PROJECT SUMMARY Hearing depends on stereocilia, which are protrusions on sensory cells in the inner ear that exist as membrane-enclosed bundles of actin filaments. If stereocilia are malformed during development or improperly maintained, then people have congenital deafness or progressive hearing loss. The composition of the stereocilia actin core is well-known and consists of a highly stable bundle of parallel actin filaments which form a stiff rod. The core dimensions dictate the length and width of stereocilia. The addition or loss of actin from the distal end of the core drives growth during development and shortening that occurs during some forms of progressive hearing loss. Our proposed experiments focus on elucidating how actin is regulated at the tips of developing stereocilia. Similar regulatory mechanisms may repair stereocilia during aging or following noise damage. Understanding these mechanisms is therefore expected to be essential for understanding many forms of hearing loss. This proposal builds on work done during the previous granting period, where we discovered differences in actin turnover at the tips of stereocilia in mechanotransducing rows. We now present evidence of a novel population of short actin filaments that exist at stereocilia tips but are not part of the actin core. Experiments in mouse models will document the key characteristics of these tip filaments, how they are regulated at the tips of mechanotransducing and non-mechanotransducing stereocilia, and how they contribute to stereocilia development and maintenance.