Cytoskeletal Regulation of Outer Hair Cell Motility

描述(申请人提供)：耳蜗外毛细胞是哺乳动物内耳中负责信号放大和频率辨别的独特的感觉-运动细胞。强有力的证据表明，内耳毛细胞对化学或电刺激的运动性反应为“耳蜗放大器”提供了机械效应器，这一活跃机制使声诱导的基底膜运动的峰值增加了近100倍。我们的长期目标是了解OHC运动调节的分子机制。基于以前对非听觉细胞群体的研究，我们假设OHC的运动是由细胞骨架的结构和功能变化调节的。最近发现的小Rho GTP酶RhoA、Rac 1和CDC42是乙酰胆碱(ACh)激活的调节OHC运动的信号通路的中介，现在提供了在分子和细胞水平上解决这一假说的机会。此外，我们的初步结果表明，属于ACh激活的信号通路的Rho GTP酶的一些下游效应对OHC的机械动态平衡是重要的。由于这些下游效应分子介导了Rho促进的过程，如肌动蛋白-血影蛋白结合和质膜-细胞骨架相互作用，这些结果为进一步研究细胞骨架在OHC运动调节中的作用提供了坚实的基础。在本方案中，我们将结合电生理、分子生物学、显微镜和图像分析技术，以：1.确定参与OHC运动调节的Rho GTP酶的下游效应分子；2.确定Rho GTP酶与其下游效应分子之间的连接模式；3.建立ACh诱导的OHC细胞骨架的结构变化。我们相信，实现这些目标将提供关于细胞骨架在OHC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): 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.