Genetic Dissection of Signaling and Cilia

项目概要/摘要 在过去的十年里，在意识到它们是一种 信号传递所需的基本细胞器。解开调节声波的特定机制 纤毛内的hedgehog（Shh）信号传导是困难的，因为如此多的破坏纤毛发生的突变体也 影响Shh信号。我们已经确定了几个小鼠突变破坏纤毛相关基因和嘘 使用正向遗传筛选进行信号传导，目的在于鉴定新基因或已知基因的新等位基因， 直接的神经模式我们的一般策略是在体内表征表型，然后衍生细胞系 以确定细胞表型。从我们的同事那里收集到的知识， 生物化学和人类遗传学，我们测试蛋白质的突变版本，然后选择一些用于体内 建模正是采用这种策略，我们长期以来一直专注于一个小的睫状体GTl 13 b，我们 假设通过不同的效应子及其相互作用整合了纤毛发生和Hh信号传导的调节， 下游路径。作为GT3，Ar 113 b的GT3结构域内的单碱基对突变是 预计会破坏个别效应途径。事实上，我们已经定义了一个Arl 13 b点突变， 破坏了Arl 13 b在纤毛发生中的作用，但使Shh反应保持完整，表明该过程可以 基因分离在接下来的五年里，使用ARL 13 B突变体，一系列基于细胞的检测， 我们可以绕过纤毛发生或敏化Hh破坏的细胞系，我们计划解开Arl 13 b在 纤毛发生，纤毛维持，蛋白质运输到纤毛/纤毛内，以及Shh信号转导， 分辨率我们期望Arl 13 b突变体将提供一个遗传切入点，我们将从中鉴定出至少一个 效应蛋白的子集，并定义其作用机制。细胞培养的工作将使我们能够 选择特定的ARL 13 B突变体，我们可以为它们生成小鼠模型，并将我们的工作带回到 体内表型表征。此外，我们正在将我们发现的新等位基因整合到我们的 分析.因此，我们的建议将产生一个分子遗传工具包，该领域将准备 区分纤毛的调节和Hh信号的调节。这对我们从根本上理解 纤毛，纤毛发生和纤毛结构，以及我们对Shh通路的基本理解。

Project Summary/Abstract Cilia have sparked phenomenal interest in the past decade, after the realization that they are 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 Shh signaling. We have identified several mouse mutants disrupting cilia-related genes and Shh signaling using forward genetic screens aimed at identifying novel genes or new alleles of known genes that direct neural patterning. Our general strategy is to characterize the in vivo phenotype and then derive cell lines from the mutant mice to define cellular phenotypes. With knowledge gleaned from our colleagues in biochemistry and human genetics, we test mutant versions of the proteins, and then select some for in vivo modeling. Employing exactly this strategy, 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. Indeed, we have defined an Arl13b point mutation that disrupts the role of Arl13b in ciliogenesis but leaves the Shh response intact, indicating that the processes can be genetically uncoupled. In the next five years, using ARL13B mutants, a series of cell-based assays, and cell lines in which we can circumvent ciliogenesis or sensitize Hh disruption, we plan to unravel Arl13b function in ciliogenesis, cilia maintenance, traffic of proteins to/within cilia, and Shh signal transduction at unprecedented resolution. We expect Arl13b mutants will provide a genetic entry point from which we will identify at least a subset of effector proteins and define their mechanisms of action. The work in cell culture will enable us to select specific ARL13B mutants for which we can generate mouse models and bring our work full circle back to in vivo phenotypic characterization. In addition, we are integrating the novel alleles we discovered into our analysis. 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 Shh pathway.