# Elucidating the architecture and composition of the hair cell mechanotransduction complex

> **NIH NIH F32** · OREGON HEALTH & SCIENCE UNIVERSITY · 2020 · $64,926

## Abstract

Project Summary
Mechanotransduction machinery located at the tips of hair bundles in the inner ear are responsible for our
sensations of movement and sound. Each bundle is composed of ~100 stereocilia organized in a staircase array
that are connected by tip-links, extracellular protein filaments composed of protocadherin 15 (PCDH15) and
cadherin-23 (CDH23). Deflection of the hair bundle by sound and fluid movement leads to opening of the
mechanotransduction channel (MEC) complex located at the lower tip-link insertion site, resulting in an electrical
signal. There are three putative components of the MEC complex aside from the tip-link proteins: the lipoma
HMGIC fusion partner-like 5 protein LHFPL5 (also known as TMHS), the transmembrane inner ear protein TMIE,
and the transmembrane-like channel proteins TMC1/2, which are the likely pore-forming subunits of the complex.
Although it is known that these proteins interact to form a mechanotransduction complex necessary for hearing,
the precise composition, stoichiometry, and structure of this complex remain elusive. The goal of this proposal
is to elucidate the composition and architecture of the MEC complex using single-molecule techniques and cryo-
electron tomography. The main challenge of the proposed work is the low abundance of the MEC complex in
native tissue. We have developed high affinity antibodies against three of the four MEC complex components
that will be key elements in our approach to overcome this issue. In the first aim, I will utilize these antibodies to
pull-down and detect the MEC complex from mouse cochlear hair cells using a single molecule pulldown method
to assess MEC complex composition. Photobleaching and single-molecule quantitation experiments will allow
me to determine MEC complex stoichiometry and measure the number of MEC complexes per cochlea. In the
second aim of this proposal, I will elucidate the architecture of the MEC complex using cryo-electron tomography.
I have developed a method to prepare cryo-EM grids with stereocilia in vitreous ice and obtained promising
preliminary tomograms. I will use our antibodies conjugated to quantum dots to label the MEC complex on cryo-
EM grids and determine a 3D structure of the complex. Illuminating the architecture and composition of the MEC
complex will provide valuable insight into the elusive mechanism of hair cell mechanotransduction and open
doors for the development of new therapeutics and treatment strategies for deaf individuals.

## Key facts

- **NIH application ID:** 9852293
- **Project number:** 5F32DC017894-02
- **Recipient organization:** OREGON HEALTH & SCIENCE UNIVERSITY
- **Principal Investigator:** Sarah A Clark
- **Activity code:** F32 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $64,926
- **Award type:** 5
- **Project period:** 2019-01-01 → 2021-12-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9852293

## Citation

> US National Institutes of Health, RePORTER application 9852293, Elucidating the architecture and composition of the hair cell mechanotransduction complex (5F32DC017894-02). Retrieved via AI Analytics 2026-05-21 from https://api.ai-analytics.org/grant/nih/9852293. Licensed CC0.

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