LOXHD1 is indispensable for maintaining TMC1 auditory mechanosensitive channels at the site of force transmission

Hearing begins in hair cells with the mechanical activation of ion channels in the hair bundle. This bundle consists of stereocilia arranged in rows of increasing heights, connected by tip links that transmit sound-induced forces to shorter stereocilia tips. Auditory mechanotransduction channel complexes, composed of proteins TMC1/2, TMIE, CIB2, and LHFPL5, are located at the tips of shorter stereocilia. While most components can interact with the tip link in vitro, their ability to maintain the channel complexes at the tip link in vivo is uncertain. Here we show that an additional component, LOXHD1, is essential for keeping TMC1-pore forming subunits at the tip link but is dispensable for TMC2. Using SUB-immunogold-SEM, we showed that TMC1 localizes near the tip link but mislocalizes without LOXHD1. LOXHD1 selectively interacts with TMC1, CIB2, LHFPL5, and tip-link protein PCDH15. Our results demonstrate that TMC1-driven mature auditory channels require LOXHD1 to stay connected to the ..., Scanning electron microscopy (SEM)
SEM sample preparation and imaging were conducted as follows: The inner ears were isolated in dissection buffer (DB) (1 X HBSS with Ca2+ and Mg2+, with osmolarity adjusted to 310 mOsm with D-glucose) and fixed in 4% PFA diluted from 32% stock in this buffer for 30 min at RT. The inner ears were then dissected to remove bone structures, the stria vascularis, and Reissner’s and tectorial membranes. The samples were refixed in 2.5% glutaraldehyde and 4% PFA in DB overnight at 4 °C, washed, dehydrated in ethanol (30%, 50%, 75%, 95%, 100%, and 100%, 5 min incubations), and processed to the critical drying point using Autosamdri-815A (Tousimis). The cochleae were mounted on studs using silver paint and coated with 4-nm palladium (sputter coater EMS150TS, Electron Microscopy Sciences). The samples were imaged with a 5-kV accelerated voltage and a 13-pA beam current using a secondary electron detector on an FEI Magellan 400 XHR Field Emission Scanning Electron..., , # Raw data of the publication: \"LOXHD1 is indispensable for maintaining TMC1 auditory mechanosensitive channels at the site of force transmission.

\[Raw data of the publication: \"LOXHD1 is indispensable for maintaining TMC1 auditory mechanosensitive channels at the site of force transmission.\"]([https://doi.org/10.5061/dryad.v15dv4256](https://doi.org/10.5061/dryad.v15dv4256))

This dataset includes raw scanning electron microscopy (SEM) images, airyscan immunofluorescent (IF) microscopy images, and SUB-Immunogold-SEM images acquired and analyzed for Figures of the publication: \"LOXHD1 is indispensable for maintaining TMC1 auditory mechanosensitive channels at the site of force transmission.\"

#### Description of the data and file structure

The data are organized in folders following the names of the Figures in the publication. Subfolders are used for better data organization based on ages, genotypes, and ID of independent experiments. Information on animal ages, genotypes, labeling, ...