Molecular Characterization of the Ankle-Link Complex in Cochlear Hair Cells and Its Role in the Hair Bundle Functioning

Abstract

Several lines of evidence indicate that very large G-protein-coupled receptor 1 (Vlgr1) makes up the ankle links that connect the stereocilia of hair cells at their base. Here, we show that the transmembrane protein usherin, the putative transmembrane protein vezatin, and the PDZ (postsynaptic density-95/Discs large/zona occludens-1) domain-containing submembrane protein whirlin are colocalized with Vlgr1 at the stereocilia base in developing cochlear hair cells and are absent in Vlgr1 Ϫ/Ϫ mice that lack the ankle links. Direct in vitro interactions between these four proteins further support their involvement in a molecular complex associated with the ankle links and scaffolded by whirlin. In addition, the delocalization of these proteins in myosin VIIa defective mutant mice as well as the myosin VIIa tail direct interactions with vezatin, whirlin, and, we show, Vlgr1 and usherin, suggest that myosin VIIa conveys proteins of the ankle-link complex to the stereocilia. Adenylyl cyclase 6, which was found at the base of stereocilia, was both overexpressed and mislocated in Vlgr1 Ϫ/Ϫ mice. In postnatal day 7 Vlgr1 Ϫ/Ϫ mice, mechanoelectrical transduction currents evoked by displacements of the hair bundle toward the tallest stereocilia (i.e., in the excitatory direction) were reduced in outer but not inner hair cells. In both cell types, stimulation of the hair bundle in the opposite direction paradoxically resulted in significant transduction currents. The absence of ankle-linkmediated cohesive forces within hair bundles lacking Vlgr1 may account for the electrophysiological results. However, because some long cadherin-23 isoforms could no longer be detected in Vlgr1 Ϫ/Ϫ mice shortly after birth, the loss of some apical links could be involved too. The premature disappearance of these cadherin isoforms in the Vlgr1 Ϫ/Ϫ mutant argues in favor of a signaling function of the ankle links in hair bundle differentiation.

Rod disc renewal occurs by evagination of the ciliary plasma membrane that makes cadherin-based contacts with the inner segment

“…7) could provide an environment that favors the formation of flattened blebs. The narrow connecting cilium is linked extracellularly to the IS periciliary ridge (32,33) by fibers containing components of the Usher Syndrome (Ush) protein network, also found in the ankle links of inner ear hair cell cilia (33)(34)(35). The cytoplasmic face of the connecting cilium plasma membrane is linked to microtubules via intraflagellar transport particles.…”

Whirlin and PDZ Domain-containing 7 (PDZD7) Proteins Are Both Required to Form the Quaternary Protein Complex Associated with Usher Syndrome Type 2

“…Whrn, or Pdzd7 gene causes disorganization and gradual degeneration of hair bundles in mice (10,11,16,21,22), which leads to reduction of mechanotransduction responses and hearing loss (10,11,16). Disruption of Whrn or Ush2a expression in the retina results in slow degeneration (21,22); vesicles and vacuoles were found to accumulate around the periciliary membrane complex in Whrn mutant photoreceptors (22).…”

“…In photoreceptors, USH2A, GPR98, and WHRN proteins show mutual dependence for normal localizations at the periciliary membrane complex, and WHRN is able to recruit USH2A and GPR98 to the periciliary membrane complex (22,26). In developing cochlear hair cells, some of the USH2A, GPR98, WHRN, and PDZD7 proteins have been demonstrated to be mutually required for normal localizations at the ankle link complex (11,16). Therefore, USH2A, GPR98, WHRN, and PDZD7 proteins are proposed to form an USH2 protein complex through direct interactions.…”

Clarin-1, Encoded by the Usher Syndrome III Causative Gene, Forms a Membranous Microdomain

“…A link between Usher gene products and actin-based organelles also has been established in vivo. In Usher syndrome I and II mouse models, the actin-enriched stereocilia are morphologically and functionally defective (11)(12)(13)(14). Because the causative gene for Usher type III was identified more recently than those of Usher types I and II, little is known about the pathogenesis of Usher syndrome III.…”