Otogelin, otogelin-like, and stereocilin form links connecting outer hair cell stereocilia to each other and the tectorial membrane

Abstract

The function of outer hair cells (OHCs), the mechanical actuators of the cochlea, involves the anchoring of their tallest stereocilia in the tectorial membrane (TM), an acellular structure overlying the sensory epithelium. Otogelin and otogelin-like are TM proteins related to secreted epithelial mucins. Defects in either cause the DFNB18B and DFNB84B genetic forms of deafness, respectively, both characterized by congenital mild-to-moderate hearing impairment. We show here that mutant mice lacking otogelin or otogelin-like have a marked OHC dysfunction, with almost no acoustic distortion products despite the persistence of some mechanoelectrical transduction. In both mutants, these cells lack the horizontal top connectors, which are fibrous links joining adjacent stereocilia, and the TM-attachment crowns coupling the tallest stereocilia to the TM. These defects are consistent with the previously unrecognized presence of otogelin and otogelin-like in the OHC hair bundle. The defective hair bundle cohesiveness and the absence of stereociliary imprints in the TM observed in these mice have also been observed in mutant mice lacking stereocilin, a model of the DFNB16 genetic form of deafness, also characterized by congenital mild-to-moderate hearing impairment. We show that the localizations of stereocilin, otogelin, and otogelin-like in the hair bundle are interdependent, indicating that these proteins interact to form the horizontal top connectors and the TM-attachment crowns. We therefore suggest that these 2 OHC-specific structures have shared mechanical properties mediating reaction forces to sound-induced shearing motion and contributing to the coordinated displacement of stereocilia.

Form and function of the apical extracellular matrix: new insights from Caenorhabditis elegans, Drosophila melanogaster, and the vertebrate inner ear

“…At its medial edge, the TM is anchored to the apical surface of the epithelium via the GPI-anchored protein otoancorin; at its lower lateral edge, the TM contacts the apical stereocilia of three rows of sensorimotor outer hair cells (OHCs). Recent genetic and morphological analysis has revealed that otogelin, otogelin-like, and the membrane-anchored stereocilin play interrelated roles in connecting the tips of OHC stereocilia to the TM 46 . While otogelin, otogelin-like, and stereocilin all localize independently within OHC hair bundles, loss of any one protein disrupts the distribution of the other two, suggesting interdependent roles in TM-OHC attachment.…”

Interaction of protocadherin-15 with the scaffold protein whirlin supports its anchoring of hair-bundle lateral links in cochlear hair cells

“…Finally, the presence of both L-whirlin and the PCDH15 isoforms CD1 and CD2 at the tips of the tall stereocilia, and their physical interaction demonstrated here in vitro, raises the possibility that L-whirlin might be required to anchor a subpopulation of apical lateral links containing PCDH15 or CDH23 to the actin cores of these stereocilia. Indeed, the top-connector-associated proteins stereocilin, otogelin and otogelin-like have an abnormally extended distribution in the absence of PCDH15 or CDH23 17 . Accordingly, the impaired DPOAEs and frequency tuning in mutant mice lacking the L-whirlin isoform could reflect a loosening of the OHC hair bundle cohesiveness due to the loss of these apical lateral links, since the loss of OHC bundle cohesiveness abolishes DPOAEs, as occurs in stereocilin-null mutant mice which lack the top-connectors 16,17,50 .…”

“…Indeed, the top-connector-associated proteins stereocilin, otogelin and otogelin-like have an abnormally extended distribution in the absence of PCDH15 or CDH23 17 . Accordingly, the impaired DPOAEs and frequency tuning in mutant mice lacking the L-whirlin isoform could reflect a loosening of the OHC hair bundle cohesiveness due to the loss of these apical lateral links, since the loss of OHC bundle cohesiveness abolishes DPOAEs, as occurs in stereocilin-null mutant mice which lack the top-connectors 16 , 17 , 50 .

Figure 5 L-whirlin-interacting networks at the ankle-links and at stereocilia tips.

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“…These proteins also form transient lateral links in the developing hair bundle 13 – 15 . Likewise, current evidence indicates that usherin and adhesion G protein-coupled receptor V1 (ADGRV1), two large transmembrane proteins, are components of the ankle links 9 , while stereocilin, otogelin and otogelin-like are associated with the OHC horizontal top connectors 16 , 17 , and protein tyrosine phosphatase receptor type Q (PTPRQ) is associated with shaft connectors 18 . By contrast, the proteins bridging the various types of links to the dense F-actin cytoskeleton of the stereocilia remain largely unknown, despite a number of putative candidates.…”

Serial scanning electron microscopy of anti-PKHD1L1 immuno-gold labeled mouse hair cell stereocilia bundles

“…Stereocilia are cross-linked by a number of transient or permanent links, collectively termed as stereociliary 'surface specializations' , which are identified with electron microscopy and ensure bundle integrity 1 . A list of proteins were shown to be important for the hair cell stereocilia bundle structure and function, many of which were localized to the bundle using variations of immunogold labeling protocols, and observed with electron microscopy (EM) techniques, mostly using transmission electron microscopy (TEM) [2][3][4][5][6][7][8][9][10][11][12][13][14][15] and more lately using scanning electron microscopy (SEM) [16][17][18][19][20][21] . In the most recent years though, serial electron microscopy techniques have become more accessible for use in biology, mounting a new body of work using 3D EM approaches 17,22,23 .…”