LMO7 deficiency reveals the significance of the cuticular plate for hearing function

Sensory hair cells, the mechanoreceptors of the auditory and vestibular systems, harbor two specialized elaborations of the apical surface, the hair bundle and the cuticular plate. In contrast to the extensively studied mechanosensory hair bundle, the cuticular plate is not as well understood. It is believed to provide a rigid foundation for stereocilia motion, but specifics about its function, especially the significance of its integrity for long-term maintenance of hair cell mechanotransduction, are not known. We discovered that a hair cell protein called LIM only protein 7 (LMO7) is specifically localized in the cuticular plate and the cell junction. Lmo7 KO mice suffer multiple cuticular plate deficiencies, including reduced filamentous actin density and abnormal stereociliar rootlets. In addition to the cuticular plate defects, older Lmo7 KO mice develop abnormalities in inner hair cell stereocilia. Together, these defects affect cochlear tuning and sensitivity and give rise to late-onset progressive hearing loss.


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Software and code
Policy information about availability of computer code Data collection Image data collection was performed using the Zen software (Zeiss) or managed through NIS-Elements software (Nikon Instruments). For ABRs, the SmartEP and for DPOAEs, the SmartOAE software were used to collect data (Intelligent Hearing Systems).

Data analysis
Scaffold viewer version 4.8.8 (free download) was used visualize and analyze Co-IP/MS data. Analysis of confocal images were performed using ImageJ or the NIS-Elements software (Nikon). Graph Pad Prism V7 for statistical analysis.
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Sample size
Sample size for the hearing tests were determined using power analysis. Historical variance of ABR thresholds in the Shin lab was used for the power analysis, with an expectation of a P-value of 0.05. In case no historical data was available, pilot experiments were performed to estimate variance (e.g. for gentamicin uptake assay in Fig.7).
Data exclusions no data were excluded Replication All findings presented in the present study either underwent rigorous statistical treatment (involving multiple independent replications) or in case of qualitative statements (e.g. differences reported in light and electron microscopy studies), findings were replicated multiple times, by multiple experimenters, and for the COS-7 expression studies, in two different laboratories (Shin and Kachar labs).
Randomization Randomization for mouse work was achieved by distributing experimental groups (Lmo7 ko or WT) across cages (in other words, cages did not indicate experimental group). For practical reasons, such randomization was not performed for sexes: mice are housed separated by sex, so when performing hearing tests, the experimenter was aware of the sex of the tested mouse.

Blinding
Data was collected and analyzed in a blinded fashion for most quantifications described in the manuscript except the functional hearing tests. For the longitudinal hearing tests that were performed over a course of 6 months, the experimenter often inadvertently "memorized" tag numbers and their corresponding genotypes, despite initial blinding, because data was analyzed at each age the hearing performance was assessed. To prevent bias, however, analysis of the data (threshold determination based on recorded traces) was confirmed by an independent experimenter.

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The protocol for care and use of animals was approved by the University of Virginia Animal Care and Use Committee. The University of Virginia is accredited by the American Association for the Accreditation of Laboratory Animal Care Note that full information on the approval of the study protocol must also be provided in the manuscript.