Molecular dissection of the ciliary gate

项目摘要 纤毛是大多数真核细胞表面的感觉元件，在细胞发育过程中起着至关重要的作用。 纤毛组装通过鞭毛内运输(IFT)和感觉转导能力在 所有有纤毛的生物。随着过去人类疾病基因定位克隆的快速发展 十年来，各种疾病，如常染色体显性遗传性多囊肾病(ADPKD) 在分子上被定性为纤毛病。与无处不在的纤毛的存在一致，许多 纤毛疾病是一种综合征，影响多个器官，包括肾脏、肝脏、四肢、眼睛和 中枢神经系统。纤毛生物学中的一个中心问题是纤毛门是如何在功能上分开的 纤毛从细胞体中分离出来，使其成为一个离散的传感细胞器。在纤毛发生过程中，远端 母中心粒的附属物转化为过渡纤维，形成一个9叶螺旋桨。 连接基体部和睫状体基底膜的结构。转录因子的独特亚细胞定位 使它成为睫状门的一个很好的候选者。尽管如此，最大的挑战是分子 关于作为睫状门的TF的建立，无论是结构上的还是功能上的，仍然没有明确的定义。 由于纤毛在哺乳动物胚胎发育中的重要作用，对纤毛之间关系的研究 纤毛和疾病在哺乳动物模型中是极其困难的。因此，替代的实验系统是 这是必要的。秀丽隐杆线虫已被建立为一种有效的模式来表征 纤毛蛋白在天然细胞环境中的生理作用与理解相关 哺乳动物生物学由于高度保守的纤毛组成和信号。我们率先推出了这一应用程序 以秀丽线虫为模型研究转铁蛋白的生物学意义。我们的初步研究表明，DYF-19 与不同的运动员进行物理联系以调节不同的纤毛门控：使用DYF-19-TALPID-3-ANK-26 功能模块用于调节IFT进口，而DYF-19-CCDC-85模块用于调节 膜蛋白。另一方面，房屋及规划地政局局长与政府助理署长8协调，以规管 睫状门。我们还找回了新的蠕虫突变体，可能在正向遗传中破坏了TF的完整性 放映。此外，我们的初步研究表明，在线虫中的关键发现非常重要 在哺乳动物细胞中保守。在这份提案中，我们将确定以下各项的全部组成部分和活动 纤毛门在纤毛和纤毛病变中的基本作用 更好地理解。我们计划通过三个具体的问题来实现这一目标：i)，纤毛门是如何 实现了吗？2)睫状门是如何建立的？以及iii)如果睫状门的核心通路是 在哺乳动物细胞中保守吗？通过将线虫与哺乳动物系统相结合，我们有信心 提供有关纤毛门的分子同一性和核心保守途径的种子信息， 并大大扩展了我们对纤毛生物学以及人类纤毛疾病发病机制的理解。

Project Summary Cilia serve as sensory devices on most eukaryotic cells surface and play an essential role in development. Ciliary assembly via intraflagellar transport (IFT) and sensory transduction capabilities are highly conserved in all ciliated organisms. With rapid advancements in the positional cloning of human disease genes in the past decade, a wide variety of disorders such as autosomal dominant polycystic kidney disease (ADPKD) have been characterized molecularly as ciliopathies. Consistent with the ubiquitous presence of cilia, many ciliopathies occur as syndromic disorders that affect multiple organs, including the kidney, liver, limb, eye, and central nervous system. One central question in cilia biology is that how the ciliary gate functionally separates the cilium from the cell body and makes it a discrete sensing organelle. During ciliogenesis, the distal appendages of the mother centriole transform to transition fibers (TFs), which form a 9-bladed propeller structure connecting the basal body to the ciliary base membrane. The distinct subcellular location of TFs makes it a good candidate for the ciliary gate. Nonetheless, the paramount challenges being that molecular insights about the establishment, either structural or functional, of TFs as the ciliary gate remain poorly defined. Due to the essential roles of cilia in mammalian embryonic development, the study of the connections between cilia and disease are extremely difficult in mammalian models. Thus, alternative experimental systems are necessary. Caenorhabditis elegans has been established as an effective model for characterizing the physiological roles of ciliary proteins in their native cellular environments that is relevant for understanding mammalian biology due to the highly conserved cilia composition and signaling. We pioneered the application of C. elegans as a model to study the biological importance of TFs. Our preliminary studies show that DYF-19 physically associates with different players to regulate distinct cilia gating: with the DYF-19-TALPID-3-ANK-26 functional module in regulating IFT import, whereas DYF-19-CCDC-85 module in regulating gating for membrane proteins. On the other hand, HYLS1 coordinate with GAS8 to regulate the establishment of the ciliary gate. We also retrieved novel worm mutants with likely disrupted TF integrity in a forward genetic screening. Furthermore, our initial studies suggested that the key discoveries made in C. elegans are highly conserved in mammalian cells. In this proposal, we will determine the full components and activities of underlying pathways so that the fundamental roles of the ciliary gate in the context of cilia and ciliopathies are better understood. We plan to achieve this goal by pursuing three specific questions: i), how cilia gating is achieved? ii) how the ciliary gate is established? and iii) if the core pathways for the ciliary gate are conserved in mammalian cells? By combining C. elegans with mammalian systems, we are confident to provide seminal information about the molecular identity and the core conserved pathways of the ciliary gate, and substantially extend our understanding of cilia biology as well as of the pathogenesis of human ciliopathies.