Defects in myosin VIIa, harmonin (a PDZ domain protein), cadherin 23, protocadherin 15 and sans (a putative scaffolding protein), underlie five forms of Usher syndrome type I (USH1). Mouse mutants for all these proteins exhibit disorganization of their hair bundle, which is the mechanotransduction receptive structure of the inner ear sensory cells, the cochlear and vestibular hair cells. We have previously demonstrated that harmonin interacts with cadherin 23 and myosin VIIa. Here we address the extent of interactions between the five known USH1 proteins. We establish the previously suggested sans-harmonin interaction and find that sans also binds to myosin VIIa. We show that sans can form homomeric structures and that harmonin b can interact with all harmonin isoforms. We reveal that harmonin also binds to protocadherin 15. Molecular characterization of these interactions indicates that through its binding to four of the five USH1 proteins, the first PDZ domain (PDZ1) of harmonin plays a central role in this network. We localize sans in the apical region of cochlear and vestibular hair cell bodies underneath the cuticular plate. In contrast to the other four known USH1 proteins, no sans labeling was detected within the stereocilia. We propose that via its binding to myosin VIIa and/or harmonin, sans controls the hair bundle cohesion and proper development by regulating the traffic of USH1 proteins en route to the stereocilia.

The neuroepithelia of the inner ear contain hair cells that function as mechanoreceptors to transduce sound and motion signals. Mutations affecting these neuroepithelia cause deafness and vestibular dysfuction in humans. Ames waltzer (av) is a recessive mutation found in mice that causes deafness and a balance disorder associated with the degeneration of inner ear neuroepithelia. Here we report that the gene that harbours the av mutation encodes a novel protocadherin. Cochlear hair cells in the av mutants show abnormal stereocilia by 10 days after birth (P10). This is the first evidence for the requirement of a protocadherin for normal function of the mammalian inner ear.

We have determined the molecular basis for Usher syndrome type 1F (USH1F) in two families segregating for this type of syndromic deafness. By fluorescence in situ hybridization, we placed the human homolog of the mouse protocadherin Pcdh15 in the linkage interval defined by the USH1F locus. We determined the genomic structure of this novel protocadherin, and found a single-base deletion in exon 10 in one USH1F family and a nonsense mutation in exon 2 in the second. Consistent with the phenotypes observed in these families, we demonstrated expression of PCDH15 in the retina and cochlea by RT-PCR and immunohistochemistry. This report shows that protocadherins are essential for maintenance of normal retinal and cochlear function.

Immunocytochemical studies have shown that protocadherin-15 (PCDH15) and cadherin-23 (CDH23) are associated with tip links, structures thought to gate the mechanotransducer channels of hair cells in the sensory epithelia of the inner ear. The present report describes functional and structural analyses of hair cells from Pcdh15av3J (av3J), Pcdh15av6J (av6J) and Cdh23v2J (v2J) mice. The av3J and v2J mice carry point mutations that are predicted to introduce premature stop codons in the transcripts for Pcdh15 and Cdh23, respectively, and av6J mice have an in-frame deletion predicted to remove most of the 9th cadherin ectodomain from PCDH15. Severe disruption of hair-bundle morphology is observed throughout the early-postnatal cochlea in av3J/av3J and v2J/v2J mice. In contrast, only mild-to-moderate bundle disruption is evident in the av6J/av6J mice. Hair cells from av3J/av3J mice are unaffected by aminoglycosides and fail to load with [3H]-gentamicin or FM1-43, compounds that permeate the hair cell's mechanotransducer channels. In contrast, hair cells from av6J/av6J mice load with both FM1-43 and [3H]-gentamicin, and are aminoglycoside sensitive. Transducer currents can be recorded from hair cells of all three mutants but are reduced in amplitude in all mutants and have abnormal directional sensitivity in the av3J/av3J and v2J/v2J mutants. Scanning electron microscopy of early postnatal cochlear hair cells reveals tip-link like links in av6J/av6J mice, substantially reduced numbers of links in the av3J/av3J mice and virtually none in the v2J/v2J mice. Analysis of mature vestibular hair bundles reveals an absence of tip links in the av3J/av3J and v2J/v2J mice and a reduction in av6J/av6J mice. These results therefore provide genetic evidence consistent with PCDH15 and CDH23 being part of the tip-link complex and necessary for normal mechanotransduction.