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 发育和成人组织的动态平衡。HH信号的错误与几种新生儿出生缺陷有关 例如骨骼畸形和头面部缺陷，并与包括基底细胞在内的多种肿瘤相关 细胞癌和髓母细胞瘤。脊椎动物HH信号的一个重要但鲜为人知的方面是 对以微管为基础的细胞器的严格要求，称为初级纤毛。虽然我们现在有一个 关于参与HH信号转导的蛋白质的“部分清单”、潜在的分子机制的大量数据 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 GAP并通过重组HH信号通路的关键反应为HH信号通路提供生化框架 从其组成来看，这是一条重要的途径。对于这项研究，我们将建立在我们整合单分子的经验基础上 结合生化分析和细胞生物学读数的成像方法，以连接生物化学特性 蛋白质组分对细胞功能的影响。在这个方案中，我们将重点放在HH信号的关键步骤 转导途径，即在纤毛的底部和顶端建立信号复合体。 是正确激活或抑制转录因子Gli所必需的，Gli是HH的主要效应因子。 路径。在这里，我们将：(1)通过一系列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与核进口机械的结合，以及(2)分析动力学 使用高分辨率实时成像技术对纤毛中的关键途径蛋白进行分析。总而言之，这些研究将 明确Gli是如何通过在特定细胞质的蛋白质复合体之间的动态转运来调节的 地点。我们预计，我们的发现不仅将促进我们对这一现象的基本生物学的理解 重要的信号转导途径，但也阐明了途径组件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.