DOI: 10.5061/dryad.4tmpg4fkw
title: Data from: Complexes of vertebrate TMC1/2 and CIB2/3 proteins form hair-cell mechanotransduction cation channels

description: Calcium and integrin-binding protein 2 (CIB2) and CIB3 bind to
 transmembrane channel-like 1 (TMC1) and TMC2, the pore-forming subunits of
 the inner-ear mechano-electrical transduction (MET) apparatus. Whether
 these interactions are functionally relevant across mechanosensory organs
 and vertebrate species is unclear. Here we show that both CIB2 and CIB3
 can form heteromeric complexes with TMC1 and TMC2 and are integral for MET
 function in mouse cochlea and vestibular end organs as well as in
 zebrafish inner ear and lateral line. Our AlphaFold 2 models suggest that
 vertebrate CIB proteins can simultaneously interact with at least two
 cytoplasmic domains of TMC1 and TMC2 as validated using nuclear magnetic
 resonance spectroscopy of TMC1 fragments interacting with CIB2 and CIB3.
 Molecular dynamics simulations of TMC1/2 complexes with CIB2/3 predict
 that TMCs are structurally stabilized by CIB proteins to form cation
 channels. Overall, our work demonstrates that intact CIB2/3 and TMC1/2
 complexes are integral to hair-cell MET function in vertebrate
 mechanosensory epithelia.
description: <strong>Labeling zebrafish hair cells with FM 1-43, FM
 4-64, and FM 1-43FX</strong> To label
 lateral-line hair cells with FM 1-43, larvae were immersed in 1 µM FM 1-43
 (ThermoFisher, T3163) in E3 for 30 s, and then washed 3 times in E3. After
 washing, larvae were mounted in 1% low melt agarose containing an
 anesthetic, 0.02% tricaine methanesulfonate (Syndel, ANADA 200-226). FM
 1-43 labeled hair cells were then imaged on an A1 Nikon upright
 laser-scanning confocal microscope with a 60× 1.0 NA water objective. FIJI
 was used to process image Z-stacks. To estimate the number of hair cells
 per neuromast, laser scanning DIC images were used to count the number of
 hair bundles per neuromast. To quantify FM 1-43 label, a 5 µm circular
 region of interest (ROI) was placed on the soma of each hair cell with a
 hair bundle in the DIC image. These ROIs were used to quantify the mean FM
 1-43 intensity per hair cell. These intensity values were averaged to
 calculate the average FM 1-43 intensity per neuromast.
 FM 4-64 (ThermoFisher, T13320) was used to label hair cells in
 the crista within the zebrafish inner ear. For this
 labeling, <em>Tg(myo6b:memGCaMP6s)<sup>idc1</sup></em>larvae were mounted on their side on Sylgard chamber in E3 embryo media containing 0.02% MESAB. Approximately 2-3 nL of 10 µM FM 4-64 in 0.1 M KCl were injected into the otic capsule as previously described (Smith et al., 2020). FM 4-64 labeled hair cells of the medial crista were then imaged on an A1 Nikon upright laser-scanning confocal microscope with a 25× 1.10 NA or 60× 1.0 NA water objective. FIJI was used to process image Z-stacks. To assess tall versus short hair cells in each crista <em>memGCaMP6s</em> label was used. To correlate hair bundle orientation in lateral line hair cells with intact mechanotransduction, larvae were immersed in 3 µM FM 1-43FX (ThermoFisher, F25255) in E3 for 30 s, and then washed 3 times in E3. After washing, larvae were fixed with 4% paraformaldehyde in PBS at 4°C overnight. After fixation, larvae were washed 4 × 5 min in 0.1% Tween in PBS (PBST). Larvae were then placed in blocking solution (2% goat serum, 1% bovine serum albumin, 2% fish skin gelatin in PBS + 0.3% triton) for 2 h at room temperature. After block Alexa Fluor 633 Phalloidin (ThermoFisher, A22284) was added at 1:100 in PBST. Larvae were incubated for 4 h at room temperature and then washed 4 × 5 min in PBST. Larvae were mounted on glass slides with Prolong Gold (ThermoFisher Scientific) using No. 1.5 coverslips. To image hair bundle orientation and FM 1-43FX label in lateral-line hair cells, a Zeiss LSM780 confocal microscope with Airyscan was used. Hair cells were imaged using a 63× 1.3 NA oil objective and processed with an Airyscan processing factor of 7.0. For analysis, Z-stacks were max-projected in FIJI. Images were background subtracted using a rolling ball correction. Hair cells with a FM 1-43FX intensity more than twice the background intensity were scored as FM 1-43FX positive. <strong>Zebrafish Ca2+ imaging and behavior</strong> For imaging of mechanosensitive Ca<sup>2+</sup> signals in neuromast hair bundles<em>, Tg(myo6b:memGCaMP6s)</em> animals were mounted on their side on Sylgard chamber in E3 embryo media containing 0.2% MESAB (Lukasz and Kindt, 2018). Larvae were then pinned and a solution containing α-bungarotoxin (125 µM, Tocris) was injected into the heart cavity for paralysis during imaging. After paralysis, larvae were immersed in extracellular imaging solution (in mM: 140 NaCl, 2 KCl, 2 CaCl<sub>2</sub>, 1 MgCl<sub>2</sub> and 10 HEPES, pH 7.3, OSM 310 +/-10). To image Ca<sup>2+</sup> signals in the hair bundles we used a Bruker Swept-Field confocal system equipped with a Rolera EM-C2 CCD camera (QImaging, Surrey, Canada) and a Nikon CFI Fluor 60×1.0 NA water immersion objective as previously described (Q. Zhang et al., 2018). The system includes a band-pass 488/561 nm filter set (59904-ET, Chroma) and is controlled using Prairie View software (Bruker Corporation). A piezoelectric motor (PICMA P-882.11-888.11 series, PI Instruments) attached to the objective and used to acquire 5-plane Z-stacks every 0.5 µm using a 35 µm slit at a 50-Hz frame rate for a 10-Hz volume rate. A fluid-jet composed of a glass capillary attached to a pressure clamp system (HSPC-2-SB, ALA) was used to deliver a 500-ms anterior and posterior step stimulation to deflect hair bundles along their axis of sensitivity. To quantify GCaMP6s fluorescent intensity changes during stimulation, we registered the Z-stacks and then average projected the Z-stacks into a single plane. We then loaded the projected images into FIJI and used the Time Series Analyzer V3 plugin to create circular ROIs with a ∼1.7 μm diameter. ROIs were placed on the center of each individual bundle. We then measured and plotted change in the mean intensity (ΔF/F<sub>0</sub>) within the region during the recording period. The mean intensity within each ROI was computed for each hair bundle. A hair bundle with a signal magnitude (peak value of intensity change upon stimulation) above 10% ΔF/F<sub>0</sub> was considered mechanosensitive. To create the AVG hair bundle GCaMP6s (ΔF/F<sub>0</sub>) per neuromast plot in Figure 5-figure supplement 1C, only traces from mechanosensitive cells were averaged. A Zantiks MWP behavioral system was used to examine startle responses. The Zantiks system tracked and monitored behavioral responses via a built-in infrared camera at 30 frames per second. A 12-well plate was used to house larvae during behavioral analysis. Each well was filled with E3 and 1 larva. All fish were acclimated in the plate within the Zantiks chamber in dark for 15 min before each test. To induce startle, an integrated stepper motor was used to drive a vibration-induced startle response. A vibrational stimulus that triggered a maximal % of animals startling in controls without any tracking artifacts (due to the vibration) was used for our analyses. Each larva was presented with the vibrational stimulus 5 times with 100 s between trials. During the tracking and stimulation, a Cisco router connected to the Zantiks system was used to relay <em>x</em>, <em>y</em> coordinates of each larva every frame. To qualify as a startle response, a distance above 4 pixels or ∼1.9 mm was required within 2 frames after stimulus onset. Animals were excluded from our analysis if no tracking data was recorded for the animal.
description: # Data from: Complexes of vertebrate TMC1/2 and CIB2/3 proteins form
 hair-cell mechanotransduction cation channels
 [https://doi.org/10.5061/dryad.4tmpg4fkw](https://doi.org/10.5061/dryad.4tmpg4fkw) ## Description of the data and file structure Title of Dataset **Complexes of vertebrate TMC1/2 and CIB2/3 proteins form hair-cell mechanotransduction cation channels** Summary Paper associated with dataset: eLife (2024) [https://doi.org/10.7554/eLife.89719.1](https://doi.org/10.7554/eLife.89719.1) This dataset includes raw data from the zebrafish portion of this paper that was collected at the NIH/NIDCD: Figure 5 and supplement Figure 6 This dataset includes live measurements of mechanosensitive function (via FM 1-43 label) of hair cells in zebrafish lateral-line hair cells (L1-L4 at 5 dpf) in sibling, cib2, cib3 and cib2;cib3 mutants (Figure 5A-G). This data revealed that hair cells in cib2;cib3 mutants fail to label with FM 1-43. Cib3 mutants label normally. Cib2 mutants have fewer hair cells labeled. Calcium imaging was used to measurement evoked mechanosensitive responses in lateral-line hair cells in sibling, cib2, cib3 and cib2;cib3 mutants (Figure 5H-L and supplement). This is data acquired from transgenic myo6b:memGCaMP6s animals. This data shows reduced mechanosensitive responses in cib2 and no responses in cib2;cib3 mutants. This dataset includes immunolabeling to link mechanosensitivity (via FM 1-43fx label) to hair bundle orientation (via phalloidin label) in zebrafish lateral-line hair cells (L1-L4 at 5 dpf) in siblings and cib2 mutants (Figure 6A-D). This data demonstrates that there are more mechanosensitive posterior-sensitive hair cells in cib2 mutants. This dataset includes live mechanosensitivity measurements (via FM 4-64 label) of hair cells in the media crista in sibling, cib2, cib3 and cib2;cib3 mutants at day 5 (Figure 6E-I). This data revealed that hair cells in cib2;cib3 mutants fail to label with FM 4-64. Cib3 mutants label normally. Cib2 mutants primarily label tall hair cells. Folders included in this submission have the following names: RAW_DATA_Figure 5 RAW_DATA_Figure 6 ### RAW\_DATA\_Figure 5 Summary of Figure 5 folder contents: This folder contains 3 subfolders named by experimental paradigm: “Calcium imaging” “Live FM 1-43” and “Startle behavior”. “ Calcium imaging” contains 76 .xml files and one excel file. “Live FM 1-43” contains 33 .nd2 files and one excel file. “Startle Behavior” contains and excel file and 3 subfolders from each experimental day. These subfolders contain video files for each plate of animals assayed and a .csv file.  Each of these folders contain named by plate ran on the day. In total there are 10 .csv files and 200 videos files. The “Calcium imaging” data contains .xml series files are the raw calcium imaging data from cib2, cib3, cib2;cib3 mutants and sibling control fish aged 5 dpf represented in Figure 5H-L and in the linked supplement. The data extracted from these files is summarized in the excel file .xlsx “calcium imaging”. The .xml files are measurements of mechanosensitive from hair bundles of neuromasts (L1-L5) in the zebrafish posterior lateral line. Responses were made using the following transgenic line: myo6b:GCaMP6scaax. To stimulate hair cells a fluidjet was used. A 500ms saturating fluid flow stimulus was used, one acquisition towards the anterior and one acquisition towards the posterior of the fish for each neuromast. The “Live FM 1-43” .nd2 files are Nikon A1R images of FM 1-43 dye labeling of neuromasts (L1-L5) in the zebrafish posterior lateral line in cib2, cib3, cib2;cib3 mutants and sibling control fish aged 5 dpf, and represents the data in Figure 5B-G. The measurements extracted from these files is summarized in the excel file .xlsx “030821_FM 1-43 neuromast”. In “Startle Behavior” the 10 files with extension .csv and 200 videos with extension .avi were generated by the Zantiks system that represent the data in Figure 5A. There is one excel file “Cib2Cib3 Startle.xlsx” denoting all data points and which experimental date, plate number, well and  genotypes. This is live behavioral data acquired from cib2, cib3, cib2;cib3 mutants and sibling control fish aged 5 dpf on 3 separate days. Video .avi files in the startle folder beginning with “S1-VIB1” correspond to the highest intensity stimulus, “S2-VIB2” the medium intensity stimulus, “S3-VIB3” the low intensity stimulus, and “S4-VIB4” the control with no stimulus. Metadata for GCaMP6s calcium imaging acquisition: Acquired on a Swept field confocal microscope using Prairie-view (Bruker) software Nikon 60x water objective 20ms per image, 0.5 microns per slice, 5 planes per Z-stack (each Z-stack or timepoint is ~0.1s) 400 tif images in total per acquisition (~8s), fluidjet stimulation at frame 150 (~3s) 70 micron slit EM gain 3900 2x2 binning Imaging ROI 128x128 Laser setting 100 Files according to date, fish # (F) and neuromast (L) are as follows: Sibling control: F1L3 090121 F1L4 090121 F1L2 071521 F1L3 071521 F1L4 071521 F4L1 071421 F4L2 071421 F4L3 071421 F1L2 062821 F1L3 062821 Cib2: F2L4 082421 F2L1 071521 F2L2 071521 F3L1 071421 F3L2 071421 F3L3 071421 F3L4 071421 F3L5 071421 F6L1 062821 F6L2 062821 Cib3: F3L2 090121 F3L4 090121 F4L2 090121 F4L3 090121 F4L4 090121 F1L3 082421 F1L4 082421 F1L2 071421 F1L3 071421 F1L4 071421 Cib2; cib3 F2L1 071421 F2L2 071421 F2L3 071421 F4L2 062821 F4L4 062821 F5L1 062821 F5L2 062821 F5L4 062821 Each acquisition is a series of 400 tif files. Each neuromast is linked to 2 acquisition files, one stimulus to the anterior and one to the posterior of the fish. Each tif series was processed using the GUI-based ImagePro. This program average projects each z-stack. Then registers the Z-stack. The first second (10 frames) of the recording was removed for baseline stability. Then the resulting 70 frames per recording was then opened in FIJI/ImageJ. The Time Series Analyzer V3 plugin was used to create size circular ROIs (6x6 pixels). ROIs were placed on each hair bundle. The multi-measure feature in the ROI manager was used to obtain the mean gray value from each ROI and each image in the series. Background subtracted plots were created. The background was the first 20 rows (F0). The calculation to subtract background was Percentage different 100*(Value-Baseline)/Baseline. From the background subtracted plots (deltaF/F0), for each neuromast the anterior and posterior responsive hair bundles were determined. For each genotype, the average response magnitude (Max deltaF/F0) and number of responding hair bundles was determined for each terminal. These analyses are summarized in Calcium imaging.xlxs file. The table “Summary data” contains the Fish, neuromast and date of each acquisition (column A), the genotype (column B), the percent of hair bundles responding per neuromast (column C), the average Max deltaF/F0 (Column D) and the images file names for each sample (columns E and F). The table “AVG response per NM plots” summarizes the average mutant and wild-type response at each time point, along with the SEM. These tables represent the data shown in Figure 5H-L and the linked supplement. Examples shown in Figure 5I-L are as follows: F2L2 071421 cib2;cib3 F1L4 071421 cib3 F3L5 071421 cib2 F4L1 071421 sibling (double het) Metadata for acoustic startle responses: A Zantiks MWP behavioral system ([https://zantiks.com](https://zantiks.com)) was used to assess acoustic startle responses in larvae at 5 dpf. The Zantiks system tracks and monitors behavioral responses using an infrared camera at 30 frames per second. During the tracking and stimulation, a Cisco router connected to the Zantiks system was used to relay x and y coordinates of each larva in every frame. The Zanzik systems creates video files (.avi) during each stimulus. In addition, from the x,y, coordinates a .cvs (vib-stim prefix) containing the distance traveled in pixels for each time point for each well of the plate (column I-T). This data is contained in the .csv files for each plate. A 12-well plate was used for behavioral analyses. Each well was filled with E3 and 1 larva. All fish were acclimated in the plate within the Zantiks chamber in the dark for 15 min before each test. A vibrational stimulus that triggered a maximal proportion of animals startling in control animals without any tracking artifacts (due to the vibration) was used for our strongest stimuli. We used 4 different levels of intensity (1-4, increasing in intensity), with level 4 as the highest intensity stimulus. To deliver the acoustic-vibrational stimulus, the solenoid motor in the Zantiks system was set to move by 7.2° (level 4: 4 full steps), 3.6° (level 3: 2 full steps), 1.8° (level 2: 1 full step), and 0.9° (level 1: 1/2 step), with a 4 x 4.25 ms motor speed moving in clockwise and anticlockwise movements. For our initial startle assay, each larva was presented with stimuli from intensity levels 1-3, 5 times, with 100 s between trials to avoid habituation. For each animal, the proportion of startle responses out of the 5 trials was plotted. The folders by date are as follows: Startle behavior: 210308 210316 210419 Each folder contains data from 2-4 plates. Each plate has 12 wells. Well position is designated by three rows A-C and 4 column 1-4; for example, the upper left well is A1 and the lower right well is C4. The data from the .csv files in the startle folder was extracted using Python code ‘zantiks processing-0.3s_eachtmpt.ipynb’. This code detects whether each animal travels more than 4 pixels or ~1.9 mm within 2 frames after stimulus onset. This is counted as a startle response. Then the number of times (out of 5 trials) the animal startles is determined. This data is summarized in the excel file “Cib2Cib3 Startle.xlxs” in table Summary data. The table Summary data outlines the date (column a), plate and well # (columns b and c) and genotype (column d) for each animal. In addition, this table lists the proportion of times (out of 5 trials) that the animal startled in response to the S1-VIB1vibrational stimulus. This is the data plotted in Figure 5A. Metadata for FM 1-43 imaging: Fish were immersed in 1 uM FM 1-43 for 30s, followed by 3 washes in embryo media. Larvae were then mounted in 1% low melt agarose with tricaine and imaged as follows: Nikon A1R 60x water objective 512x1042 Zoom 3x Averaging 4x Laser power: 488 and 561 2% with a laser scanning transmitted detector (channel 3) Z stacks were acquired every 0.4 um The following images were acquired: Sibling: F6L1_030821_FM143.nd2 F6L3_030821_FM143.nd2 F6L2_030821_FM143.nd2 F7L2_030821_FM143.nd2 F7L3_030821_FM143.nd2 F7L4_030821_FM143.nd2 F14L1_030821_FM143.nd2 F14L2_030821_FM143.nd2  Cib2: F9L3_030821_FM143.nd2 F9L4_030821_FM143.nd2 F9L5_030821_FM143.nd2 F13L1_030821_FM143.nd2 F13L2_030821_FM143.nd2 F13L4_030821_FM143.nd2 F19L1_030821_FM143.nd2 F19L2_030821_FM143.nd2 Cib3: F3L1_030821_FM143.nd2 F3L2_030821_FM143.nd2 F3L3_030821_FM143.nd2 F8L2_030821_FM143.nd2 F8L3_030821_FM143.nd2 F8L4_030821_FM143.nd2 F18L3_030821_FM143.nd2 Cib2; cib3: F2L1_030821_FM143.nd2 F2L3_030821_FM143.nd2 F4L1_030821_FM143.nd2 F4L2_030821_FM143.nd2 F4L4_030821_FM143.nd2 F16L1_030821_FM143.nd2 F16L3_030821_FM143.nd2 F20L3_030821_FM143.nd2 To quantify FM 1-43 label, Z-stacks were processed in FIJI. To estimate the number of hair cells per neuromast, laser scanning DIC images (channel 3) were used to count the number of hair bundles per neuromast. To quantify FM 1-43 label, Z-stacks were. Max-projected and a 5 µm circular region of interest (ROI) was placed on the soma of each hair cell with a hair bundle in the DIC image. These ROIs were used to quantify the mean FM 1-43 intensity per hair cell. These intensity values were averaged to calculate the average FM 1-43 intensity per neuromast. These measurements are summarized in the excel file “030821_FM 1-43 neuromast.xlxs” in table live FM 1-43. The table “live FM 1-43” outlines the filename (column a), genotype (column b), the average FM 1-43 intensity per neuromast (columns c), the number of hair cells per neuromast (column d), the number of hair cells and percent of hair cells labeling with FM 1-43  (column e and f). This is the data plotted in Figure 5B-G. The following filed were partially z-projected in FIJI and displayed in the figure: F11L3_030821_FM143.nd2 F16L3_030821_FM143.nd2 F13L1_030821_FM143.nd2 F18L1_030821_FM143.nd2 ### RAW\_DATA\_Figure 6 Summary of Figure 6 folder contents: This folder contains 2 subfolders named by experimental paradigm: “FM 1-43fx phalloidin” and “FM 4-64 medial cristae”. “FM 1-43fx phalloidin” contains 35 .czi files and one excel file. “FM 4-64 medial cristae” contains 36 .nd2 files and one excel file. The “FM 1-43fx phalloidin” data contains .czi files of immunostains of hair bundle orientation (phalloidin) and mechanosensitivity (FM 1-43fx) from cib2 mutants and sibling control fish aged 5 dpf represented in Figure 6A-D. The data extracted from these files is summarized in the excel file .xlsx “FM 1-43fx”. The .czi files are measurements of hair cells of neuromasts (L1-L5) in the zebrafish posterior lateral line. The “FM 4-64 medial cristae” data contains .nd2 files that show live images of mechanosensitivity (FM 4-64) of hair cells in the cristae of cib2, cib3, cib2;cib3 mutants and sibling control fish aged 5 dpf represented in Figure E-I. The files used for this analysis are summarized in the excel file .xlsx “Medial cristae_FM 4-64”. The .nd2 files are measurements of mechanosensitive from hair cells in the medial cristae of zebrafish. Information on FM 1-43fx immunostaining: To correlate hair bundle orientation in lateral line hair cells with intact mechanotransduction, larvae were immersed in 3 µM FM 1-43FX (ThermoFisher, F25255) in E3 for 30 s, and then washed 3 times in E3. After washing, larvae were fixed with 4% paraformaldehyde in PBS at 4°C overnight. After fixation, larvae were washed 4 × 5 min in 0.1% Tween in PBS (PBST). Larvae were then placed in blocking solution (2% goat serum, 1% bovine serum albumin, 2% fish skin gelatin in PBS + 0.3% triton) for 2 h at room temperature. After block Alexa Fluor 633 Phalloidin (ThermoFisher, A22284) was added at 1:100 in PBST. Larvae were incubated for 4 h at room temperature and then washed 4 × 5 min in PBST. Larvae were mounted on glass slides with Prolong Gold (ThermoFisher Scientific). Metadata for FM 1-43fx imaging: Fixed samples were imaged on a Zeiss LSM780 confocal microscope with Airyscan 63x 1.3 NA oil objective 632x632 Zoom 4x Laser power: 488 and 633 1% gain 800 Z stacks were acquired every 0.3 um Images were processed with an Airyscan processing factor of 7.0 The following images were acquired: Siblings: S1R1_F1L1_040921_cib2_3_d5_FMfx_ph647-Airyscan Processing-01.czi S1R1_F1L3_040921_cib2_3_d5_FMfx_ph647-Airyscan Processing-02.czi S1R1_F6L1_040921_cib2_3_d5_FMfx_ph647-Airyscan Processing-11.czi S1CS2R1_F1L1_041421_cib2_3_d5_FMfx_ph647-Airyscan Processing-01.czi S1CS2R1_F2L3_041421_cib2_3_d5_FMfx_ph647-Airyscan Processing-05.czi S1CS2R1_F3L2_041421_cib2_3_d5_FMfx_ph647-Airyscan Processing-06.czi S1CS2R1_F3L3_041421_cib2_3_d5_FMfx_ph647-Airyscan Processing-07.czi S1CS2R1_F5L1_041421_cib2_3_d5_FMfx_ph647-Airyscan Processing-12.czi S1CS2R2_F6L3_041421_cib2_3_d5_FMfx_ph647-Airyscan Processing-13.czi Cib2 mutants: S1R1_F3L1_040921_cib2_3_d5_FMfx_ph647-Airyscan Processing-02.czi S1R1_F3L2_040921_cib2_3_d5_FMfx_ph647-Airyscan Processing-05.czi S1R1_F3L4_040921_cib2_3_d5_FMfx_ph647-Airyscan Processing-06.czi S1R1_F4L1_040921_cib2_3_d5_FMfx_ph647-Airyscan Processing-03.czi S1R1_F4L2_040921_cib2_3_d5_FMfx_ph647-Airyscan Processing-08.czi S2C12R2_F10earNM3_042121_cib2_3_d5_FMfx_ph647-Airyscan Processing-14.czi S2C12R2_F10L1_042121_cib2_3_d5_FMfx_ph647-Airyscan Processing-15.czi S2C12R2_F10L2_042121_cib2_3_d5_FMfx_ph647-Airyscan Processing-16.czi S2C12R2_F10L3_042121_cib2_3_d5_FMfx_ph647-Airyscan Processing-17.czi S2C22R1_F12earNM3_042121_cib2_3_d5_FMfx_ph647-Airyscan Processing-18.czi S2C22R1_F12L1_042121_cib2_3_d5_FMfx_ph647-Airyscan Processing-19.czi S2C22R1_F12L2_042121_cib2_3_d5_FMfx_ph647-Airyscan Processing-20.czi S2C22R1_F12L3_042121_cib2_3_d5_FMfx_ph647-Airyscan Processing-21.czi S2C2R1_F14L1_042121_cib2_3_d5_FMfx_ph647-Airyscan Processing-02.czi S2C2R1_F14L2_042121_cib2_3_d5_FMfx_ph647-Airyscan Processing-03.czi S2C2R1_F14L4_042121_cib2_3_d5_FMfx_ph647-Airyscan Processing-04.czi S2C2R1_F14earNM3_042121_cib2_3_d5_FMfx_ph647-Airyscan Processing-01.czi S3C12R1_F1L2_042121_cib2_3_d5_FMfx_ph647-Airyscan Processing-01.czi S3C12R1_F1L3_042121_cib2_3_d5_FMfx_ph647-Airyscan Processing-02.czi S3C12R1_F1L4_042121_cib2_3_d5_FMfx_ph647-Airyscan Processing-03.czi S3C12R2_F1L5_042121_cib2_3_d5_FMfx_ph647-Airyscan Processing-07.czi S3C22R2_F8L1_042121_cib2_3_d5_FMfx_ph647-Airyscan Processing-08.czi S3C22R2_F8L2_042121_cib2_3_d5_FMfx_ph647-Airyscan Processing-09.czi S3C22R2_F8L3_042121_cib2_3_d5_FMfx_ph647-Airyscan Processing-10.czi S3C22R2_F8L4_042121_cib2_3_d5_FMfx_ph647-Airyscan Processing-11.czi S3C22R2_F8L5_042121_cib2_3_d5_FMfx_ph647-Airyscan Processing-12.czi For analysis, Z-stacks were max-projected in FIJI. Images were background subtracted using a rolling ball correction. Hair cells with a FM 1-43FX intensity more than twice the background intensity were scored as FM 1-43FX positive. This data is represented in the excel file FM 1-43fx in sheet FM 1-43fx labeling. In this file the filename is listed (column a) and the genotype (column b). The percentage of posterior- and anterior -sensitive hair cells with FM 1-43 fx labeled are indicated in columns c and d. The following filed were partially z-projected in FIJI and displayed in the figure: S1CS2R2_F6L3_041421_cib2_3_d5_FMfx_ph647-Airyscan Processing-13 S1R1_F3L2_040921_cib2_3_d5_FMfx_ph647-Airyscan Processing-05 Information on FM 4-64 labeling: FM 4-64 (ThermoFisher, T13320) was used to label hair cells in the crista within the zebrafish inner ear. For this labeling, myo6b:memGCaMP6s transgenic larvae were pinned on their side on Sylgard chamber in E3 embryo media containing 0.02% MESAB. Approximately 2-3 nL of 10 µM FM 4-64 in 0.1 M KCl was injected into the otic capsule. Metadata for FM 4-64 imaging: FM 4-64 labeled hair cells of the medial crista were then imaged on an A1R Nikon upright laser-scanning confocal microscope with either:  25× 1.10 NA water immersion objective 1024x1024 4x averaging Every 0.375 um 488 and 561 lasers with a power of 3 With a Transmitted detector  or 60× 1.0 NA water objective 1024x1024 16x averaging Every 0.4 um 488 and 561 lasers with a power of 4 and 6 With a Transmitted detector Channel = 488/GCaMP6s; Channel 2 = 561/FM 4-64; Channel 3 = transmitted detector The following images were acquired: Siblings (double hets): 082422_Fish9_MC1-2.nd2 082422_Fish1_MC1.nd2 082422_Fish4_MC1-2.nd2 082422_Fish6_MC1.nd2 090722_cib2_3_F1_MC-2.nd2 090722_cib2_3_F5_MC.nd2 090722_cib2_3_F13_MC.nd2 090722_cib2_3_F14_MC.nd2 110922_fish3_MC.nd2 110922_fish7_MC.nd2 110922_fish10_MC.nd2   Cib2: 082422_Fish3_MC1.nd2 082422_Fish5_MC1.nd2 082422_Fish7_MC1-2.nd2 082422_Fish13_MC1.nd2 090722_cib2_3_F3_MC.nd2 090722_cib2_3_F6_MC.nd2 090722_cib2_3_F11_MC.nd2 090722_cib2_3_F12_MC.nd2 090722_cib2_3_F15_MC.nd2 090722_cib2_3_F16_MC.nd2 110922_fish4_MC.nd2 110922_fish12_MC.nd2 110922_fish15_MC.nd2 Cib3: 082422_Fish11_MC1-2.nd2 082422_Fish15_MC1.nd2 090722_cib2_3_F10_MC.nd2 110922_fish5_MC.nd2 110922_fish6_MC.nd2 110922_fish13_MC.nd2 110922_fish14_MC.nd2 Cib2; cib3 082422_Fish8_MC1.nd2 090722_cib2_3_F4_MC.nd2 090722_cib2_3_F8_MC.nd2 110922_fish2MC.nd2 FIJI was used to process image Z-stacks. To assess tall versus short hair cells in each crista *memGCaMP6s* label was used. The following filed were partially z-projected in FIJI and displayed in the figure: 090722_cib2_3_F14_MC.nd2 090722_cib2_3_F9_MC.nd2 090722_cib2_3_F10_MC.nd2 090722_cib2_3_F8_MC.nd2 ### Files and variables #### File: RAW\_DATA\_FIGURE\_5.zip **Description:** Live FM 1-43 labeling of zebrafish neuromasts, mechanosensitive calcium responses in neuromast hair cells and acoustic startle behavior in control, cib2, cib3 and cib2;cib3 mutants #### File: RAW\_DATA\_FIGURE\_6.zip **Description:** Fixed FM 1-43fx and phalloidin label of control and cib2 mutant neuromasts; Live FM 4-64 label of hair cells in the medial crista of control, cib2, cib3 and cib2;cib3 mutants. ## Code/software To open Nikon .nd2 files, Zeiss .czi files and Prairie view .xml files FIJI with appropriate Bio-Formats plugins are needed. To open .csv or .xlxs files Microsoft excel is needed. To open .avi files Quicktime is needed. To use the code to analyze behavioral data Anaconda is needed.