1)Discovery of the rapid renewal of the stereocilia actin core. We exploited the preferential localization of beta actin in the stereocilia to determine the locus of actin polymerization during stereocilia development and to assess the rate of actin turnover in mature stereocilia in hair cells of the rat organ of Corti. Using the gene gun procedure to transfect green fluorescence protein-tagged beta actin (beta actin-GFP), we showed that the semicrystalline actin filament bundle at the core of the stereocilia is continuously renewed. Renewal occurs by the addition of actin monomers at the tips and removal at the base such that the entire stereocilium is renewed every 48 hours. Recognition of this dynamic aspect of stereocilia construction is essential to understand the development, repair, and maintenance of normal sensory function. 2) Localization of , Myosin XVA at the tips of stereocilia. Mutations of myosin XVA gene cause recessive, non-syndromic congenital deafness (DFNB3) in humans.. In mice, myosin XVA mutations cause the shaker-2 phenotype, characterized by deafness and vestibular disorders, and abnormal development of stereocilia. Myosin XVA encodes a 390 kDa protein that contains a unique 1200 aa N-terminal extension that precedes the motor domain. We used affinity-purified antibodies to different domains of myosin XVA to show that it is present at the tips of the stereocilia in auditory and vestibular hair cells. In collaboration with Dr. T. Friedman?s lab (LMG, NIDCD) we used in vitro transfection with myosin XVA-GFP to confirm that it is targeted to the tips of stereocilia, suggesting that it may be involved in either the transduction machinery or the actin renewal mechanism.
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