内耳毛细胞肌球蛋白VI相互作用蛋白筛选及在维持静纤毛结构中的作用

Myosin superfamily play a critical role in the development, structure maintenance and function performance of stereocilia, myosin VI is the only motor in the superfamily that moves towards the plus end of actin filaments, and acts as both anchor and transporter. Mutations in myosin VI (MYO6) underlie nonsyndromic hearing loss DFNA22 and DFNB37.Myo6 mutation snell's mice show that stereociliary bundles are irregular and disorganized and the apical surface of mutant hair cells abnormally protrudes above the epithelium and the membrane near the base of the stereocilia is raised, suggesting myosin VI is required for pulling the membranes down between each stereocilium. In addtion, myosin VI is required for the proper maturation and function of inner hair cell ribbon synapses. However, the exact mechanism for the anchoring and transporting still needs to be explored. In this project, we are going to screen myosin VI interactive proteins in hair cells by co-immunoprecipitation and mass spectrometry, and to testify the screened proteins by western blot and to select the proteins that localize in the cytoplasm membrane between each stereocilium with immunofluorescence technique. By down regulating Myo6b in zebrafish, we want to investigate the distribution of of Myosin VI interactive protreins and what structure changes happen in the apical structure of hair cells. This project is aimed to screen the proteins interactive with Myosin VI in hair cells and explore the role of myosin VI in maintaining the stability of hair cells apical structure, and the implementation of this project helps to uncover the role of myosin VI in stereocilia stability, which will provide cues for the prevention and treatment of stereocilia related sensorineural hearing loss.

肌球蛋白VI是肌球蛋白超家族中唯一朝肌动蛋白丝负端单向运动的分子马达，具有锚定和囊泡运输的功能。肌球蛋白VI基因（MYO6）突变导致DFNA22和DFNB37。Myo6基因突变小鼠静纤毛和突触发育异常，超微结构示毛细胞静纤毛根部细胞膜上抬，导致纤毛的融合、巨静纤毛的产生，和内毛细胞突触减少等，提示肌球蛋白VI参与静纤毛和突触的发育和维持。肌球蛋白VI这些功能的实现依赖于与其它蛋白相互作用，但是哪些蛋白参与毛细胞静纤毛的锚定和囊泡的运输有待进一步研究。本项目拟通过免疫共沉淀联合质谱分析技术筛选毛细胞肌球蛋白VI相互作用蛋白，通过western blot和荧光共定位技术确认相互作用的蛋白；在此基础上，下调Myo6b基因，观察态Myo6b下调对相互作用蛋白在毛细胞内分布的影响以及毛细胞顶部结构的影响。本项目旨在筛选毛细胞肌球蛋白VI相互作用蛋白，探讨肌球蛋白VI维持毛细胞顶端结构稳定的分子机制。

肌球蛋白VI 是肌球蛋白超家族中唯一朝肌动蛋白丝负端单向运动的分子马达，具有锚定和囊泡运输的功能。肌球蛋白VI 基因（MYO6）突变导致DFNA22 和DFNB37。Myo6基因突变小鼠静纤毛和突触发育异常，超微结构示毛细胞静纤毛根部细胞膜上抬，导致纤毛的融合、巨静纤毛的产生，和内毛细胞突触减少等，提示肌球蛋白VI 参与静纤毛和突触的发育和维持。肌球蛋白VI 这些功能的实现依赖于与其它蛋白相互作用，但是哪些蛋白参与毛细胞静纤毛的锚定和囊泡的运输有待进一步研究。本项目通过出生3天C57小鼠和SD大鼠内耳蛋白，结合免疫共沉淀、质谱分析技术筛选出20余种可能与肌球蛋白VI 相互作用蛋白。通过Western blot证实肌球蛋白VI与血影蛋白αⅡ，通过免疫荧光技术发现肌球蛋白VI在整个内毛细胞和外毛细胞的细胞体表达，其中在表皮板的周围强表达，细胞核和纤毛无表达；血影蛋白αⅡ在内、外毛细胞的表皮板强表达，在细胞膜较弱表达，在细胞体的其它部位和细胞核无表达；在表皮板，血影蛋白αⅡ和F-肌动蛋白表达高度一致，纤毛根部位于两种蛋白形成的机构中；此外血影蛋白αⅡ在纤毛的根部强表达，起到加固纤毛根部的作用；肌球蛋白VI和血影蛋白αⅡ均在表皮板表达，其中在毛细胞根部的位置可见部分共表达。本项目研究结果提示血影蛋白αⅡ是肌球蛋白VI的一种相互作用蛋白，在静纤毛结构稳定方面具有重要作用。