DOI: 10.5061/dryad.v15dv4256
title: Data from: LOXHD1 is indispensable for maintaining TMC1 auditory mechanosensitive channels at the site of force transmission

description: 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.
description: 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.<br>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:<br>SEM: Scanning
 Electron Microscopy<br>IF: Immunofluorescence<br>IHC: Inner
 Hair Cell<br>OHC: Outter Hair Cell<br>TL:
 Tip-Link<br>P7: Postnatal Day 7. The same "P (Postnatal Day)
 number" format was also used to describe other ages.<br>Homo:
 Homozygotes<br>WT: Wildtype<br>anti-HA: antibody against HA
 tag. The same "anti-epitope" format was also used to describe
 other immunostainings.
description: # 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, and position was also included in the title of each file. To have a better understanding of the data, we suggest future users of this dataset to read our publication. All IF data files are in a *.czi format. Please use Zeiss Zen software or Oxford Instruments Imaris software to open and analyze. All SEM and SUB-Immunogold-SEM files are in *.tiff or *.jpg format. We suggest using ImageJ to open and analyze. Please find the detailed folder and subfolder organization structure and description below: Figure 1h-k and Supp. Figure 2a-c----SEM pictures IHC: It contains 4 subfolders: ``` Figure 1h-k and Supp. Figure 2a-c----SEM pictures IHC/P7: It contains 2 Excel files and 23 subfolders. Each Excel file is a summary of the quantification of P7 stereocilia width for Loxhd1-WT or Delta homo. Each subfolder represents one IHC used for P7 stereocilia width measurement and contains 3 images that show the measurement for each row and the corresponding *.csv files with width data. Figure 1h-k and Supp. Figure 2a-c----SEM pictures IHC/P11: It contains 2 Excel files and 25 subfolders. Each Excel file is a summary of the quantification of P11 stereocilia width for Loxhd1-WT or Delta homo. Each subfolder represents one IHC used for P11 stereocilia width measurement and contains 3 images that show the measurement for each row and the corresponding *.csv files with width data. Figure 1h-k and Supp. Figure 2a-c----SEM pictures IHC/P21: It contains 2 Excel files and 21 subfolders. Each Excel file is a summary of the quantification of P21 stereocilia width for Loxhd1-WT or Delta homo. Each subfolder represents one IHC used for P21 stereocilia width measurement and contains 3 images that show the measurement for each row and the corresponding *.csv files with width data. Figure 1h-k and Supp. Figure 2a-c----SEM pictures IHC/P60: It contains Excel files and 12 subfolders. Each Excel file is a summary of the quantification of P60 stereocilia width for Loxhd1-WT or Delta homo. Each subfolder represents one IHC used for P60 stereocilia width measurement and contains 3 images that show the measurement for each row and the corresponding *.csv files with width data. ``` Figure 3----Airy scan IF pictures IHC: It contains 3 subfolders: ``` Figure 3----Airy scan IF pictures IHC/TMC1-HA: It contains 8 subfolders representing each independent immunofluorescent staining experiment, and each subfolder contains 4-27 Zeiss airyscan high-resolution microscopy files. Figure 3----Airy scan IF pictures IHC/TMC2-Myc: It contains 4 subfolders representing each independent immunofluorescent staining experiment, and each subfolder contains 4-7 Zeiss airyscan high-resolution microscopy files. Figure 3----Airy scan IF pictures IHC/TMIE-HA: It contains 2 subfolders representing each independent immunofluorescent staining experiment, and each subfolder contains 4-7 Zeiss airyscan high-resolution microscopy files. ``` Figure 4----SEM pictures IHC: It contains 3 subfolders organized based on mouse ages: ``` Figure 4----SEM pictures IHC/P7 IHC SUB anti-HA: It contains 3 subfolders organized by mouse genotypes, each containing 16-46 P7 IHC anti-HA SUB-Immunogold-SEM images. Figure 4----SEM pictures IHC/P11 IHC SUB anti-HA: It contains 3 subfolders organized by mouse genotypes, each containing 13-25 P11 IHC anti-HA SUB-Immunogold-SEM images. Figure 4----SEM pictures IHC/P21 IHC SUB anti-HA: It contains 4 subfolders organized by mouse genotypes, each containing 13-18 21 IHC anti-HA SUB-Immunogold-SEM images. ``` Figure 5ab----Airy scan IF pictures OHC: It contains 2 subfolders representing each independent immunofluorescent staining experiment, and each subfolder contains 15-27 Zeiss airyscan high-resolution microscopy files. Figure 5c-f----SEM pictures OHC: It contains 3 subfolders organized by mouse genotypes, and each contains 36-39 P7 OHC anti-HA SUB-Immunogold-SEM images. Figure 6b----Airy scan IF pictures IHC BAIAP2L2: It contains 1 Excel file which summarizes the BAIAP2L2 pattern-based quantification results and 2 subfolders representing each independent immunofluorescent staining experiment: ``` Figure 6b----Airy scan IF pictures IHC BAIAP2L2/HCIF51 P21 anti-BAIAP2L2: It contains 6 Zeiss airyscan high-resolution microscopy files and 8 subfolders for each cell used for the BAIAP2L2 pattern-based quantification, with channel composite and separate images in these subfolders. Figure 6b----Airy scan IF pictures IHC BAIAP2L2/HCIF57 P21 anti-BAIAP2L2: It contains 4 Zeiss airyscan high-resolution microscopy files and 13 subfolders for each cell used for the BAIAP2L2 pattern-based quantification, with channel composite and separate images in these subfolders. ``` Figure 6c-g----SEM pictures IHC BAIAP2L2: It contains 2 subfolders organized by mouse genotypes, each containing 33-39 P21 IHC anti-BAIAP2L2 SUB-Immunogold-SEM images. Figure 8a and Supp Figure 9----SEM pictures for detached TL: It contains 4 subfolders: ``` Figure 8a and Supp Figure 9----SEM pictures for detached TL/Detached TL SUB anti-BAIAP2L2 P21 IHC: It contains 14 P21 WT IHC anti-BAIAP2L2 SUB-Immunogold-SEM images used to quantify the percentage of detached tip-links with BAIAP2L2 gold beads. Figure 8a and Supp Figure 9----SEM pictures for detached TL/Detached TL SUB anti-HA P18 TMC1 HA/HA IHC: It contains 9 P18 TMC1-HA/HA IHC anti-HA SUB-Immunogold-SEM images used to quantify the percentage of detached tip-links with TMC1 gold beads. Figure 8a and Supp Figure 9----SEM pictures for detached TL/Detached TL SUB anti-HA P21 LOXHD1 HA/HA IHC: It contains 18 P21 LOXHD1-HA/HA IHC anti-HA SUB-Immunogold-SEM images used to quantify the percentage of detached tip-links with LOXHD1 gold beads. Figure 8a and Supp Figure 9----SEM pictures for detached TL/Detached TL SUB anti-HA P22 WT IHC: It contains 5 P22 WT IHC anti-HA SUB-Immunogold-SEM images used to quantify the percentage of detached tip-links with gold beads. ``` Supp Figure 2f----SEM pictures OHC: It contains 4 subfolders: ``` Supp Figure 2f----SEM pictures OHC/P7: It contains 1 Excel file and 21 subfolders. The Excel file is a summary of the quantification of P7 OHC row2 and row3 stereocilia length for Loxhd1-WT or Delta homo. Each subfolder represents one OHC used for P7 stereocilia length measurement and contains 3 images that show the measurement for row3, row2-row3, and row1-row2 length and the corresponding *.csv files with width data. Supp Figure 2f----SEM pictures OHC/P11: It contains 1 Excel file and 15 subfolders. The Excel file is a summary of the quantification of P11 OHC row2 and row3 stereocilia length for Loxhd1-WT or Delta homo. Each subfolder represents one OHC used for P11 stereocilia length measurement and contains 3 images that show the measurement for row3, row2-row3, and row1-row2 length and the corresponding *.csv files with width data. Supp Figure 2f----SEM pictures OHC/P21: It contains 1 Excel file and 14 subfolders. The Excel file is a summary of the quantification of P21 OHC row2 and row3 stereocilia length for Loxhd1-WT or Delta homo. Each subfolder represents one OHC used for P21 stereocilia length measurement and contains 2 images that show the measurement for row3, row2-row3 length, and the corresponding *.csv files with width data. Supp Figure 2f----SEM pictures OHC/P60: It contains 1 Excel file and 13 subfolders. The Excel file is a summary of the quantification of P60 OHC row2 and row3 stereocilia length for Loxhd1-WT or Delta homo. Each subfolder represents one OHC used for P60 stereocilia length measurement and contains 2 images that show the measurement for row3, row2-row3 length, and the corresponding *.csv files with width data. ``` Supp Figure 4c----Airy scan IF pictures IHC LHFPL5: It contains 2 subfolders representing each independent anti-LHFPL5 immunofluorescent P11 IHC staining experiment. Each subfolder contains 6-9 Zeiss airyscan high-resolution microscopy files.