The long-term objective of our research is to understand the molecular mechanisms involved in the regulation of outer hair cell (OHC) motility and cochlear amplification, a critical knowledge for developing strategies aimed at preventing cochlear damage and sensorineural hearing loss. We have identified Rho GTPases (RhoA, Rac and Cdc42) as key signaling molecules in the homeostatic regulation of prestindependent and prestin-independent OHC motility. Homeostatic control would be ultimately achieved through Rho-mediated changes in actin-filament dynamics, in the integrity of the actin-spectrin network, in the plasma membrane-cytoskeleton connection, or in all of the above in a complex network of molecular reactions at different levels of the cell cortex. Our previous studies assigned an important role to the RhoA/ROCK/LIMK2/cofilin and RhoA/ROCK/adducin signaling cascades in the regulation of OHC motility, and suggested that Rac1/Cdc42-mediated signals could also be crucial for this process. These results have led us to fully investigate the molecular signaling pathways underlying cytoskeleton-mediated regulation of OHC motility towards an understanding of the molecular basis of OHCs' mechanical homeostasis. As a short-term objective, we propose to use electrophysiological, pharmacological, microscopy and molecular biology techniques [unreadable]as well as a novel methodological approach and an innovative image analysis system based on recordings with an ultra high-speed video camera[unreadable] to elucidate the regulatory pathways of prestin-dependent and prestin-independent OHC motility at low and auditory frequencies, by addressing the following Specific Aims: 1 - Determine the role of the Rac1-Cdc42/PAK1/LIMK1/cofilin signaling pathway in the regulation of OHC motility, and its interaction with the RhoA/ROCK/LIMK2/cofilin pathway. 2 - Determine the role of the RhoA/mDia1/profilin signaling pathway in the regulation of OHC motility; and 3 - Determine the role of the RhoA/ROCK-PKC/adducin signaling pathway in the regulation of OHC motility. We are confident that accomplishing these aims will provide essential information about the role of the cytoskeleton in the regulation of OHC motility. Damage of OHCs and the mechanism of cochlear amplification is the most common cause of sensorineural hearing loss, a condition afflicting millions of people around the world. Understanding the cell and molecular basis of OHC motility and cochlear amplification will provide critical insights into the basic mechanisms of both normal human hearing and deafness, a critical step for developing clinical strategies aimed at preventing or ameliorating sensorineural hearing loss.