Sorting Nexin 9 (SNX9)在听毛细胞表皮板中的功能研究

The cuticular palte is a F-actin-based subcellular structure in auditory hair cells. It occupies the apical cytoplasm of hair cells and serves as an anchor for stereocilia, hence plays an important role in hearing transduction. At present the mechanisms of how this struture is developed and maintained are largely unknown. In previous study we've shown that a WASP-Arp2/3-dependent F-actin regulatory protein, Sorting Nexin 9 (SNX9), is expressed in the cuticular plate of mouse auditory hair cells, and might play an important role in the development and/or maintenance of this structure. We will further investigate the expression patterns of SNX9 and other F-actin regulatory proteins such as WASP and Arp2/3 in the cuticular plate. We will identify SNX9-interacting proetins in hair cells by performing yeast two-hybrid screenings and co-immunoprecipitation, and study the expression and function of some important SNX9-interacting proetins in the cuticular plate. We will also create conditional SNX9 knockout mice and explore the role of SNX9 in the cuticular plate and hearing transduction. This work will help us to further understand the mechanisms of the development and maintainence of the cuticular plate.

表皮板（cuticular plate）是听觉感受器细胞－毛细胞中以F-actin为主体的亚细胞结构，承担了把听纤毛（stereocilia）固定在毛细胞上表面的作用，对听觉传导的发生至关重要。目前人们对调控表皮板发育及维持的机制还不很清楚。申请人发现依赖WASP和Arp2/3的F-actin调控蛋白Sorting Nexin 9 (SNX9) 在小鼠听毛细胞表皮板表达，可能在表皮板的发育或组装中发挥重要作用。我们计划进一步检测SNX9以及WASP、Arp2/3等F-actin调控蛋白在表皮板的表达模式，同时利用酵母双杂交、免疫共沉淀等方法鉴定毛细胞中与SNX9作用的蛋白，并对其中部分重要蛋白在表皮板中的表达和功能进行探索。最后，申请人会构建条件性SNX9基因敲除小鼠，深入探讨SNX9在表皮板发育和维持以及听觉传导中的功能。这一工作将帮助我们进一步认识听毛细胞表皮板发育及维持的机制。

Sorting nexin 9（SNX9）在调控肌动蛋白骨架与细胞膜成管及膜泡形成过程中发挥重要作用。在前期工作中我们发现SNX9在小鼠听觉毛细胞表达，并与FCHSD1协同调控肌动蛋白聚合。在本项目中，我们详细检测了SNX9在毛细胞发育过程中的表达模式，并鉴定了在内耳中表达的SNX9作用蛋白。为进一步深入探讨SNX9的生理功能，我们利用同源重组方法获得了Snx9基因敲除小鼠。Snx9敲除小鼠可以正常存活及繁殖，形态学和行为学方面也与对照小鼠没有差别。进一步研究显示Snx9敲除对听觉毛细胞的形态和功能也没有影响，Snx9基因敲除小鼠的听力水平正常。考虑到FCHSD1与SNX9相互作用并共同调控F-actin聚合，而FCHSD1也在小鼠毛细胞表达，我们计划将Snx9敲除小鼠与Fchsd1敲除小鼠交配，对得到的Fchsd1/Snx9双敲小鼠毛细胞发育、功能及听力水平等进行深入研究。Snx9和Fchsd1基因敲除小鼠的构建和研究对深入认识这两个蛋白在听觉中的生物学功能具有重要意义。