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 tip link and remain functional, while TMC2-driven developmental channels do not. Since both tip links and TMC1 are present in hair bundles lacking LOXHD1, there is potential for reconnecting them and restoring hearing in this form of genetic deafness.

Methods
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 Microscope at the Stanford Nano Shared Facilities. The microscope is periodically calibrated for measurements using a SIRA-type calibration specimen for ultra-high-resolution modes with 2% error between 50- and 350-k magnification at our imaging settings.

Whole-mount immunofluorescence (IF) staining and imaging

The temporal bones were removed from the skull and put in a dish with ice-cold dissection buffer (1 × HBSS with Ca2+ and Mg2+, with osmolarity adjusted to 310 mOsm with D-glucose) as follows: The inner ears were then dissected out and transferred to a dish with a fixative (4% PFA in dissection buffer), a hole was poked on the bony cochlear shell at the apex, and the fixative was perfused slowly through round and oval windows. The perfused inner ears were incubated in the fixative for 40 min (20 min for the TMIE-HA samples) at RT. The fixed inner ear samples were transferred to new dishes with 1 × PBS, and the bony cochlear shell, stria vascularis, Reissner’s membrane, tectorial membrane, and the modiolus were sequentially removed. The finely dissected organs of Corti were transferred to a glass well plate with PBS containing 0.05% Triton X-100 and permeabilized for 40 min at RT. After permeabilization, the samples were blocked in PBS with 0.05% Tween 20 (PBST) containing 4% bovine serum albumin Fraction V (BSA) overnight or at least 6 hours at 4 °C. The tissues were then incubated with primary antibodies in PBST with 1% BSA (incubation buffer) overnight at 4°C. The samples were then washed 4 times, 5–10 min per wash, in the incubation buffer at RT. Subsequently, the tissues were incubated with fluorescent dye-conjugated secondary antibodies (see below) in the incubation buffer at RT for 1–2 hours. After one wash with the incubation buffer, the samples were incubated with fluorescent dye-conjugated phalloidin (see below) in incubation buffer at RT for 25 min. The samples were then washed thrice, 5–10 min per wash, with incubation buffer. For the TMIE-HA samples, the detergent of permeabilization buffer, blocking buffer, and incubation buffer was changed to 0.05% saponin. The glass well plate was on a horizontal shaker with a 60-rpm speed during permeabilization, incubation, and washing steps. For TMC1-HA, TMC2-MYC, TMIE-HA, and LOXHD1-HA, each experiment contained at least one parallel stained cochlea sample from a similar age mouse without any tag as a background control.
After washing, each sample was mounted on a glass slide under a coverslip by using ProLong Gold Antifade Mountant (Thermo Fisher Scientific). Z-stacks were captured using the Airyscan Super-resolution mode of a Zeiss LSM880 microscope with Objective C Plan-Apochromat 63x/1.4 Oil DIC M27 lens and Zen black software (Zeiss). The image acquisition parameters were determined as the best X*Y and Z axis resolution possible for the shortest wavelength used channel.

SUB-immunogold-SEM labeling, processing, and imaging

The fixed inner ear samples were transferred to new dishes with dissection buffer to dissect the organs of Corti out. Thereafter, the samples were transferred to 2-ml tubes with TBST (150-mM NaCl, 10-mM Tris-HCl, 0.05% Tween-20, pH 7.5) containing 0.5% Triton X-100, permeabilized for 1 hour at RT. After permeabilization, the samples were washed with TBST once and then blocked in TBST containing 4% BSA for at least 6 hours or overnight at 4°C. The samples were transferred to 0.3-ml PELCO mini vials (TED PELLA, #21441) with primary antibodies in TBST with 1% BSA overnight at 4 °C. Subsequently, the samples were transferred into 2-ml tubes, rinsed once, and washed thrice (15 min per wash) with 1% BSA TBST. The samples were then transferred to 0.3-ml PELCO mini vials with 10-nm gold conjugated goat anti-rabbit IgG (BBI: 1:200 in 1% BSA TBST) and incubated overnight at 4 °C. After 2nd antibody incubation, the samples were rinsed once and washed thrice (15 min per wash) with 1% BSA TBST in 2-ml tubes. The samples were then rinsed twice with 0.1-M sodium cacodylate buffer (pH 7.2) and fixed with 10% glutaraldehyde, 4% PFA in 0.1-M sodium cacodylate buffer for at least 24 hours at 4 °C, then washed with 0.1-M sodium cacodylate buffer, dehydrated in ethanol (30%, 50%, 75%, 95%, 100%, and 100%, 5-min incubations), and then processed to the critical drying point using Autosamdri-815A (Tousimis). The cochleae were mounted on studs using silver paint and coated with 2- to 3-nm of palladium (sputter coater EMS150TS, Electron Microscopy Sciences). The samples were imaged with a 5-kV accelerated voltage and a 100-pA beam current using a concentric backscattered electron detector on an FEI Magellan 400 XHR Field Emission SEM. The gold beads, characterized by their circular shape with an approximate diameter of 10 nm, were easily described as sources of backscattered electrons, and they were distinguishable from the signal originating from the stereocilia surface. 

Abbreviations:
SEM: Scanning Electron Microscopy
IF: Immunofluorescence
IHC: Inner Hair Cell
OHC: Outter Hair Cell
TL: Tip-Link
P7: Postnatal Day 7. The same "P (Postnatal Day) number" format was also used to describe other ages.
Homo: Homozygotes
WT: Wildtype
anti-HA: antibody against HA tag. The same "anti-epitope" format was also used to describe other immunostainings.