Class III myosins shape the auditory hair bundles by limiting microvilli and stereocilia growth

Abstract

The precise architecture of hair bundles, the arrays of mechanosensitive microvilli-like stereocilia crowning the auditory hair cells, is essential to hearing. Myosin IIIa, defective in the late-onset deafness form DFNB30, has been proposed to transport espin-1 to the tips of stereocilia, thereby promoting their elongation. We show that Myo3a(-/-)Myo3b(-/-) mice lacking myosin IIIa and myosin IIIb are profoundly deaf, whereas Myo3a-cKO Myo3b(-/-) mice lacking myosin IIIb and losing myosin IIIa postnatally have normal hearing. Myo3a(-/-)Myo3b(-/-) cochlear hair bundles display robust mechanoelectrical transduction currents with normal kinetics but show severe embryonic abnormalities whose features rapidly change. These include abnormally tall and numerous microvilli or stereocilia, ungraded stereocilia bundles, and bundle rounding and closure. Surprisingly, espin-1 is properly targeted to Myo3a(-/-)Myo3b(-/-) stereocilia tips. Our results uncover the critical role that class III myosins play redundantly in hair-bundle morphogenesis; they unexpectedly limit the elongation of stereocilia and of subsequently regressing microvilli, thus contributing to the early hair bundle shaping.

Figure 1.

Hearing sensitivity in myosin III–deficient mice and distribution of class III myosins in the hair bundle. (A and B) ABR thresholds for pure tones between 5 and 32 kHz (A), and DPOAE amplitudes and thresholds for primary tone frequencies f₁ = 15 kHz and f₂ = 1.2 f₁ (B) in Myo3a^+/−, Myo3a^−/−, and Myo3b^−/− mice. Plotted curves represent averages over the period 2–4 mo. (C and D) ABR thresholds (C) and DPOAE amplitudes and thresholds for the same primary frequencies (D) in Myo3a^+/−Myo3b^−/− (dark purple) and Myo3a^−/−Myo3b^−/− (red) mice. Averages (mean ± SEM) are plotted over the age period 1–3 mo. The dashed line indicates the noise floor of the microphone (−8 dB SPL); 0 dB SPL marks a sound pressure level at the human hearing threshold at 1 kHz (20 µPa). Asterisks indicate significance (***, P < 0.001; ns, nonsignificant difference; Welsch's t test). (E and F) Myosin IIIa (E) and myosin IIIb (F) in F-actin–labeled (red) IHC hair bundles. On E16.5, both myosins display robust expression in the stereocilia. On P5, they are located at the stereocilia apices but excluded from their extreme tips (right). Bars: (left and middle) 2 µm; (right) 500 nm.

Figure 2.

Myosin IIIa distribution and hearing sensitivity in Myo3a-cKO mice. (A) Cochleas of Myo3a-cKO mice immunostained with anti–myosin IIIa antibodies (green) on P8 and P13. Note the disappearance of myosin IIIa from the F-actin–labeled stereocilia (red) on P13. Bars, 5 µm. (B) ABR thresholds of Myo3a^fl/flMyo3b^−/− (controls, gray), Myo3a-cKO (orange), and Myo3a-cKO Myo3b^−/− (yellow) mice, averaged over the age period of 1–3 mo (mean ± SEM).

Figure 3.

Architecture of cochlear hair bundles in wild-type and Myo3a^−/−Myo3b^−/− mice. (A) On P0, more pronounced defects are observed at the cochlear apex in Myo3a^−/−Myo3b^−/− mice than at the cochlear base. (B) Examples of IHC and OHC hair bundle defects in Myo3a^−/−Myo3b^−/− mice on E16.5, P0, and P9. The shape abnormalities are grouped as long amorphous bundles (a), V-like–shaped bundles (b), and others (c; hair bundles showing a well-defined but abnormally shaped staircase of stereocilia, e.g., rounded or closed, or harboring lateral “wings”). An example of central-exuberant protrusions in an E16.5 OHC bundle (arrow) is shown. On P0, a few IHC bundles displayed base-to-tip tapering of the stereocilia (arrowheads). (C) Fractions of the various categories of hair bundle shape abnormalities in Myo3a^−/−Myo3b^−/− cochleas, estimated for 26 IHCs and 91 OHCs on E16.5 and for 33 IHCs and 117 OHCs on P0. 95% confidence intervals are indicated in parentheses. Bars: (A) 5 µm; (B and C) 2 µm.

Figure 4.

Geometric characteristics of IHC and OHC hair bundles in Myo3a^−/−Myo3b^−/− mice. (A) Projected height of the tallest stereocilia row in the IHC and OHC hair bundles of control and Myo3a^−/−Myo3b^−/− mice, with illustrative P0 hair bundles. Mean ± SEM for bundles with a distinct tallest row (types b or c) is plotted on P0 and P9. (B) The mean number of stereocilia, the interstereocilia distances within the tallest row, and between the three tallest rows are plotted (mean ± SEM) for P0 and P9 mice. Representative hair bundles illustrating the measurements performed, e.g., the interstereocilia distances and the total length of the tallest row of stereocilia (used to estimate the total number of stereocilia) are shown. Cell numbers for the various characteristics averaged are given in Table 1. Bars, 2 µm. Asterisks indicate significance as in Table 1.

Figure 5.

Supernumerary and abnormal distribution of stereocilia rootlets in the hair bundles of Myo3a^−/−Myo3b^−/− mice. (A) Immunostaining for TRIOBP labels the actin filament rootlets at the bases of stereocilia in P4 Myo3a^+/−Myo3b^−/− (controls) and Myo3a^−/−Myo3b^−/− mice and the quantification of the number of stereocilia rootlets at the apical surfaces of the IHCs and OHCs. (B) Mean values (mean ± SEM) of interrow and intrarow spacing between rootlets. Bars, 2 µm. Asterisks indicate significance as in Table 1.

Figure 6.

Cochlear hair cells of P7 Myo3a^−/−Myo3b^−/− mice display normal MET features. (A and F) The mechanical stimulation protocol (upper traces), with examples of MET currents in a Myo3a^−/−Myo3b^−/− IHC and a Myo3a^−/−Myo3b^−/− OHC (red traces). (B and G) The mean amplitude–displacement relationships [I(X)] in both IHCs (n = 6) and OHCs (n = 11) from Myo3a^−/−Myo3b^−/− mice show a reduced response sensitivity compared with IHCs (n = 25) and OHCs (n = 19) from control mice (wild-type or Myo3a^+/−Myo3b^−/− littermates). (C–E and H–J) Adaptation characteristics of MET currents (extent, C and H; fast rate, D and I; slow rate, E and J) in either IHCs or OHCs from Myo3a^−/−Myo3b^−/− mice do not differ significantly from those measured in control IHCs or OHCs.

Figure 7.

Supernumerary stereocilia in Myo3a^−/−Myo3b^−/− mice display inward MET currents. MET currents recorded from a P7 OHC with a circular hair bundle. The thin glass probe used to stimulate the bundle is illustrated in the insets (bar, 2 µm). (A) Response to a sine wave deflection (protocol shown in the upper gray trace). The phase of stimulation directed toward the kinocilium, and that directed in the opposite (negative) direction, both elicit depolarizing inward MET currents (the latter of smaller amplitude, arrows) that are sensitive to treatment with 5 mM BAPTA (lower trace). (B) Response to displacement steps in the negative direction (protocol shown above). The fast and slow adaptation time constants indicated were estimated by fitting a double exponential y = y₀ + A₁ exp(−Δx/τ₁)+A₂ exp(−Δx/τ₂) to the maximum displacement response (black curve; fit parameters: y₀ = −36.1 pA, A₁ = −27.8 pA, A₂ = −13.5 pA; x₀ = 50.6 ms). (C) The activation time constants of the MET current traces shown in B, estimated from a single exponential fit, are similar to those of MET currents recorded in control OHCs.

Figure 8.

Espin-1 and MORN4, both binding to myosin IIIa, are properly targeted to the stereocilia tips in Myo3a^−/−Myo3b^−/− mice. (A) Espin-1 immunostaining (green) in F-actin–labeled IHC and OHC hair bundles (red) from P0 Myo3a^+/−Myo3b^−/− (control) and Myo3a^−/−Myo3b^−/− mice. (B) MORN4 immunostaining (green) in control and Myo3a^−/−Myo3b^−/− mice. (left) A transient pattern of MORN4 expression during development is observed from the base to the apex of the cochlea between E17.5 and P3. (middle and right) MORN4 is detected at the stereocilia tips in IHC and OHC hair bundles in control and Myo3a^−/−Myo3b^−/− mice. Bars: (B, left) 500 µm; (other panels) 2 µm.

Figure 9.

Schematic representations illustrating the role of class III myosins in the developing cochlear hair bundles. (A) OHC hair bundle formation in wild-type and Myo3a^−/−Myo3b^−/− mice. On E14, undifferentiated microvilli cover the apical surface of the differentiating hair cell. E14–E16.5 period: the first phase of stereocilia growth after the kinocilium migration; in Myo3a^−/−Myo3b^−/− mice, unstable growth of stereocilia perturbs their initial organization and selection, resulting in “long amorphous” bundles (a) and other shape abnormalities including exuberant F-actin protrusions (c). E16.5–P0 period: stabilization of the V shape; in Myo3a^−/−Myo3b^−/− mice, hair bundles stabilize but remain affected by various shape abnormalities. P0–P9 period: second phase of stereocilia growth. Past this stage, only myosin IIIa is expressed in wild-type hair cells. (B) Suggested action of myosins IIIa and IIIb in early hair bundle shaping; by forming a ring containing membrane-stabilizing proteins (e.g., MORN4), the two myosins regulate the membrane tension near stereocilia tips and/or the transport and function of proteins involved in the stereocilia actin turnover.