Protein dynamics underlying cilium-dependent Hedgehog signaling

摘要 Hedgehog（Hh）信号通路是胚胎发育过程中重要的细胞间信号通路 发育和成体组织内稳态。Hh信号错误与几种新生儿出生缺陷有关 例如骨骼畸形和颅面缺损，并与多种肿瘤相关，包括基底动脉瘤， 细胞癌和髓母细胞瘤。脊椎动物Hh信号传导的一个重要但知之甚少的方面是 严格要求一个微管为基础的细胞器称为初级纤毛。虽然我们现在有一个 丰富的数据“零件清单”的蛋白质参与Hh信号，分子机制的基础 纤毛介导的信号转导仍然知之甚少。我们的总体目标是填补这一主要知识 间隙，并通过重建该间隙的关键反应，为Hh信号通路提供生物化学框架。 从其组成部分的路径。对于这项研究，我们将建立在我们的经验，整合单分子 成像方法与生物化学测定和细胞生物学读数，以连接的生化特性， 蛋白质成分对细胞功能的影响。在这个方案中，我们关注Hh信号的一个关键步骤 转导途径，即在纤毛的基部和尖端建立信号复合物，如 是转录因子Gli（Hh的主要效应子）的适当激活或抑制所必需的。 通路在这里，我们将：（1）通过一系列的重构研究来定义这些蛋白质-蛋白质相互作用 并确定他们如何限制Gli与核进口机制的结合，以及（2）分析动力学 纤毛中的关键通路蛋白质的高分辨率实时成像。这些研究将 定义Gli如何通过蛋白质复合物之间的动态转运进行调节， 地点我们希望我们的发现不仅能促进我们对这一基础生物学的理解， 重要的信号转导通路，但也阐明了通路组分中的突变如何起作用 发育障碍和人类癌症的联系

ABSTRACT The Hedgehog (Hh) signaling pathway is a major intercellular signaling pathway important for embryonic development and adult tissue homeostasis. Errors in Hh signaling are linked to several newborn birth defects such as skeletal malformations and craniofacial defects, and associated with multiple tumors including basal cell carcinoma and medulloblastoma. An important but poorly understood aspect of vertebrate Hh signaling is the strict requirement of a microtubule-based organelle known as the primary cilium. While we now have a wealth of data on the “parts-list” of proteins involved in the Hh signaling, the molecular mechanisms underlying cilia-mediated signal transduction remains poorly understood. Our overall goal is to fill this major knowledge gap and provide a biochemical framework for the Hh signaling pathway by reconstituting key reaction of this pathway from its components. For this research, we will build on our experience in integrating single-molecule imaging methods with biochemical assays and cell biological readouts to connect the biochemical properties of the protein components to their cellular function. In this proposal, we focus a key step of the Hh signal transduction pathway which is the establishment of signaling complexes at the base and the tip of the cilia, as is needed for the proper activation or repression of the transcription factor Gli, the major effector of the Hh pathway. Here, we will: (1) define these protein-protein interactions through a series of reconstitution studies and determine how they restrict the Gli binding to the nuclear import machinery and (2) analyze the dynamics of key pathway proteins in the cilium using high-resolution real-time imaging. Together, these studies will define how Gli is regulated through dynamic transit between protein complexes at defined cytoplasmic locations. We expect that our findings will not only advance our understanding of the basic biology of this important signal transduction pathway but also shed light on how mutations in pathway components contribute to developmental disorders and human cancers.