Project Summary The long-term goal of this laboratory is reveal the structure of the hair cell's mechanosensitive organelle, the hair bundle, and to determine how structural features of the bundle are responsible for its mechanotransduction function. In this proposal, we focus on the under-appreciated process of actin-core widening, which occurs in stereocilia during development of the bundle. We utilize inner hair cells of the mouse cochlea as our model system; not only do their stereocilia rows show unique diameters and lengths, but all of the tools we deploy in studying bundle function can be deployed with these cells. In Aim 1, we will extend our cryo-electron tomography program to developing inner hair cells, asking specifically when and where peripheral actin filaments are added to the actin core during the widening process. In Aim 2, we will isolate inner hair cells marked with GFP using Fgf8-Gfp;Atoh1-Cre mice, and then subject them to protein mass spectrometry. In conjunction with our collaborators at the Pacific Northwest National Laboratory, we have defined the proteomes of single inner hair cells isolated by fluorescence-activated cell sorting. While we will not analyze single cells in the present project, we will exploit the sensitivity of the new techniques to analyze small pools of cells isolated from a specific region of the cochlea at precise development times. In addition, we will isolate inner hair cell stereocilia at the same time points using pipette aspiration, allowing us to also determine the stereocilia proteome over development. By comparing the whole-cell and stereocilia proteomics data, we will track when each protein enters stereocilia, and mine these data to identify new candidates for complexes that control stereocilia widening. Finally, in Aim 3, we will study three mutant mouse lines (Espn, Capzb, Grxcr1) that have thin stereocilia, using the techniques developed for Aims 1 and 2 to characterize the structural and temporal features of stereocilia. Together, the experiments developed in this project will allow us to determine how the hair cell widens its stereocilia, which is one of the critical steps in development of the hair bundle.