Molecular Basis Of Transduction In Auditory Sensory Orga

1)静纤毛肌动蛋白核心快速更新的发现。 我们利用β-肌动蛋白在静纤毛的优先定位，以确定在静纤毛发展过程中的肌动蛋白聚合的轨迹，并评估在成熟的静纤毛在大鼠Corti器官的毛细胞的肌动蛋白周转率。利用基因枪技术检测绿色荧光蛋白标记的β-肌动蛋白（betaactin-GFP），我们发现静纤毛核心的半结晶肌动蛋白纤维束是不断更新的。更新是通过在顶端添加肌动蛋白单体并在基部去除而发生的，使得整个静纤毛每48小时更新一次。认识到静纤毛结构的动态方面对于理解正常感觉功能的发育、修复和维持至关重要。 2)肌球蛋白XVA在静纤毛顶端的定位。 肌球蛋白XVA基因突变导致人类隐性、非综合征型先天性耳聋（DFNB 3）。在小鼠中，肌球蛋白XVA突变导致shaker-2表型，其特征在于耳聋和前庭障碍，以及静纤毛的异常发育。肌球蛋白XVA编码一个390 kDa的蛋白，包含一个独特的1200 aa的N-末端延伸之前的电机域。我们使用亲和纯化的抗体，肌球蛋白XVA的不同领域，以表明它是目前在听觉和前庭毛细胞的静纤毛的尖端。与T博士合作。弗里德曼？我们使用肌球蛋白XVA-GFP体外转染证实它靶向静纤毛的尖端，这表明它可能参与转导机制或肌动蛋白更新机制。

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.