Anna Underhill

Myosin-VIIA (MYO7A) is an unconventional myosin responsible for syndromic (Usher 1B) or nonsyndromic forms of deafness in humans when mutated. In the cochlea, MYO7A is expressed in hair cells, where it is believed to act as the motor protein tensioning the mechanoelectrical transducer (MET) channels, thus setting their resting open probability ( P _o ). However, direct evidence for this unique role for an unconventional myosin in mature hair cells is lacking. Here, we show that MYO7A has a distinct role in hair cells, being crucial for the structural integrity of hair bundles. Postnatal deletion of Myo7a leads to 87 to 96% reduction in MYO7A from hair cells by postnatal day 20 (P20), without affecting hearing function. During the following week, mice showed progressive decline in both hearing function and MET current amplitude in hair cells without affecting the resting P _o and calcium sensitivity of the MET channel. Hair-bundle stiffness was normal at P20 but halved at P30, despite it having a normal staircase morphology and tip links. The reduction of MYO7A in the stereocilia (>87%) increased their vulnerability to sound-induced damage, with significantly more hearing loss and hair bundle deterioration than in control mice. RNA-sequencing identified a downregulation of several stereociliary genes in the Myo7a -deficient cochlea, indicating the presence of indirect compensatory mechanisms. This study reveals that mature hair cells seem to use a MYO7A-independent mechanism to maintain the resting P _o of the MET channels. Instead, MYO7A is essential for maintaining the structural and functional integrity of the hair bundles.

Sensory‐independent Ca ²⁺ spiking regulates the development of mammalian sensory systems. In the immature cochlea, inner hair cells (IHCs) fire spontaneous Ca ²⁺ action potentials (APs) that are generated either intrinsically or by intercellular Ca ²⁺ waves in the nonsensory cells. The extent to which either or both of these Ca ²⁺ signalling mechansims are required for IHC maturation is unknown. We find that intrinsic Ca ²⁺ APs in IHCs, but not those elicited by Ca ²⁺ waves, regulate the maturation and maintenance of the stereociliary hair bundles. Using a mouse model in which the potassium channel Kir2.1 is reversibly overexpressed in IHCs ( Kir2.1‐ OE), we find that IHC membrane hyperpolarization prevents IHCs from generating intrinsic Ca ²⁺ APs but not APs induced by Ca ²⁺ waves. Absence of intrinsic Ca ²⁺ APs leads to the loss of mechanoelectrical transduction in IHCs prior to hearing onset due to progressive loss or fusion of stereocilia. RNA‐sequencing data show that pathways involved in morphogenesis, actin filament‐based processes, and Rho‐GTPase signaling are upregulated in Kir2.1‐ OE mice. By manipulating in vivo expression of Kir2.1 channels, we identify a “critical time period” during which intrinsic Ca ²⁺ APs in IHCs regulate hair‐bundle function.

Key points: Mechanoelectrical transduction at auditory hair cells requires highly specialized stereociliary bundles that project from their apical surface, forming a characteristic graded ‘staircase’ structure. The morphogenesis and maintenance of these stereociliary bundles is a tightly regulated process requiring the involvement of several actin‐binding proteins, many of which are still unidentified. We identify a new stereociliary protein, the I‐BAR protein BAIAP2L2, which localizes to the tips of the shorter transducing stereocilia in both inner and outer hair cells (IHCs and OHCs). We find that Baiap2l2 deficient mice lose their second and third rows of stereocilia, their mechanoelectrical transducer current, and develop progressive hearing loss, becoming deaf by 8 months of age. We demonstrate that BAIAP2L2 localization to stereocilia tips is dependent on the motor protein MYO15A and its cargo EPS8. We propose that BAIAP2L2 is a new key protein required for the maintenance of the transducing stereocilia in mature cochlear hair cells. The transduction of sound waves into electrical signals depends upon mechanosensitive stereociliary bundles that project from the apical surface of hair cells within the cochlea. The height and width of these actin‐based stereocilia is tightly regulated throughout life to establish and maintain their characteristic staircase‐like structure, which is essential for normal mechanoelectrical transduction. Here, we show that BAIAP2L2, a member of the I‐BAR protein family, is a newly identified hair bundle protein that is localized to the tips of the shorter rows of transducing stereocilia in mouse cochlear hair cells. BAIAP2L2 was detected by immunohistochemistry from postnatal day 2.5 (P2.5) throughout adulthood. In Baiap2l2 deficient mice, outer hair cells (OHCs), but not inner hair cells (IHCs), began to lose their third row of stereocilia and showed a reduction in the size of the mechanoelectrical transducer current from just after P9. Over the following post‐hearing weeks, the ordered staircase structure of the bundle progressively deteriorates, such that, by 8 months of age, both OHCs and IHCs of Baiap2l2 deficient mice have lost most of the second and third rows of stereocilia and become deaf. We also found that BAIAP2L2 interacts with other key stereociliary proteins involved in normal hair bundle morphogenesis, such as CDC42, RAC1, EPS8 and ESPNL. Furthermore, we show that BAIAP2L2 localization to the stereocilia tips depends on the motor protein MYO15A and its cargo EPS8. We propose that BAIAP2L2 is key to maintenance of the normal actin structure of the transducing stereocilia in mature mouse cochlear hair cells.