Genetic Dissection of Signaling and Cilia

信号传导和纤毛的基因剖析

基本信息

批准号：
9892778
负责人：
TAMARA J. CASPARY
金额：
$ 9.54万
依托单位：
EMORY UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2017
资助国家：
美国
起止时间：
2017-09-20 至 2022-08-31
项目状态：
已结题

项目摘要

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.

项目概要/摘要纤毛在过去十年中引起了人们极大的兴趣，因为人们意识到它们是一种信号传导所需的基本细胞器。解开调节索尼克的具体机制纤毛内的刺猬 (Shh) 信号传导很困难，因为许多突变体也会破坏纤毛发生影响Shh信号传导。我们已经鉴定出几种破坏纤毛相关基因和“嘘”的小鼠突变体使用正向遗传筛选进行信号传导，旨在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表型，然后衍生细胞系来自突变小鼠来定义细胞表型。凭借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造型。正是采用这种策略，我们长期以来一直专注于一种小的纤毛 GTP 酶，Arl13b，我们假设通过不同的效应器及其它们整合了纤毛发生和 Hh 信号传导的调节下游途径。作为 GTP 酶，Arl13b 的 GTP 酶结构域内的单碱基对突变是预计会破坏个体效应通路。事实上，我们已经定义了 Arl13b 点突变破坏 Arl13b 在纤毛发生中的作用，但保持 Shh 反应完整，表明该过程可以基因上是解偶联的。在接下来的五年中，使用ARL13B突变体、一系列基于细胞的测定以及细胞我们可以在其中规避纤毛发生或使 Hh 破坏敏感，我们计划解开 Arl13b 的功能纤毛发生、纤毛维持、纤毛内/纤毛内的蛋白质运输以及 Shh 信号转导以前所未有的速度进行解决。我们预计 Arl13b 突变体将提供一个基因切入点，从中我们至少可以鉴定出效应蛋白的子集并定义其作用机制。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，并使我们的工作回到原点体内表型表征。此外，我们正在将我们发现的新颖等位基因整合到我们的基因中分析。因此，我们的建议将产生一个分子遗传工具包，该领域将准备好区分纤毛的调节和 Hh 信号传导的调节。这对于我们的基本理解很重要纤毛、纤毛发生和纤毛结构，以及我们对 Shh 通路的基本理解。