Molecular Basis Of Transduction In Auditory Sensory Orga

1)发现了立体纤毛肌动蛋白核心的快速更新。 我们利用β-肌动蛋白在立体纤毛中的优先定位来确定在立体纤毛发育过程中肌动蛋白聚合的位置，并评估成熟的立体纤毛中Corti器官毛细胞中肌动蛋白的周转率。通过基因枪法将绿色荧光蛋白标记的β-肌动蛋白(β-肌动蛋白-GFP)转化为绿色荧光蛋白-β-肌动蛋白(β-肌动蛋白-GFP)，我们发现位于立体纤毛中心的半晶肌动蛋白细丝束不断更新。更新是通过在顶端添加肌动蛋白单体并在底部移除，从而使整个立体纤毛每48小时更新一次。认识到立体纤毛结构的这一动态方面对于理解正常感觉功能的发展、修复和维持是至关重要的。 2)肌球蛋白XVA在纤毛顶端的定位。 肌球蛋白XVA基因突变导致人类隐性非综合征先天性耳聋(DFNB3)。在小鼠中，肌球蛋白XVA突变导致Shaker-2表型，特征是耳聋和前庭障碍，以及立体纤毛的异常发展。肌球蛋白XVA编码一个390 kDa的蛋白质，它包含一个位于运动域之前的独特的1200aa N末端延伸。我们使用针对肌球蛋白XVA不同结构域的亲和纯化抗体来证明它存在于听觉和前庭毛细胞的纤毛末端。我们与T.Friedman？S博士合作，通过肌球蛋白XVA-GFP的体外转染，证实其靶向于立体纤毛的尖端，提示它可能参与了转导机制或肌动蛋白的更新机制。

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.