gms | German Medical Science

Research background: OHC stereocilia (in contrast to inner hair cells (IHCs)), are linked to each other by horizontal top connectors (HTCs) and the tallest stereocilia are physically connected to the overlaying tectorial membrane (TM) by tectorial membrane attachment crowns (TM-ACs). Both protein complexes comprise the secreted proteins stereocilin, otogelin, otogelin-like and colocalize with the intracellular adaptor protein, tubby. The absence or mutations of either of the extracellular components results in the loss of both types of stereociliary links and results in hearing loss in humans (DFNB16; DFNB18B; DFNB84B, respectively) and mice. Strikingly, mutation or genetic ablation of tubby similarly disrupts both links. The complete composition and assembly of both protein complexes is unknown. Moreover, how the extracellular components can interact functionally or structurally with the cytoplasmic tubby protein is unclear. Here, we describe the identification of a novel transmembrane component of these complexes that is indispensable for the formation of HTCs and TM-ACs.

Methods: Expression and subcellular localization of hair cell proteins was determined using immunohistochemistry with fluorescence-labelled secondary antibodies in whole-mount preparations of the mouse organ of Corti. Three-dimensional distribution of proteins was imaged by confocal and by STED super-resolution microscopy. Scanning electron microscopy was used to examine the structural integrity of hair bundles.

Results: We identified a so far uncharacterized membrane protein, which we provisionally term TM_OHC that is selectively expressed in outer hair cells. TM_OHC immunoreactivity was restricted to the tips of OHC stereocilia, where it closely co-localized with both tubby and stereocilin in confocal imaging. Super-resolution STED microscopy revealed a ring-like distribution of both TM_OHC and tubby around the tips of the longest stereocilia, indicating their close spatial association with TM-ACs as outlined by previous electron microscopy studies. Additionally, TM_OHC labeling co-localized with tubby to the sites of HTCs. In TM_OHC knockout mice, both stereocilin and tubby were lost from developing and mature OHC hair bundles, and stereocilin immunoreactivity at the stereocilia contact sites of the tectorial membrane (imprints) was abolished. The structural integrity of OHC stereocilia bundles was compromised in the KO mice, as shown by SEM.

Conclusions: We propose that TM_OHC is a novel integral component of the OHC stereocilia TM-AC and HTC complexes. Future studies will reveal the precise role of TM_OHC in structure, assembly, and maintenance of the OHC stereociliary connectors, and help to understand the specific mechanical framework of the OHC hair bundle and its role for OHC transduction and cochlear mechanics.