Control of stereocilia length during development of hair bundles

Abstract

Assembly of the hair bundle, the sensory organelle of the inner ear, depends on differential growth of actin-based stereocilia. Separate rows of stereocilia, labeled 1 through 3 from tallest to shortest, lengthen or shorten during discrete time intervals during development. We used lattice structured illumination microscopy and surface rendering to measure dimensions of stereocilia from mouse apical inner hair cells during early postnatal development; these measurements revealed a sharp transition at postnatal day 8 between stage III (row 1 and 2 widening; row 2 shortening) and stage IV (final row 1 lengthening and widening). Tip proteins that determine row 1 lengthening did not accumulate simultaneously during stages III and IV; while the actin-bundling protein EPS8 peaked at the end of stage III, GNAI3 peaked several days later—in early stage IV—and GPSM2 peaked near the end of stage IV. To establish the contributions of key macromolecular assemblies to bundle structure, we examined mouse mutants that eliminated tip links (Cdh23v2J or Pcdh15av3J), transduction channels (TmieKO), or the row 1 tip complex (Myo15ash2). Cdh23v2J/v2J and Pcdh15av3J/av3J bundles had adjacent stereocilia in the same row that were not matched in length, revealing that a major role of these cadherins is to synchronize lengths of side-by-side stereocilia. Use of the tip-link mutants also allowed us to distinguish the role of transduction from effects of transduction proteins themselves. While levels of GNAI3 and GPSM2, which stimulate stereocilia elongation, were greatly attenuated at the tips of TmieKO/KO row 1 stereocilia, they accumulated normally in Cdh23v2J/v2J and Pcdh15av3J/av3J stereocilia. These results reinforced the suggestion that the transduction proteins themselves facilitate localization of proteins in the row 1 complex. By contrast, EPS8 concentrates at tips of all TmieKO/KO, Cdh23v2J/v2J, and Pcdh15av3J/av3J stereocilia, correlating with the less polarized distribution of stereocilia lengths in these bundles. These latter results indicated that in wild-type hair cells, the transduction complex prevents accumulation of EPS8 at the tips of shorter stereocilia, causing them to shrink (rows 2 and 3) or disappear (row 4 and microvilli). Reduced rhodamine-actin labeling at row 2 stereocilia tips of tip-link and transduction mutants suggests that transduction’s role is to destabilize actin filaments there. These results suggest that regulation of stereocilia length occurs through EPS8 and that CDH23 and PCDH15 regulate stereocilia lengthening beyond their role in gating mechanotransduction channels.

Spontaneous allelic variant inUsh1gresulting in an expanded phenotype

“…While localization of GPSM2 and GNAI3 was only modestly affected, EPS8 shifted from being primarily found at row 1 tips to being equally distributed between row 1 and 2 tips. We also showed that CDH23 and PCDH15 play an underappreciated role in coordinating the lengths of adjacent stereocilia 28 . Row 1 proteins showed similar altered distribution in Ush1g bw/bw , and adjacent stereocilia lengths were less coordinated, albeit not as profoundly disrupted as in Cdh23 v2J/v2J and Pcdh15 av3J/av3J hair cells.…”

“…Homozygous null mutations in Cdh23 and Pcdh15 lead to altered distribution of proteins that concentrate at the tips of row 1 stereocilia in postnatal hair bundle development 28 . While localization of GPSM2 and GNAI3 was only modestly affected, EPS8 shifted from being primarily found at row 1 tips to being equally distributed between row 1 and 2 tips.…”

Inhibitory G proteins play multiple roles to polarize sensory hair cell morphogenesis

“…Potential differences in data distribution between genotypes were tested for significance using nested (hierarchical) t-test except for ABR thresholds ( Figure 3 and Figure 3—figure supplement 1 ) where a two-way ANOVA with Sidak’s multiple comparison post hoc test was used. Nested t-tests help avoid pseudoreplication by taking into consideration the data structure, here specifically variance in each animal ( Eisner, 2021 ; Krey et al, 2023 ). OHC half-bundle lengths ( Figure 2G , Figure 2—figure supplement 1G ) were plotted as paired left and right values in the same HC and a potential difference in data variance between genotypes was tested using an F-test.…”

Inhibitory G proteins play multiple roles to polarize sensory hair cell morphogenesis

“…1) where a 2-way ANOVA with Sidak’s multiple comparison post-hoc test was used. Nested t-tests help avoid pseudoreplication by taking into consideration the data structure, here specifically variance in each animal (Eisner, 2021; Krey et al, 2023). OHC half-bundle lengths (Figure 2G, Figure 2 Supp.…”