Hedgehog signaling and intracellular transport

Hedgehog 信号传导和细胞内运输

• 批准号: 6960741
• 负责人: JONATHAN T EGGENSCHWILER
• 金额: $ 32.4万
• 依托单位: PRINCETON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-07-15 至 2010-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6960741
• 关键词: biological signal transduction; cell line; flagellum; gene mutation; genetic regulation; genetically modified animals; immunocytochemistry; inhibitor /antagonist; intracellular transport; kinesin; laboratory mouse; laboratory rabbit; molecular /cellular imaging; myosins; neuronal transport; protein localization; protein protein interaction; protein structure function; transcription factor; transport proteins

DESCRIPTION (provided by applicant): Embryonic cells use the Hedgehog signaling pathway to convey information that controls cell fate, proliferation, and survival. Misregulation of this pathway leads to birth defects and a variety of cancers in humans. Recent data point to key mechanisms controlling the Hedgehog pathway in mammals that do not appear to be evolutionary conserved in invertebrate model organisms. The primary objective of the proposed research is to understand how Hedgehog signal transduction functions at the mechanistic level using the mouse as a model system. Specifically, the roles of 2 novel antagonists of the mammalian pathway, Wdr10 and Tulp3, will be studied using genetic, cellular, and biochemical approaches outlined in 3 specific aims. The Wdr10 protein, as well as its invertebrate homologs, functions in intraflagellar transport, a process used for building cilia and flagella. The experiments proposed in Aim 1 will confirm the requirement for Wdr10 in regulating the Hedgehog pathway through targeted mutagenesis and transgenic rescue approaches. Other experiments will address the subcellular localization of Wdr10 and test whether it acts in retrograde transport using immunocytochemistry and live cell imaging techniques. The mechanism of Wdr10 function in the Hedgehog pathway will be addressed in Aim 2. The hypothesis that Wdr10 acts cell autonomously will be tested through chimera analysis and the step in the Hedgehog pathway at which Wdr10 acts will be clarified through epistasis analysis. In addition, the hypothesis that Wdr10 acts directly or indirectly in controlling nuclear localization of the Gli transcription factors will be pursued using immunohistochemical and biochemical methods. Finally, the experiments in Aim 3 will test 2 hypotheses regarding the function of Tulp3 in Hedgehog signaling. First, the importance of the interaction between Tulp3 and myosin Vb will be functionally tested by blocking myosin Vb activity in vivo. Second, the significance of regulated Tulp3 nuclear translocation in Hedgehog signaling will be examined in tissue culture cells.

描述（由申请人提供）：胚胎细胞使用 Hedgehog 信号通路来传递控制细胞命运、增殖和存活的信息。该通路的失调会导致人类出生缺陷和多种癌症。最近的数据表明，哺乳动物中控制 Hedgehog 通路的关键机制在无脊椎动物模型生物中似乎并不具有进化保守性。本研究的主要目的是使用小鼠作为模型系统来了解 Hedgehog 信号转导在机械层面上的功能。具体而言，将使用 3 个具体目标中概述的遗传、细胞和生化方法来研究哺乳动物途径的 2 种新型拮抗剂 Wdr10 和 Tulp3 的作用。 Wdr10 蛋白及其无脊椎动物同源物在鞭毛内运输中发挥作用，这是用于构建纤毛和鞭毛的过程。目标 1 中提出的实验将通过定向诱变和转基因拯救方法确认 Wdr10 在调节 Hedgehog 通路中的需求。其他实验将解决 Wdr10 的亚细胞定位，并使用免疫细胞化学和活细胞成像技术测试它是否在逆行运输中发挥作用。 Wdr10 在 Hedgehog 通路中的作用机制将在目标 2 中阐述。将通过嵌合体分析检验 Wdr10 自主作用细胞的假设，并通过上位性分析阐明 Wdr10 在 Hedgehog 通路中作用的步骤。此外，Wdr10 直接或间接控制 Gli 转录因子的核定位的假设将使用免疫组织化学和生化方法进行研究。最后，目标 3 中的实验将测试关于 Tulp3 在 Hedgehog 信号传导中的功能的 2 个假设。首先，Tulp3 和肌球蛋白 Vb 之间相互作用的重要性将通过体内阻断肌球蛋白 Vb 活性进行功能测试。其次，将在组织培养细胞中检查 Hedgehog 信号传导中受调节的 Tulp3 核转位的重要性。