PROJECT SUMMARY/ABSTRACT Inner ear hair cells have actin-filled projections known as stereocilia on the apical surface. These stereocilia are microvilli-like structures that are arranged in highly organized rows of increasing heights known as “hair bundles”. The stereocilia possess mechano-electrical transduction (MET) channels at their tips where sound-induced vibration and head motion are converted into electrical signals. Previously, Velez-Ortega et al. (Elife, 2017) demonstrated that the actin cytoskeleton in stereocilia exhibits MET-dependent remodeling as the stability of the stereocilia was crucially affected by alterations to the constant entry of calcium through MET channels (which are partially open at rest). The actin cytoskeleton within stereocilia is comprised of two actin isoforms, beta and gamma, which are produced by two separate genes. Prior studies have elucidated that although hair cells can develop hair bundles in the absence of either actin isoform, these bundles quickly degenerate resulting in hearing loss (Perrin et al., PLoS Genet, 2010). Thus, it is apparent that both actin isoforms are required for proper stereocilia stability and maintenance. Here, we hypothesize that a specific mutation in the gamma actin gene leads to progressive hearing loss (in both human and mouse models) through the exaggeration of MET- dependent remodeling of the aforementioned actin cytoskeleton. To investigate this hypothesis, Aim 1 will analyze MET-dependent changes to the morphology of stereocilia through scanning electron microscopy after exposing gamma actin mutant mice and littermate controls to MET channel blockers or an increase in intracellular calcium buffering. Given that beta and gamma actin exhibit different rates of polymerization and depolymerization in the presence of calcium (Bergeron et al., J Biol Chem, 2010), Aim 2 will evaluate how MET-dependent alterations affect the ratio of the beta and gamma actin isoforms in both the stereocilia and cuticular plate of auditory hair cells in mutant mice and littermate controls using immunohistochemistry and high-resolution confocal microscopy. Overall, results from this supplement may elucidate the molecular mechanism that triggers stereocilia degeneration in sensorineural progressive hearing loss when a mutation in the gamma actin gene is present.