RabL在纤毛相关信号转导中的功能研究

Cilia are microtubule-based hair-like organelles present on nearly all vertebrate cells. Cilia can be classified as motile and primary cilia. Motile cilia are important for cell locomotion, removing the materials from cell surface and generating fluid flow, whereas primary cilia act as cell sensors for extracellular signals and platforms for signal transdunction including Sonnic Hedgehog pathway. Ciliary defects lead to a series of genetic disorders, called ciliopathies, and the patients display a series of disorders, including polycystic disease, polydactyly, skeletal abnormalities and mental retardation. In our previous studies, we identified a noval cilia-related small GTPase, called RabL, whose expression level is increased during ciliogenesis. Its ciliary localization is correlated with its GTP/GDP switch and only GTP-bound RabL associates with cilia. Knockdown of RabL in zebrafish results in ciliopathies-like pheotypes, such as bent bodies. Although the cilia structure is not affected, Hedgehog signaling pathway is suppressed upon the ablation of RabL in zebrafish. More interestingly, RabL knockout mice display a series of ciliopathies-related disorders, including polydactyly and skeletal abnormalities. In this proposal, we will work with cell cultures, tissues and knockout mice and a combination of molecular cell biological methods will be used. Our aim is to explore the function and underlying molecular mechanism of RabL in cilia and try to find its link to ciliopathies.

纤毛是一种广泛分布在动物体内突出于细胞表面的毛状细胞器，可分为动纤毛和初级纤毛。动纤毛通过摆动驱使细胞运动、排除异物和形成液体流，而初级纤毛主要作为细胞的"天线"感知胞外环境和介导Hedgehog等信号通路。纤毛功能的异常会导致一类被称为"纤毛病"的遗传疾病，它的病征包括脑水肿、多囊肾、多指/趾、骨骼异常、肥胖和智力低下等。在前期的研究中，我们发现了一个新的纤毛相关的小GTP酶，RabL，它的表达量随着纤毛发生逐渐升高，并且只有激活形式的RabL才能定位到纤毛轴丝上。RabL敲低的斑马鱼具有身体弯曲等一系列类似纤毛病的表型，虽然其纤毛发生未受影响，但是Hedgehog信号通路被强烈抑制。更有趣的是，RabL的基因敲除小鼠具有多趾、骨骼发育异常和肥胖等纤毛病相关的表型。本项目拟通过多种分子细胞生物学手段，利用细胞、组织和基因敲除小鼠等模型，针对RabL在纤毛中的功能和作用机理进行深入的研究。

初级纤毛是一种突出于细胞表面的毛状细胞器。由于纤毛膜上富含各种信号分子，纤毛作为细胞的“天线”，感知胞外信号并介导信号转导。Shh信号通路是广为人知的纤毛相关的信号通路，多个Shh信号分子都依赖纤毛发挥功能。有研究表明BBSome对于Shh信号分子的纤毛运出至关重要，但是这个过程是如何调控的却并不清楚。通过本项目的研究，我们发现了一个纤毛特异的小GTPase，Rabl2，它通过调控鞭毛内运输复合物B（IFT-B）和BBSome的结合，从而调控BBSome和Shh信号分子的纤毛运出过程。Rabl2的基因敲除小鼠具有典型的纤毛病的表型，包括多趾、肥胖、渐进式失明和雄性不育等。BBSome的组分和Shh信号分子都会富集在表达了Rabl2-GTP的突变体的纤毛中，提示Rabl2的GTP/GDP的转换对于BBSome及其相关的信号分子的纤毛运输非常重要。进一步的分子机制的研究，我们发现Rabl2-GTP突变体可能是通过Ift27/Ift25异源二聚体来调控BBSome的纤毛运出的。Rabl2-GTP突变体的替换小鼠出生后致死，并伴随多器官的发育异常。我们的研究不但发现了Rabl2调控了BBSome及其相关的信号分子的纤毛运输，并且提示Rabl2极有可能是一个潜在的纤毛病的致病基因。此外，在本项目中，我们还研究了多个TTC蛋白和一个内动力蛋白臂(dynein-f)的调控基因Wdr78在纤毛中的功能，论文分别发表在PLoS One和JMCB上。