Hair Bundle Structure and Dynamics

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.

Grant Number: 5R01DC011034-15
NIH Institute/Center: NIH
Principal Investigator: Peter Barr-Gillespie