Protein dynamics underlying cilium-dependent Hedgehog signaling

摘要 Hedgehog(HH)信号通路是一条重要的细胞间信号通路 胚胎发育和成人组织动态平衡。HH信令中的错误与 一些新生儿出生缺陷，如骨骼畸形和头面部缺陷，以及 与多种肿瘤相关，包括基底细胞癌和髓母细胞瘤。一个 脊椎动物HH信号的重要但鲜为人知的方面是严格要求 以微管为基础的细胞器，称为初级纤毛。虽然我们现在拥有丰富的数据 在参与HH信号转导的蛋白质“部分清单”上，其分子机制 纤毛介导的信号转导仍然知之甚少。我们的总体目标是攻读这个专业 知识鸿沟，并通过以下方式为HH信号通路提供生化框架 从其组分重构这一途径的关键反应。对于这项研究，我们将建立 关于我们将单分子成像方法与生化分析相结合的经验 细胞生物读数将蛋白质组分的生化特性与它们的 细胞功能。在这个提议中，我们关注HH信号转导途径的一个关键步骤 它是在纤毛的底部和顶端建立信号复合体，如 是转录因子Gli的适当激活或抑制所必需的，Gli是主要的效应因子 在HH途径上。在这里，我们将：(1)通过一系列的 重建研究并确定他们如何限制GLI对核进口的约束 机械和(2)分析纤毛中关键途径蛋白的动态。 分辨率实时成像。总而言之，这些研究将定义GLI是如何通过 在确定的细胞质位置的蛋白质复合体之间的动态传递。我们希望我们的 这些发现不仅将促进我们对这一重要信号的基本生物学的理解 转导途径，但也阐明了途径成分的突变是如何起作用的 到发育障碍和人类癌症。

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.