Structural Basis of Outer Hair Cell Function

描述（由申请人提供）：过去二十年见证了我们对外毛细胞（OHC）结构和功能的理解的巨大增长。例如，我们知道耳蜗放大与OHC快速运动有关，OHC快速运动是由质膜中数千个完整膜蛋白普雷斯廷拷贝的构象变化产生的力驱动的。我们知道，这些力量是利用和定向平行于OHC的主轴由皮质细胞骨架，一个二维各向异性网络的肌动蛋白丝交联的血影蛋白，在整个OHC侧质膜，我们知道，这是可能的，因为质膜是连接到细胞骨架肌动蛋白丝的数千个结构的“支柱”。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prestin产生的力量中起作用。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。本提案的目标是填补我们对OHC结构和功能的了解中的这些空白，这是我们寻求了解耳蜗功能的关键一步，涉及OHC形状变化的故障以及对过度刺激的保护机制，通过解决以下具体目标：1）证明蛋白质GLUT 5、脱蛋白和内收蛋白是OHC柱的主要组分，2）确定由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，和3）确定RhoA/ROCK-PKC途径在调节OHC长度和运动性中的作用。Corti器官的结构和/或功能损伤是影响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合于支持巨大范围的输入声压，并且外毛细胞（OHC）的独特之处在于其体长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节细胞静纤毛和/或基底膜的局部共振中托管的机械感觉装置的操作点。OHC长度的变化，称为“OHC运动性”，被认为是耳蜗放大所必需的。尽管人道主义事务协调员十分重要，但我们对它们的结构和职能的了解仍存在许多空白。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响应过度刺激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结构和细胞骨架在OHC长度和运动性调节中的作用的重要信息，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.