PROJECT SUMMARY/ABSTRACT Mechanosensitivity of the inner ear hair cells depends on actin-filled cellular projections known as stereocilia. We recently found that the stability of the stereocilia cytoskeleton depends on the constant influx of calcium ions through the mechano-electrical transduction (MET) channels that are located at the tips of stereocilia (Velez-Ortega, et al., eLife 2017). We showed that the blockage of MET channels or the breakage of the tip links that gate these channels leads to the selective shortening of transducing stereocilia in the bundle (i.e. only the middle and short row stereocilia harbor MET channels). Once the MET blockage is removed or the tip links regenerate, the shortened stereocilia regrow. An increase in intracellular calcium buffering also led to the selective remodeling of transducing stereocilia, indicating that calcium ions are the main component of the MET current regulating the stereocilia morphology. Stereocilia remodeling in response to variations in the resting MET current represents an activity- dependent plasticity of the stereocilia actin cytoskeleton, which may initiate profound changes in the hair bundle morphology in various types of hereditary or acquired hearing losses. Therefore, this project begins the search for the molecular players involved in the MET-dependent remodeling of the stereocilia cytoskeleton. Aim 1 will determine the MET-dependent changes in the actin incorporation into the stereocilia, potential actin treadmill, and actin isoform composition. Given that myosin XV delivers the elongation machinery to the tips of stereocilia, Aim 2 will test for the contribution of myosin XV isoforms in the MET-dependent regulation of the stereocilia height. This study is significant, because it may uncover the molecular mechanisms of the fine adjustments of the staircase architecture of the hair cell bundles. Moreover, stereocilia shortening?and perhaps eventual stereocilia disappearance?could occur after noise exposure (when the MET current is reduced due to tip link breakage) or in certain cases of congenital deafness (due to impaired MET current). Therefore, this study will expand our knowledge of the molecular mechanisms of various types of hearing loss.