Cytoskeletal Regulation of Outer Hair Cell Motility

描述（由申请人提供）：耳蜗外毛细胞（OHC）是哺乳动物内耳中负责信号放大和频率辨别的独特感觉运动细胞。强有力的证据表明，OHC对化学或电刺激的运动反应为“耳蜗放大器”提供了机械效应器，这是一种主动机制，可将声音诱导的基底膜运动的峰值提高约100倍。我们的长期目标是了解参与OHC运动调节的分子机制。基于以前在非听觉细胞群的研究，我们假设OHC运动是由细胞骨架的结构和功能的变化。最近鉴定的小Rho GTPases RhoA，Rac 1和Cdc 42作为调节OHC运动的乙酰胆碱（ACh）激活的信号通路中的介质，现在提供了在分子和细胞水平上解决这一假设的机会。此外，我们的初步研究结果表明，一些下游效应的Rho GTP酶-属于乙酰胆碱激活的信号通路-是重要的OHC的机械稳态。由于这些下游效应介导的Rho促进的过程，如肌动蛋白血影蛋白协会和质膜细胞骨架的相互作用，这些结果提供了一个坚实的基础，为进一步调查的作用，细胞骨架在OHC运动的调节。在这项研究中，我们将结合电生理学、分子生物学、显微镜和图像分析技术，以便：1。鉴定参与OHC运动调节的Rho GTP酶的下游效应子，2.确定Rho GTP酶与其下游效应物之间的连接模式，以及3.建立乙酰胆碱诱导的OHC细胞骨架结构变化。我们相信，实现这些目标将提供有关细胞骨架在OHC运动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): Cochlear outer hair cells (OHCs) are unique sensory-motor cells responsible for signal amplification and frequency discrimination in the mammalian inner ear. Strong evidence suggests that the motile response of OHCs to chemical or electrical stimulation provides the mechanical effectors to the "cochlear amplifier," the active mechanism that enhances approximately 100-fold the peak of the sound-induced basilar membrane motion. Our long-term goal is to understand the molecular mechanisms involved in the regulation of OHC motility. Based on previous studies in non-auditory cell populations, we hypothesize that OHC motility is regulated by structural and functional changes in the cytoskeleton. The recent identification of the small Rho GTPases RhoA, Rac 1 and Cdc42 as mediators in an acetyicholine (ACh)-activated signaling pathway that modulates OHC motility, offers now the opportunity to address this hypothesis at molecular and cellular levels. Moreover, our preliminary results indicate that some downstream effectors of Rho GTPases -belonging to the ACh-activated signaling pathway- are important for OHCs' mechanical homeostasis. Since these downstream effectors mediate Rho-promoted processes such as actin-spectrin association and plasma membrane-cytoskeleton interaction, these results provide a solid foundation for further investigation of the role of the cytoskeleton in the regulation of OHC motility. In this proposal, we will use a combination of electrophysiological, molecular biology, microscopical and image analysis techniques in order to: 1. Identify downstream effectors of Rho GTPases involved in the regulation of OHC motility, 2. Determine the connection pattern between Rho GTPases and their downstream effectors, and 3. Establish the structural changes induced by ACh on the OHC cytoskeleton. We are confident that accomplishing these aims will provide essential information about the role of the cytoskeleton in the regulation of OHC motility as well as critical insights into the basic mechanisms of both normal human hearing and deafness.