Mechanotransduction-dependent changes in the contributions of actin isoforms to the auditory stereocilia cytoskeleton

Abstract

ABSTRACT: Stereocilia, mechanosensitive projections on top of the inner ear hair cells, are made of filaments from both cytoplasmatic actin isoforms: β and γ- (Furness et al., 2005; Perrin et al., 2010). The absence of either of these isoforms results in the normal growth of stereocilia but they eventually degenerate (Belyantseva et al., 2009; Perrin et al., 2010). Moreover, a decrease in the resting influx of Ca2+ through the mechanotransduction (MET) channels, disrupts the stability of the stereocilia cytoskeleton, leading to thinning and shrinking of stereocilia (Vélez-Ortega et al., 2017). When bound to Ca2+, β- actin has faster polymerization and depolymerization kinetics than γ- actin (Bergeron et al., 2010) so changes in Ca2+ influx to the hair cell through MET channels might affect both isoforms in a different way. Therefore, the existence of MET-dependent changes in the distribution of β- and γ- actin along stereocilia lengths in mouse auditory hair cells was evaluated. Organ of Corti explants from wildtype mice were isolated at early postnatal days and cultured in control conditions or in the presence of the MET channel blocker tubocurarine (60 µM). Explants were fixed when freshly isolated or at specific time points after MET channel blockage. Depending on the stage of the project, samples were either immunostained with fluorescently-labeled antibodies against β- and γ-actin isoforms and imaged via confocal microscopy, or prepared for scanning electron microscopy (SEM) imaging. Obtained images from confocal microscopy were processed and measured using the created algorithm. It was found that the ratios of γ- to β- actin along the length of stereocilia were not affected in culture conditions up to 48 hours. Evidence was provided on the thinning and shortening of second and third row of stereocilia after the incubation with tubocurarine. Nevertheless, according to the presented data on the fluorescence quantification, no changes in the ratio of β- and γ- actin were found during these MET-dependent cytoskeleton rearrangements. Therefore, it can be affirmed that Ca2+-dependent differences in polymerization and depolymerization rates between β- and γ- actin are not the main mechanism driving stereocilia cytoskeleton rearrangements upon changes in intracellular Ca2+ concentrations in mammalian auditory hair cells, since the proportions of both actin isoforms were maintained in the stereocilia cytoskeleton during this process.