Protein dynamics underlying cilium-dependent Hedgehog signaling

摘要 Hedgehog（Hh）信号通路是一种重要的细胞间信号通路， 胚胎发育和成体组织稳态。Hh信号中的错误与 几种新生儿出生缺陷，如骨骼畸形和颅面缺陷，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信号传导的一个重要但知之甚少的方面是， 以微管为基础的细胞器，称为初级纤毛。虽然我们现在有大量的数据 在参与Hh信号传导的蛋白质的“部分列表”上， 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个主要 知识差距，并提供一个生化框架的Hh信号通路， 从其组分中重建该途径的关键反应。为了这项研究，我们将建立 基于我们在将单分子成像方法与生化分析相结合方面的经验， 细胞生物学读数，以将蛋白质组分的生化特性与其 细胞功能在本研究中，我们重点研究了Hh信号转导途径中的一个关键步骤 这是在纤毛的基部和尖端建立信号复合物， 是转录因子Gli的适当激活或抑制所必需的， Hh途径。在这里，我们将：（1）通过一系列的定义这些蛋白质-蛋白质相互作用 重组研究，并确定他们如何限制Gli与核输入的结合 机械和（2）分析纤毛中关键途径蛋白质的动态，使用高- 分辨率实时成像。总之，这些研究将确定Gli是如何通过以下方式进行调节的： 在确定的细胞质位置处蛋白质复合物之间的动态转运。我们希望我们 这些发现不仅将促进我们对这一重要信号的基础生物学的理解， 转导通路，但也阐明了通路组分中的突变如何有助于 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.