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. 2014 Jan 27;9(1):e87331.
doi: 10.1371/journal.pone.0087331. eCollection 2014.

The actin-binding proteins eps8 and gelsolin have complementary roles in regulating the growth and stability of mechanosensory hair bundles of mammalian cochlear outer hair cells

Jennifer Olt  1 , Philomena Mburu  2 , Stuart L Johnson  1 , Andy Parker  2 , Stephanie Kuhn  1 , Mike Bowl  2 , Walter Marcotti  1 , Steve D M Brown  2
Affiliations

The actin-binding proteins eps8 and gelsolin have complementary roles in regulating the growth and stability of mechanosensory hair bundles of mammalian cochlear outer hair cells

Jennifer Olt et al. PLoS One. .

Abstract

Sound transduction depends upon mechanosensitive channels localized on the hair-like bundles that project from the apical surface of cochlear hair cells. Hair bundles show a stair-case structure composed of rows of stereocilia, and each stereocilium contains a core of tightly-packed and uniformly-polarized actin filaments. The growth and maintenance of the stereociliary actin core are dynamically regulated. Recently, it was shown that the actin-binding protein gelsolin is expressed in the stereocilia of outer hair cells (OHCs) and in its absence they become long and straggly. Gelsolin is part of a whirlin scaffolding protein complex at the stereocilia tip, which has been shown to interact with other actin regulatory molecules such as Eps8. Here we investigated the physiological effects associated with the absence of gelsolin and its possible overlapping role with Eps8. We found that, in contrast to Eps8, gelsolin does not affect mechanoelectrical transduction during immature stages of development. Moreover, OHCs from gelsolin knockout mice were able to mature into fully functional sensory receptors as judged by the normal resting membrane potential and basolateral membrane currents. Mechanoelectrical transducer current in gelsolin-Eps8 double knockout mice showed a profile similar to that observed in the single mutants for Eps8. We propose that gelsolin has a non-overlapping role with Eps8. While Eps8 is mainly involved in the initial growth of stereocilia in both inner hair cells (IHCs) and OHCs, gelsolin is required for the maintenance of mature hair bundles of low-frequency OHCs after the onset of hearing.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Mechanotransducer currents in outer hair cells from gelsolin knockout mice (Gsntm1Djk/Gsntm1Djk). A
and B, Saturating mechanotransducer (MET) currents recorded from a control (A) and a gelsolin knockout (B) P6 apical-coil OHC. MET currents (bottom panels) were elicited by applying sinusoidal force stimuli to the hair bundles while changing the membrane potential between −121 mV and +99 mV in 20 mV nominal increments from the holding potential of −81 mV (middle panel). For clarity only responses at −121 mV are shown. The driver voltage (DV) signal of ±40 V at 50 Hz to the fluid jet is shown above the traces (negative deflections of the DV are inhibitory). The arrows indicate the closure of the transducer channels, i.e. disappearance of the resting current, during inhibitory bundle displacements. Dashed lines indicate the holding or resting current. C, Peak-to-peak current-voltage curves were obtained from four control and three knockout OHCs (P6) using 1.3 mM extracellular Ca2+. The fits through the data are according to eqn.1 (see Methods) with values: control k = 494±51, V r = 2.3±0.4 mV, V s = 40±3 mV, and γ = 0.41±0.01; gelsolin knockout k = 537±40, V r = 1.6±0.3 mV, V s = 40±2 mV, and γ = 0.42±0.01. D, Changes in the resting transducer current at −81 mV and +99 mV in control and knockout OHCs. The resting current is given by the holding current minus the current present during inhibitory bundle deflection. E, Fluorescence images with the DIC image superimposed from the control and knockout P11 cochleae taken after exposure to FM1-43 (both OHCs and IHCs were labelled by the dye). Scale bars: 20 µm. F and G, Driver voltages to the fluid jet (top) and transducer currents recorded at –81 mV (left panels) and +99 mV (right) from a control and a knockout gelsolin OHC respectively. At –81 mV, positive driver voltages (excitatory direction) elicited inward transducer currents that declined or adapted over time in control and knockout OHCs (arrows). A small transducer current was present at rest (dashed line) and inhibitory bundle displacements turned this off. Upon termination of the inhibitory stimulus, the transducer current in control and knockout OHCs showed evidence of rebound adaptation (arrowheads). These manifestations of MET current adaptation were absent at positive membrane potentials (e.g. +99 mV) and the resting current increased.
Figure 2
Figure 2. Gelsolin is not involved in the development of OHC basolateral currents in knockout mice (Gsntm1Djk/Gsntm1Djk). A
and B, K+ currents recorded from mature control and knockout OHCs, respectively, elicited by depolarizing voltage steps (10 mV nominal increments) from –124 mV to more depolarized values from the holding potential of –64 mV. The K+ current characteristic of adult OHCs, I K,n, was similarly expressed in OHCs from control and knockout gelsolin mice. C, Steady-state current-voltage curves for the total K+ current in control (n = 6) and gelsolin knockout (n = 4) OHCs. D, Size of the total outward K+ current measured at 0 mV (left) and the isolated adult-type current I K,n, which was measured as the deactivating tail currents (difference between instantaneous and steady-state inward currents) at –124 mV .
Figure 3
Figure 3. Eps8 localization in cochlear hair cells of gelsolin knockout mice (Gsntm1Djk/Gsntm1Djk).
Cochlear whole mounts from 3 month old gelsolin knockout mice were stained with antibody to Eps8 (green) and phalloidin (red) to detect actin. Right hand panels show merge of Eps8 and actin. Eps8 localizes at the tip of stereocilia in both inner (IHC) and outer (OHC) hair cells. In IHC stereocilia Eps8 is present in the tips of both the taller and shorter rows. There was no obvious difference in localization pattern between wild type and gelsolin knockout mice. Scale bars: 10 µm.
Figure 4
Figure 4. Gelsolin localization in cochlear hair cells of Eps8 knockout mice (Eps8tm1Ppdf/Eps8tm1Ppdf).
Cochlear whole mounts from P8 mice were stained with antibody to gelsolin (green) and phalloidin (red) to detect actin. Right hand panels for OHC and IHC cells show merge of gelsolin and actin. Note the absence of IHC labeling. Gelsolin localizes to stereocilia of OHCs (domain D2) and is also detected outside of the stereocilia bundle in strial (D1) and neural (D3) domains of the apical hair cell surface. Gelsolin localization was not affected in the Eps8 knockout mice. Similar results were seen at P6 (data not shown). Scale bars: 10 µm.
Figure 5
Figure 5. Ultrastructural analysis of cochlear hair cell stereocilia in Eps8-gelsolin double heterozygous mutant mice (Eps8tm1Ppdf/+; Gsntm1Djk/+).
SEM analysis of hair cell stereocilia bundles in Eps8-gelsolin double heterozygous mice and wild type litter mate controls at P27. OHCs and IHCs at the apex, mid and basal turns of the cochlea from Eps8-gelsolin double heterozygous mice showed similar hair bundles to those in wild type litter mate controls. Scale bars: 10 µm.
Figure 6
Figure 6. Mechanotransducer currents in OHCs from Eps8-gelsolin double mutant mice (Eps8tm1Ppdf/+; Gsntm1Djk/+). A
and B, Saturating MET currents recorded from apical-coil OHCs of mice that were all knockout for Eps8 (Eps8/Eps8) and either heterozygous (Gsn/+: A, black lines) or homozygous (Gsn/Gsn: B, grey lines) for the gelsolin mutant allele. MET currents were elicited by applying sinusoidal force stimuli as described in Figure 1 . The arrows indicate the resting MET current at hyperpolarized and depolarized membrane potentials. Dashed lines indicate the holding current. C, Peak-to-peak current-voltage curves were obtained from four Eps8/Eps8; Gsn/+ mutants and five Eps8/Eps8; Gsn/Gsn mutants (P6). The fits through the data are according to eqn.1 (see Methods) with values: Eps8/Eps8; Gsn/+k = 372±23, V r = 3.0±0.2 mV, V s = 43 ±2 mV, and γ = 0.47±0.01; Eps8/Eps8; Gsn/Gsn k = 321±20, V r = 0.1±0.3 mV, V s = 45±2 mV, and γ = 0.46±0.01. D, Size of the resting transducer current measured at −81 mV and +99 mV in Eps8/Eps8; Gsn/+ and Eps8/Eps8; Gsn/Gsn OHCs.
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