NRSA Fellowship for Dragich: Mechanisms of Tip Link Tensioning in Mammalian Auditory Hair Cells

Abstract: Mammalian auditory hair cells detect sound through deflections of stereocilia that are organized in precise staircase-like bundles and interconnected by extracellular tip links. Sound-induced deflections modulate the tension of tip links and convey these forces to mechano-electrical transduction (MET) channels located at the tips of the shorter rows of stereocilia. Even at rest, there is a certain amount of tension on the tip links, which ensures detection of the softest sounds and results in some resting amount of MET current continuously entering the cell. We and other groups demonstrated that this resting MET current regulates the height of transducing stereocilia, thereby providing a plausible mechanism for long-term maintenance of the shape of stereocilia bundle. Furthermore, my recent study revealed that MET-dependent retraction of stereocilia in mammalian auditory hair cells increases the tension within MET machinery, which could only occur if, in contrast to the classical models, the upper end of the tip link is not freely moved by myosin motors but instead somehow locked to the stereocilia core (Dragich et. al., in review). The proposed project will explore a potential molecular mechanism of this phenomenon. We hypothesize that Gα-Interacting Protein, C-terminus-3 (GIPC3) is involved in locking the upper end of the tip link to the stereocilia actin core. Several mutations in GIPC3 have been linked to hearing loss in humans, but the exact function of this protein in the mammalian cochlea is yet unknown. Data from our collaborator (Dr. Craig Vander Kooi) show that GIPC3 interacts with myosin VI (MYO6) and the upper tip link density (UTLD) proteins, cadherin-23 (CDH23) and potentially myosin VIIa (MYO7a). My preliminary data also show that GIPC3 deficiency results in the loss of resting MET current and “slipping adaptation” of the MET responses in cochlear outer hair cells of mice carrying the p.W301X mutation in Gipc3, recapitulating a known human deafness. In this project, we will use this Gipc3W301X mouse strain as well as Gipc3 knockout strain to determine: (a) the role of GIPC3 in regulating the tension within the MET machinery and adaptation in mammalian auditory hair cells; (b) the role of GIPC3 in UTLD assembly and maintenance; and (c) the potential for restoring wildtype MET responses in Gipc3-deficient mice. This project will not only identify the specific role of GIPC3 in the mammalian auditory hair cells but also elucidate the mechanisms behind the maintenance of stereocilia bundle structure and tensioning of the MET machinery. Approaching this project using electrophysiology, advanced electron microscopy, and cell biology techniques will help me to develop a unique set of scientific skills in preparation for a career as a future principal investigator in basic auditory research.