Genetic Dissection of Signaling and Cilia

项目摘要/摘要 纤毛在过去的十年里引起了人们的极大兴趣，因为人们意识到它们是一种 信号传递所需的基本细胞器。解开规范Sonic的具体机制 Hedgehog(Shh)在纤毛内的信号是困难的，因为如此多的突变体也扰乱了纤毛的发生 影响Shh信号。我们已经确定了几个破坏纤毛相关基因和Shh的小鼠突变体 使用正向遗传筛选的信号，目的是识别新基因或已知基因的新等位基因 直接神经模式。我们的总体策略是表征体内的表型，然后衍生出细胞系 从突变小鼠身上提取，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 模特儿。正是采用这种策略，我们长期以来一直专注于一个小的纤毛状GTP酶，Arl13b，我们 假说整合了纤毛发生和HH信号的调节，通过不同的效应器和他们的 下游的小路。作为一种GTP酶，Arl13b的GTP酶结构域中的单个碱基突变是 预计会扰乱单个效应器的通路。事实上，我们已经定义了一种Arl13b点突变 干扰Arl13b在纤毛发生中的作用，但保持Shh反应不变，表明这些过程可以 在基因上是分离的。在接下来的五年里，使用Arl13b突变体，一系列基于细胞的分析，以及细胞 我们可以绕过纤毛发生或使HH破坏敏感的系列，我们计划在 纤毛发生、纤毛维持、纤毛内/纤毛内蛋白质运输以及Shh信号转导都是前所未有的 决议。我们预计arl13b突变将提供一个遗传切入点，从这个切入点我们将识别至少一个 效应蛋白的子集，并定义它们的作用机制。细胞培养方面的工作将使我们能够 选择特定的Arl13b突变体，我们可以为其生成小鼠模型，并将我们的工作带回原点 体内表型特征。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，我们的提议将产生一个分子遗传工具包，场将从该工具包中稳定下来 区分纤毛和HH信号的调节。这对我们的基本认识很重要 纤毛、纤毛发生和纤毛结构，以及我们对Shh途径的基本理解。

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.