ANKRD24 organizes TRIOBP to reinforce stereocilia insertion points

Abstract

The stereocilia rootlet is a key structure in vertebrate hair cells, anchoring stereocilia firmly into the cell’s cuticular plate and protecting them from overstimulation. Using superresolution microscopy, we show that the ankyrin-repeat protein ANKRD24 concentrates at the stereocilia insertion point, forming a ring at the junction between the lower and upper rootlets. Annular ANKRD24 continues into the lower rootlet, where it surrounds and binds TRIOBP-5, which itself bundles rootlet F-actin. TRIOBP-5 is mislocalized in Ankrd24KO/KO hair cells, and ANKRD24 no longer localizes with rootlets in mice lacking TRIOBP-5; exogenous DsRed–TRIOBP-5 restores endogenous ANKRD24 to rootlets in these mice. Ankrd24KO/KO mice show progressive hearing loss and diminished recovery of auditory function after noise damage, as well as increased susceptibility to overstimulation of the hair bundle. We propose that ANKRD24 bridges the apical plasma membrane with the lower rootlet, maintaining a normal distribution of TRIOBP-5. Together with TRIOBP-5, ANKRD24 organizes rootlets to enable hearing with long-term resilience.

Control of stereocilia length during development of hair bundles

“…Contrast was manually adjusted to retain both dim and bright structures due to the high dynamic range of the phalloidin signal. Verification of channel alignment and measurement of the microscope point-spread function was carried out as previously described [46].…”

“…Our measurements should reflect native lengths and widths; dimensions of mildly-fixed, phalloidin-labeled stereocilia are not significantly different from dimensions of live stereocilia labeled with membrane dyes [43]. We improved resolution over conventional confocal microscopy by using lattice structured illumination microscopy (lattice SIM) [44][45][46], which has a point-spread function (PSF) of ~150 nm under our conditions [46]; typical confocal microscopy PSFs are ~230 nm [47]. We rendered image surfaces from each phalloidin-stained hair bundle (Fig 1A -F); rendered surfaces are three-dimensional models of specific structures computed from stacks of images by pre-processing, segmentation, and connectedcomponent labeling steps.…”

“…Contrast was manually adjusted to retain both dim and bright structures due to the high dynamic range of the phalloidin signal. Verification of channel alignment and measurement of the microscope point-spread function was carried out as previously described [46]. Airyscan images were acquired at room temperature using a 63x, 1.4 NA Plan-Apochromat objective on a Zeiss 3-channel LSM980 laser-scanning confocal microscope equipped with an Airyscan.2 detector and run under ZEISS ZEN (v3.1, 64-bit software; Zeiss) acquisition software.…”

Unbalanced bidirectional radial stiffness gradients within the organ of Corti promoted by TRIOBP

“…However, localization of TRIOBP-1 was not previously investigated for lack of a TRIOBP-1–specific antibody since most of the sequence of TRIOBP-1 is identical to the C-terminal part of TRIOBP-5. We recently validated an anti-TARA antibody using our Triobp ΔEx9-10/ΔEx9-10 mice ( 29 ) and showed that the antibody signal in stereocilia rootlets solely represents the TRIOBP-5 isoform. Consequently, to investigate the correlation of our expression data of Triobp-1 , Triobp-4 , and Triobp-5 mRNAs with the corresponding protein isoform localizations, we used TRIOBP4/5-specific and anti-TARA antibodies in Triobp +/+ and Triobp ΔEx9-10/ΔEx9-10 mice.…”

“…P8 and P20 wild-type Triobp +/+ and homozygous Triobp ΔEx9-10/ΔEx9-10 mice were used for immunostaining of the organ of Corti with anti-TARA antibody (Proteintech, 16124-1-AP) which was developed against an antigen corresponding to the C-terminal sequence of TRIOBP-5 identical to TRIOBP-1, as reported ( 29 ). We also used antibodies against TRIOBP-4/5 and against TRIOBP-5 as previously described ( 9 , 10 ).…”