Usher type 1G protein sans is a critical component of the tip-link complex, a structure controlling actin polymerization in stereocilia

Abstract

The mechanotransducer channels of auditory hair cells are gated by tip-links, oblique filaments that interconnect the stereocilia of the hair bundle. Tip-links stretch from the tips of stereocilia in the short and middle rows to the sides of neighboring, taller stereocilia. They are made of cadherin-23 and protocadherin-15, products of the Usher syndrome type 1 genes USH1D and USH1F, respectively. In this study we address the role of sans, a putative scaffold protein and product of the USH1G gene. In Ush1g(-/-) mice, the cohesion of stereocilia is disrupted, and both the amplitude and the sensitivity of the transduction currents are reduced. In Ush1g(fl/fl)Myo15-cre(+/-) mice, the loss of sans occurs postnatally and the stereocilia remain cohesive. In these mice, there is a decrease in the amplitude of the total transducer current with no loss in sensitivity, and the tips of the stereocilia in the short and middle rows lose their prolate shape, features that can be attributed to the loss of tip-links. Furthermore, stereocilia from these rows undergo a dramatic reduction in length, suggesting that the mechanotransduction machinery has a positive effect on F-actin polymerization. Sans interacts with the cytoplasmic domains of cadherin-23 and protocadherin-15 in vitro and is absent from the hair bundle in mice defective for either of the two cadherins. Because sans localizes mainly to the tips of short- and middle-row stereocilia in vivo, we conclude that it belongs to a molecular complex at the lower end of the tip-link and plays a critical role in the maintenance of this link.

Fig. 1.

(A) Mechanoelectrical transduction current recordings in UHCs from Ush1g^−/− P1 mice. In the graduated shading of green, examples of transduction current recordings in UHCs from Ush1g^fl/fl and Ush1g^−/− P1 mice are shown. Mean maximum amplitudes are 295 ± 30 pA and 88 ± 23 pA in Ush1g^fl/fl and Ush1g^−/− UHCs, respectively (unpaired t test, P < 10⁻⁴). P_o(X) curves plotted for Ush1g^fl/fl and Ush1g^−/− UHCs can be superimposed, with average sensitivity values of 0.88 ± 0.11 μm⁻¹ and 1.18 ± 0.21 μm⁻¹ for Ush1g^fl/fl and Ush1g^−/− UHCs, respectively (unpaired t test, P = 0.08). No change in X_0.5 can be detected in the mutant UHCs (X_0.5 = 686 ± 138 nm and 620 ± 75 nm for Ush1g^fl/fl and Ush1g^−/− UHCs, respectively; unpaired t test, P = 0.06). (B) Mechanoelectrical transduction currents were recorded in IHCs from Ush1g^fl/flMyo15-cre^+/− P8 mice. We analyzed mechanoelectrical transduction currents in cochlear inner hair cells from the apical region (∼35% of the total length of the cochlea from the apex) and the middle region (∼55% of the total length) of the cochlea in Ush1g^fl/fl and Ush1g^fl/flMyo15-cre^+/− P8 mice. (Left) Examples of transduction currents in apical IHCs from Ush1g^fl/fl (black) and Ush1g^fl/flMyo15-cre^+/− (blue) mice. Mean maximum current amplitude is 584 ± 52 pA and 346 ± 71 pA for Ush1g^fl/fl and Ush1g^fl/flMyo15-cre^+/− IHCs, respectively (unpaired t test, P = 0.015). The P_o(X) curves, however, can be superimposed with average sensitivity values of 1.85 ± 0.16 μm⁻¹ and 1.72 ± 0.15 μm⁻¹ for Ush1g^fl/fl and Ush1g^fl/flMyo15-cre^+/− IHCs, respectively (unpaired t test, P = 0.58). In addition, no change in X_0.5 can be detected in the mutant IHCs (X_0.5 = 218 ± 13 nm and 421 ± 109 nm for Ush1g^fl/fl and Ush1g^fl/flMyo15-cre^+/− IHCs, respectively; unpaired t test, P = 0.08). (Right) Examples of transduction currents in Ush1g^fl/fl and Ush1g^fl/flMyo15-cre^+/− IHCs from the middle of the cochlea at P8. Mean maximum current amplitude is 652 ± 44 pA and 84 ± 44 pA for Ush1g^fl/fl and Ush1g^fl/flMyo15-cre^+/− IHCs, respectively (unpaired t test, P < 10⁻⁴). The P_o(X) curves, however, can be superimposed with average sensitivity values of 1.95 ± 0.12 μm⁻¹ and 2.00 ± 0.61 μm⁻¹ for Ush1g^fl/fl and Ush1g^fl/flMyo15-cre^+/− IHCs, respectively (unpaired t test, P = 0.94). In addition, no change in X_0.5 can be detected in the mutant IHCs (X_0.5 =220 ± 19 nm and 489 ± 122 nm in Ush1g^fl/fl and Ush1g^fl/flMyo15-cre^+/− mice, respectively; unpaired t test, P = 0.07).

Fig. 2.

Hair bundle morphology in OHCs and IHCs from Ush1g^fl/flMyo15-cre^+/− P8 mice. Scanning electron microscopy analysis of OHCs and IHCs from the apex (Upper) and from the middle region of the cochlea (Lower) in Ush1g^fl/fl and Ush1g^fl/flMyo15-cre^+/− P8 mice. In the apical region, the hair bundles of Ush1g^fl/flMyo15-cre^+/− IHCs and OHCs are cohesive. In the apical region, the hair bundles of Ush1g^fl/flMyo15-cre^+/− IHCs and OHCs are cohesive, the tip-links are present, and prolate-shaped stereocilia tips are systematically observed. In the middle region, no differences were detected between Ush1g^fl/fl and Ush1g^fl/flMyo15-cre^+/− OHCs. In contrast, the presence of nonprolate-shaped stereocilia tips within the middle row of stereocilia was frequently detected in Ush1g^fl/flMyo15-cre^+/− IHCs, and the number of tip-links that could be detected in these cells was reduced. At P9, additional hair bundle anomalies appeared in the Ush1g^fl/flMyo15-cre^+/− IHCs from the mid to the apex of the cochlea; specifically, some stereocilia within the small and middle rows had reduced heights. No defects were found in OHCs from the cochlear apex at this stage, but in the middle region of the cochlea, we found the same anomalies as in IHCs. At P22, the reduction of the stereocilia length dramatically worsened, and some of the stereocilia from the small row had even disappeared in both IHCs and OHCs. Notably, the size of the stereocilia that compose the tall row was unchanged in Ush1g^fl/flMyo15-cre^+/− mutants. (Scale bar: 1 μm.)

Fig. 3.

Analysis of stereocilia length in IHCs and OHCs from P8 and P9 Ush1g^fl/flMyo15-cre^+/− mice. Data corresponding to Ush1g^fl/fl and Ush1g^fl/flMyo15-cre^+/− mice are indicated in blue and in red, respectively. Five cells were analyzed in each group. The length of every measurable stereocilium from the middle and small rows was normalized to the mean length of stereocilia in the tall row (L₂/L₁ and L₃/L₁, respectively; mean ± SEM). The numbers (mean ± SEM) of tip-links detected in the apical (apex) and middle (mid) regions of the cochlea are indicated by histograms (Right panels). In Ush1g^fl/flMyo15-cre^+/− IHCs (Upper panels), there is a progressive reduction of the stereocilia length in the middle and small rows and a parallel decrease of the number of tip-links detected, compared with Ush1g^fl/fl IHCs (two-way ANOVA, P < 10⁻⁴ for both comparisons). In Ush1g^fl/flMyo15-cre^+/− OHCs (Lower panels), a decrease of the stereocilia length and number of tip-links is also observed (two-way ANOVA, P < 10⁻⁴ for both comparisons). In Ush1g^fl/flMyo15-cre^+/− P9 mice, note that some stereocilia have completely disappeared in both IHCs and OHCs (red dots on the x axis), specifically, 4% of stereocilia from the small row in IHCs of the mid cochlear region, 3% of stereocilia from the small row in OHCs of the apical region, and 15% and 32% of the stereocilia from the middle and small rows in OHCs of the mid region, respectively.

Fig. 4.

(A) Distribution of sans in mouse cochlear and vestibular hair cells. Hair bundles from E16.5 and P1 mouse cochlear and utricular sensory cells stained using phalloidin to detect F-actin (red) and the antibody to sans (green). From E16.5, sans is detected in the actin-rich protrusions that grow on top of the newly differentiated cochlear hair cells, with a particular concentration at their actin-free distal end. In the P1 cochlea, sans immunoreactivity becomes restricted to stereocilia tips. The stereocilia immunoreactivity is specific to sans as it is absent in Ush1g^−/− P1 mice. In P1 vestibular hair cells, the sans signal is also restricted to stereocilia tips. (B) Distribution of sans in different Ush1 mutant mice at P1. Hair bundles of sensory cells from the cochlear basal turn of Cdh23^−/−, Pcdh15^av3J/av3J, Myo7a^{4626SB/4626SB}, and Ush1c^−/− P1 mice are shown. F-actin (red) and sans (green) stainings as in A. Note the absence of immunoreactivity in the hair bundles of Cdh23^−/− and Pcdh15^av3J/av3J mice. (C) Distribution of sans in cochlear hair cells from P8 Ush1g^fl/fl, Ush1g^fl/flMyo15-cre^+/−, and Ush1g^−/− mice. F-actin (red) and sans (green) stainings as in A. In OHCs from Ush1g^fl/fl mice, sans is detected in the apical region of the stereocilia. In IHCs from Ush1g^fl/fl mice, sans is detected at the distal ends of the stereocilia from the short, medium, and tall rows. Occasional subapical labeling of some tall stereocilia is compatible with a presence of sans at the upper insertion point of the tip-link. In Ush1g^fl/flMyo15-cre^+/− P8 mice, sans is still detected in OHCs, but is no longer present in IHCs. The immunoreactivity of stereocilia is specific as it is absent in Ush1g^−/− P8 mice. Note the nonspecific immunolabeling of the kinocilium. (D) Sans immunogold labeling of a stereocilium from the intermediate stereocilia row in an Ush1g^fl/fl P8 OHC (shown in a transmission electron micrograph). The gold particles (5-nm gold particles, arrow.) are located at the tip of the stereocilium. (Scale bars: 2 μm in A–C and 100 nm in D.)