Investigating the role of mechanotransduction machinery and the rootlet in modulating stereocilia motion.

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Project Summary/Abstract Auditory sensory hair cells transduce sound using a bundle of actin-filled cellular protrusions called stereocilia which are coupled together by tip links, top connectors and side connectors, and fluid forces. Activity of mechanoelectrical transduction (MET) channels, located near the tops of the shorter stereocilia, are modulated by the differential motion of stereocilia as conveyed via the tip link connection. Thus, stereocilia motion regulates the open probability of MET channels which drives communication of sound to the brain. The fundamental goal of this proposal is to characterize the mechanical underpinnings of the stereociliary connections that shape the force applied to MET channels. Many human deafness genes affect the molecular components of the MET machinery, including tip links and MET channels. The biophysical characteristics of components coupling the bundle dictate how they filter stimuli. Understanding the mechanical properties of coupling in mammalian hair bundles is essential to our understanding of hair cell function and hearing (Aim 1, 2). We hypothesize that channel open probability reflects tension in the tip link and that changes in hair bundle stiffness associated with channel gating will be present in mammalian cochlear hair bundles. To test these hypotheses, hair bundle mechanics will be investigated using newly developed technology that uses a ~1 µm diameter stiff probe to push on 1-3 stereocilia which will displace the remaining stereocilia through the connections coupling them. High- speed motion tracking will be used to reveal the rapid (<100 μs) movements of individual stereocilia in rows 1 and 2, allowing for characterization of stereociliary connectivity while whole cell voltage clamp provides the MET current response. The MET machinery and its regulation by calcium will be examined by raising or lowering open probability by changing intracellular free calcium levels, disrupting the tip link connections (Aim 1), and with channel blockade (Aim 2). The experiments in this proposal, their analyses, and the dissemination of their findings will serve as strong technical training for the applicant, providing the tools necessary to become an internationally competitive, rigorous, and independent research scientist. Professional development will be provided by experiences within the laboratory setting, the department, as well as by the environment and resources provided by Stanford University. Technical and career development are provided through excellent workshops, seminars, conferences, and collaborations with outstanding researchers inside and outside Stanford. The research training plan outlined in this proposal is designed to create a pathway to independence where both the technical expertise and foundational data will provide the cornerstone for independent work.