Dataset DOI: 10.5061/dryad.mcvdnckcd

Description of the data and file structure

"Compressed_Fig_1-Fig_3-Fig_6_and_Fig_S5-Fig_7_folders.zip" zipped file that contained four folders with data analysis is uploaded.

Each folder within this zipped file is named corresponding to the figure from the manuscript and its panels for which analysis was performed, as well as what was measured. Inside each folder are the analysis files. The names of the folders are:

Fig 1_ring diameter and linescan_O_P_Q

Fig 3_negative stain_filament distances and diameter_E_F_G

Fig 6 and Fig S5 Stats_ABR and VsEP

Fig 7_fluorescence spot diameter and intensity_C_D_E_H_I_J

Folder: Fig_1_ring_diameter_and_linescan_O_P_Q

Description: this folder contains the following file:

Ring_diameters_and_linescans.xlsx

This dataset contains measurement data for ANKRD24 (Ankyrin repeat domain-containing protein 24), TRIOBP-4 (TRIO and F-Actin Binding Protein, isoform 4), TRIOBP-5 (TRIO and F-Actin Binding Protein, isoform 5), and TPRN (Taperin) proteins that formed rings surrounding stereocilia rootlets of mouse inner ear hair cells. Measurements were taken from Row 1 stereocilia of outer hair cells (OHCs).

The Excel spreadsheet contains quantitative data obtained using Fiji/ImageJ program to open for quantification the STED (Stimulated Emission Depletion) microscopy images of OHCs stained with anti-ANKRD24, anti-TRIOBP-4, anti-TRIOBP-5, and anti-TPRN antibodies. Measurements of ANKRD24, TRIOBP-4, TRIOBP-5, and TPRN rings formed around stereocilia rootlets were taken from Row 1 stereocilia of OHCs.

For convenience in the Excel sheets, all proteins’ names are in lowercase (no caps and no dash line used).

Sheet "Fig_1O"

Diameters of ANKRD24, TRIOBP-4, and TRIOBP-5 rings. Each column lists individual measurements of ring diameter in µm (micrometers).

Sheets "Fig_1P" and "Fig_1Q"

Fluorescence intensity line profiles measured along the ring diameters. Sheets contain the X and Y interpolated coordinates of the points used to plot the line profiles and full width at half maximum (FWHM) cutoff lines. The X-coordinate unit is µm, and the Y-coordinate unit is arbitrary units, representing fluorescence intensity. To distinguish the interpolated X and Y coordinates from the original data points, in the column names of sheets Fig_1P and Fig_1Q, “ix” or “iy” are used specifically for interpolated X- or Y-coordinates of an original data points in Fiji/ImageJ.

Sheet "Fig_1P Cut-off lines"

The columns A, C, and E represent X-coordinates of edges in µm for ANKRD24 (ankrd24_edge_x), TRIOBP-4 (triobp4_edge_x), TRIOBP-5 (triobp5_edge_x), respectively. The columns B, D, and F contain Y-coordinates in arbitrary units, representing fluorescence intensity measurements for line profiles of ANKRD24 (ankrd24_edge_y), TRIOBP-4 (triobp4_edge_y), and TRIOBP-5 (triobp5_edge_y), respectively.

Sheet “Fig_1Q Cut-off lines”

Column A represents X-coordinates of edges in µm for TPRN (tprn_edge_x), while column B contains Y-coordinates in arbitrary units, representing fluorescence intensity measurements for line profiles of TPRN (tprn_edge_y).

Folder: Fig_3_negative_stain_filament_distances_and_diameter_E_F_G

Description: this folder contains the following files:

ESPN_1uM.DM4; 

ESPN_300nM.DM4;

TRX_100nM.DM4;

TRX-TPRN_100nM.DM4;

TRX-TPRN_300nM. DM4;

Negative_staining_data_diameters.csv;

Negative_staining_data_distances.csv

This dataset contains transmission electron microscopy (TEM) images of negatively stained actin filaments polymerized with the actin-bundling proteins TPRN (Taperin) and ESPN (Espin). The accompanying measurement data include actin bundle diameters and inter-filament distances.

The original TEM images used for Fig. 3 are stored in the .dm4 format files. These files can be opened using the Bio-Formats plugin in Fiji/ImageJ. Each image shows 3 µM (micromoles) of actin monomers polymerized with 1.5 µM (micromoles) phalloidin. In file names: TRX (thioredoxin tag, is a solubilization tag used in protein purification. It helps in the proper folding of recombinant proteins and prevents aggregation. The TRX tag can also enhance the solubility of proteins, making it easier to purify them); nM (nanomole) and uM  = µM (micromole) are the units of concentration measurements.

• TRX_100nM.dm4 - Polymerized with 100 nM TRX (control)

• TRX-TPRN_100nM.dm4 - Polymerized with 100 nM TRX-TPRN

• TRX-TPRN_300nM.dm4 - Polymerized with 300 nM TRX-TPRN

• ESPN_300nM.dm4 - Polymerized with 300 nM ESPN

• ESPN_1µM.dm4 - Polymerized with 1 µM ESPN

The two csv files contain quantitative measurements of actin bundle diameters measured in micrometers (µm) and inter-filament distances measured in nanometers (nm). These data were obtained from a set of TEM images, including those listed above, and were used for the graphs in Fig. 3. For actin filaments polymerized with 100 nM TRX (control), bundle diameters and inter-filament distances were measured at sites where three or more filaments were running in parallel. The different number of measurements present in each Excel column is expected because of the different bundling ability of different proteins, which depends on presence of bundling protein, its concentration, and number of actin bundles formed and present in the field of view in each image.

• Negative_staining_data_diameters.csv

• Negative_staining_data_distances.csv

Method used to generate dm4 file data:

To observe actin filaments and bundles using negative stain TEM, 3 µM of β-actin from human platelets (APHL99; Cytoskeleton, Inc) was polymerized in 100 μl of a solution containing 100 nM TRX, 100 nM TRX-TPRN, 300 nM TRX-TPRN, 300 nM ESPN, or 1 µM ESPN. Using mouse Espn cDNA (RefSeq: NM_207687.3), recombinant full-length Espin (FL ESPN) was subcloned with BamHI and NotI restriction enzymes into a pGEX-6P-1 vector containing a GST tag (28954648; Cytiva). Using the described method (Miyoshi et al., 2021), GST-ESPN was expressed and purified using a bacterial expression system. In brief, GST-ESPN was expressed in BL21 (DE3) cells (EC0114; Invitrogen) and purified using a Glutathione Agarose Resin (P306010; Ubpbio) and PreScission protease (Z02799; GenScript) in a buffer containing 20 mM Tris-HCl, pH 7.4, 150 mM NaCl, and 1 mM DTT. For incubation with TRN and TPX-TPRN, β-actin was polymerized in a buffer containing 20 mM Tris-HCl, pH 7.4, 50 mM KCl, 50 mM NaCl, 2 mM MgCl2, 1 mM ATP, 1 mM DTT, and 1.5 µM unconjugated phalloidin (P2141; Sigma-Aldrich), of which 10 mM Tris-HCl, pH 7.4 and 50 mM NaCl were derived from the buffer dissolving the TRX-TPRN or TRX. For β-actin incubated with 300 nM and 1 µM ESPN, the polymerization buffer contained 15 and 50 mM NaCl, respectively. After incubating for 1.5 h at RT, 4 μl of the solution was applied to 400 mesh copper grids with a continuous carbon film (CF400-Cu-50; Electron Microscopy Sciences), pre-treated using a Fischione Instruments Model 1020 Plasma Cleaner for 13 s to make them hydrophilic. Grids were incubated for 30 s at RT, washed briefly in distilled water three times, and stained with 0.5% uranyl acetate dissolved in molecular biology grade water (351-029-101; Quality Biological) for 30 s at RT. Images were acquired on a JEM 2100 transmission electron microscope (JEOL) using a OneView Camera (Gatan) at nominal magnifications of 20,000 × and 100,000 ×. Diameters of bundles were measured using the line tool on the Fiji platform (Schindelin et al., 2012) drawing cross sections for each bundle. As described (Volkmann et al., 2001), distances between filaments were measured detecting the center line of each filament using a bandpass filter on the Fiji platform (Schindelin et al., 2012), the profile_line function of scikit-image (https://scikit-image.org/), and the interp1 and find_peaks functions of SciPy (https://scipy.org/). For 100 nM TRX-TPRN, diameters and distances between filaments were measured, avoiding the segments where actin filaments coalesce. For 100 nM TRX, these parameters were measured using pseudo-bundles consisting of at least three filaments running in contact with each other.

Folder: Fig_6_and_Fig_S5_Stats_ABR_and_VsEP

Description: this folder contains the following files: 

TPRN KO post hocs.xlsx;

TPRN KO stat summaries.csv

File: TPRN KO post hocs.xlsx

Description: This file contains post hoc analysis statistics for analyses of auditory brainstem response (ABR) hearing thresholds in mutant and wild type (WT) mice and analyses of vestibular evoked potentials (VsEP) for Fig 6B and Fig S5A-F.

Column Descriptions:

• Age Group: Age group for VsEP measures taken on either postnatal day 30 (P30) or P60

• Age group: Age group for ABR measures taken on P18, P30, or P60

• Genotype: Genotype of mice - WT, heterozygous (het), or homozygous mutant (mut)

• contrast: Comparison being made between group averages

• estimate: Estimated size of the mean difference between groups

• Std Error: Standard error of the mean difference

• df: Degrees of freedom for the analysis, calculated using the Satterthwaite method

• t value: t statistic for the analysis

• p value: P-value for the analysis

Model Name: Name of the model starting with the figure it’s related to, including interaction terms, and frequency (either 8 kHz, 16 kHz, or 32 kHz) when appropriate. 

File: TPRN KO stat summaries.csv

Description: Statistical summaries for ABR and VsEP analyses included in Figure 6B and Figure S5A-F.

Column Descriptions:

• Variable: Name of the variable included in the model - interacting variables are separated by a “:”

• Estimate: Estimated regression coefficient

• Std Error: Standard error of the mean estimated regression coefficient

• df: Degrees of freedom for the analysis, calculated using the Satterthwaite method

• t value: T-statistic for the analysis

• p value: P-value for the analysis

• Lower CI: Lower limit of the 95% confidence interval for the estimate

• Upper CI: Upper limit of the 95% confidence interval for the estimate

• Model Name: Model name given during the analysis in Studio. Includes the figure for which the model was run, interaction terms separated by a “:”, and frequency (8, 16, or 32 kHz) where appropriate

• Model: The formula used for the model.

Statistical Analysis for Figure 6B and Figure S5A-F:

We used a linear mixed effects regression model to assess the effect of manipulations of the Tprn(Taperin) gene on hearing and vestibular function in mice. All models included a random effect for subjects and fixed effects for age, genotype, the interaction of age and genotype, and frequency (only for ABR - Auditory Brainstem Response threshold models). The youngest age of testing Tprn^+/+ mice and the lowest frequency tested were set as reference levels for their respective variables. Post hoc pairwise comparisons used Tukey’s adjustment for multiple comparisons. For post hoc analyses assessing hearing effects at specific frequencies, a separate model was fit for measurements of ABR threshold at each frequency. In the case of Tprn^in103/in103, individual mice were only assessed at a single time point with the control group only being assessed at P60 (Postnatal day 60). Consequently, we fitted a mixed effects linear regression model with a random effect for subject ID and a fixed effect for genotype/age group and frequency that compared ABR threshold from the three distinct Tprn^in103/in103^ ^{age groups to that of *Tprn}+/+^* mice at P60. Linear mixed effects models were run in R version 4.4.2 (https://www.r-project.org/) using the lme4 and emmeans packages. All graphs display means ± SD (mean values + Standard Deviation).

Folder: Fig_7_fluorescence_spot_diameter_and_intensity_C_D_E_H_I_J

Description: this folder contains the following files:

analysis_Fig_7.xlsx;

analysis.prism;

Subfolders:

"P21 apical IHCs" contains 2 png files:

21_01_11_Taperin_IHCS_ptprq_mouse_2024-10-31T17-10-41.750.png; 21_01_11_Taperin_IHCS_ptprq_mouse_2024-10-31T17-11-29.239.png

"P21 apical OHC" contains 2 png files:

21_01_11_Taperin_OHCS_ptprq_mouse_2024-10-31T17-32-10.929.png; 21_01_11_Taperin_OHCS_ptprq_mouse_2024-10-31T17-32-28.465.png

This dataset contains the quantification of the number and intensity of puncta the inner and outer hair cells of Ptprq^+/- het (heterozygous) and Ptprq^-/- mut (mutant) mice. This folder contains image files analyzed using Imaris software (version 9.8). The Spot function was applied to label individual puncta within each image. For each hair cell, the number of puncta and the mean intensity of each punctum were quantified. Subsequently, the average of the mean intensities per cell was computed. These values were used for genotype comparisons. Mean intensity of hair cells puncta was measured using ImageJ. The “set measurement” function in Fiji was setup to limit mean gray value measurements to threshold. This allows the software to examine the mean intensity at specified areas with fluorescent signal and ignoring the regions without signal. With this, the mean intensity value for the puncta (collectively) in each hair cell was calculated and compared within the genotypes using unpaired t test.

Description of the data and file structure:

The original Microsoft Excel and GraphPad Prism files from which the figures were generated were named as analysis_Fig_7.xlsx and analysis.prism, respectively.

The Microsoft Excel file "Analysis_Fig_7.xlsx" contains the intensity of each punctum from which the number (Sheet = # Puncta per cell) and average intensities of the IHC puncta (Sheet = IHC-puncta-intensity, Sheet = IHC-fiji-cell-intensity) were calculated.

The folder "P21 apical IHCs" contains two image files: an original png image and an image with spots showing each punctum of the IHCs (21_01_11_Taperin_IHCS_ptprq_mouse_2024-10-31T17-10-41.750.png) generated in Imaris from the original png image (21_01_11_Taperin_IHCS_ptprq_mouse_2024-10-31T17-11-29.239.png).

The folder "P21 apical OHC" contains two image files: an original png image and an image with spots showing each punctum of the OHCs (21_01_11_Taperin_OHCS_ptprq_mouse_2024-10-31T17-32-10.929.png) generated in Imaris from the original png image (21_01_11_Taperin_OHCS_ptprq_mouse_2024-10-31T17-32-28.465.png). The Microsoft Excel file "Analysis_Fig_7.xlsx" contains the intensity of each punctum from which the number (Sheet = # Puncta per cell) and average intensities of the OHC puncta (Sheet = OHC-puncta-Intensity, Sheet = OHC-fiji-cell-intensity) were calculated.

Note: Empty cells in .xlsx or .csv files with no data are indicated as “null”, where applicable. The different number of measurements for each condition is expected and reflects the natural variability of biological structures present in cells or in vitro that were analyzed. So, “null” indicates natural absence of a particular variable to measure.

Code/software

Fiji/ImageJ (version 1.54p)

GraphPad Prism (version 10)

Imaris software (version 9.8)

R (version 4.4.2)

Access information

Other publicly accessible locations of the data:

• Figures related to these data and additional related data are located at: Taperin bundles F-actin at stereocilia pivot points enabling optimal lifelong mechanosensitivity - PubMed; DOI: 10.1083/jcb.202408026

Data was derived from the following sources:

• The data of this study are original and generated by the authors.