Genetic Dissection of Signaling and Cilia

项目摘要/摘要 当科学家们认识到纤毛是一种基本的细胞器时，纤毛引起了人们极大的兴趣 信令所需的。解开内部调节Sonic Hedgehog(Shh)信号的具体机制 纤毛是困难的，因为许多破坏纤毛发生的突变也影响HH信号。我们有很长的时间 聚焦于一个小的纤毛GTP酶，Arl13b，我们假设整合了纤毛发生和 HH信号通过不同的效应器及其下游通路。作为GTP酶，单一碱基对 Arl13b的GTPase结构域内的突变被预测会扰乱单个效应器通路。规则第13B条 富含纤毛。通过使鼠标仅表达未本地化到的Arl13b变体 纤毛，我们从基因上分离了Arl13b在纤毛发生中的作用和它在信号转导中的作用。我们专注于 哺乳动物的神经发育和通过正向遗传筛选发现的小鼠突变体有几个 与Ar13b相关的蛋白质。此外，ARL家族的GTP酶中的其他蛋白质也与纤毛有关 并通过似乎与Arl13b相关的机制发出信号。在未来五年，我们提出 利用小鼠突变体来确定体内和特定细胞中这些角色之间的调控关系 类型。这些实验将揭示Arl13b在纤毛发生、蛋白质到纤毛/纤毛内的运输以及 以前所未有的分辨率进行Shh信号转导。因此，我们的提议将产生一种分子遗传 该领域将准备好区分纤毛调控和HH信号调控的工具包。这是 对于我们对纤毛、纤毛发生和纤毛结构的基本理解以及我们的基本 HH途径的理解。

Project Summary/Abstract Cilia sparked phenomenal interest as scientists recognized them as a fundamental cellular organelle required for signaling. Untangling the specific mechanisms that regulate Sonic hedgehog (Shh) signaling within the cilium is difficult since so many mutants that disrupt ciliogenesis also affect Hh signaling. We have long focused on a small ciliary GTPase, ARL13B, that we hypothesize integrates the regulation of ciliogenesis and Hh signaling through distinct effectors and their downstream pathways. As a GTPase, single basepair mutations within the GTPase domain of ARL13B are predicted to disrupt individual effector pathways. ARL13B is highly enriched in cilia. By engineering mouse expressing only an ARL13B variant that does not localize to cilia, we genetically uncoupled the role of ARL13B in ciliogenesis from its role in signaling. We focus on mammalian neural development and through forward genetic screens identified mouse mutants in several proteins related to ARL13B. Additionally, other proteins in the ARL family of GTPases are implicated in cilia and signaling through what appear to be ARL13B related mechanisms. In the next five years, we propose using mouse mutants to define the regulatory relationships among these players in vivo and in specific cell types. These experiments will unravel ARL13B function in ciliogenesis, traffic of proteins to/within cilia, and Shh signal transduction at unprecedented resolution. Thus, our proposal will generate a molecular genetic toolkit from which the field will be poised to distinguish the regulation of cilia from that of Hh signaling. This is important to our fundamental understanding of cilia, ciliogenesis and cilia structure, as well as our basic comprehension of the Hh pathway.