应的保护机制的关键一步：1)证明GLUT5、Dematin和Adducin是OHC支柱的主要组成部分；2)确定Profilin和Cofilin介导的信号诱导的肌动蛋白聚合和解聚在OHC长度和运动性调节中的作用；以及3)确定RhoA/Rock-PKC通路在OHC长度和运动性调节中的作用。Corti器官的结构和/或功能损害是困扰世界各地数百万人的感音神经性听力损失的主要原因。Corti器官的精致结构独特地适用于支持巨大范围的输入声压，而外毛细胞(OHC)的独特之处在于其身体长度直接影响该器官的微观力学的重要方面。例如，OHC长度的微小变化可以动态地调整细胞立体纤毛中承载的机械感觉器的操作点和/或基底膜的局部共振。OHC长度的变化被称为“OHC运动性”，被认为是耳蜗放大所必需的。尽管它们很重要，但我们对人权委员会的结构和功能的了解仍有许多空白。填补这些空白将是更好地理解耳蜗功能、涉及OHC形状变化的功能障碍以及过度刺激时激活的保护机制的重要一步。我们相信，实现本提案的目标将提供有关OHC的结构和细胞骨架在OHC长度和运动调节中的作用的基本信息，以及对正常人类听力和耳聋的基本机制的重要见解。

DESCRIPTION (provided by applicant): The last two decades witnessed a huge increase in our understanding of outer hair cell (OHC) structure and function. We know, for example, that cochlear amplification is associated with OHC fast motility, which is driven by forces generated in the plasma membrane by conformational changes in thousands of copies of the integral-membrane protein prestin. We know that these forces are harnessed and oriented parallel to the OHC's major axis by the cortical cytoskeleton, a two-dimensional anisotropic network of actin filaments cross-linked by spectrin that underlies the entire OHC lateral plasma membrane, and we know that that is possible because the plasma membrane is linked to cytoskeletal actin filaments by thousands of structural "pillars". We still don't know, however, the nature of these pillars. We don't know how they work, or whether they have any role in the regulation of the prestin-generated force. We don't know either the molecular mechanisms that control OHC length and motility by regulating cytoskeletal organization and its pillar-mediated connection to the plasma membrane. The goal of this proposal is to fill these gaps in our knowledge of OHC's structure and function, a crucial step in our quest to understand cochlear function, malfunctions that involve changes in OHC shape, and protective mechanisms in response to over stimulation, by addressing the following Specific Aims: 1) Demonstrate that proteins GLUT5, Dematin and Adducin are major components of OHC pillars, 2) Determine the role of actin polymerization and depolymerization induced by profilin- and cofilin-mediated signals in the regulation of OHC length and motility, and 3) Determine the role of the RhoA/ROCK-PKC pathway in the modulation of OHC length and motility. Structural and/or functional damage of the organ of Corti is the major cause of sensorineural hearing loss afflicting millions of people around the world. The exquisite architecture of the organ of Corti is uniquely adapted to support an enormous range of input sound pressures, and outer hair cells (OHCs) are unique in having their body length directly influencing important aspects of the micromechanics of this organ. For example, minute changes in OHC length can dynamically adjust the operating point of the mechano-sensory apparatus hosted in the cell stereocilia and/or the local resonance of the basilar membrane. Changes in OHC length, known as "OHC motility", are thought to be essential for cochlear amplification. Despite their importance, many gaps exist in our knowledge of the structure and function of OHCs. Filling these gaps would be an important step towards a better understanding of cochlear function, malfunctions that involve changes in OHC shape, and protective mechanisms activated in response to overstimulation. We are confident that accomplishing the aims of the present proposal will provide essential information about the structure of OHCs and the role of the cytoskeleton in the regulation of OHC length and motility, as well as critical insights into the basic mechanisms of both normal human hearing and deafness.