Signal Transduction in the Primary Cilium: Hedgehog and Beyond

项目摘要/摘要：初级纤毛是一个微小的、不活动的天线状突起。 几乎存在于人体的每一个细胞上。这种细胞器对于器官的发育和功能是必不可少的。 作为几个关键细胞信号通路的枢纽。扰乱纤毛会引发一系列纤毛病，包括 发育、感觉、代谢和再生障碍。而纤毛的生物学作用是 已知的是，在生化水平上，尚不清楚纤毛如何或为什么需要纤毛才能使其组成的级联发挥作用。 这妨碍了人们对纤毛如何发挥其生物学功能的理解。它也阻碍了我们的能力 从治疗的角度控制纤毛信号。这里提出的工作的目标是了解生物化学 以及控制纤毛信号转导的生物物理原理。而不是依赖于传统的遗传和 细胞生物学方法在该领域，我们正在利用非传统的生化和生理工具 生物学的其他领域；这包括重建系统和关键转导事件的活细胞传感器。 该建议使用Hedgehog(HH)途径，一个模型睫状级联和基本调节器 胚胎发生和干细胞生物学，以解决这些悬而未决的问题。在睫状膜上， 七跨膜(7TM)蛋白平滑(SMO)是控制几乎所有 HH途径的生物学活性。以下三个项目将共同揭示分子和 SMO和相关纤毛7TM蛋白发出信号的机制：1)什么是生化机制 调节SMO激活？这是建立在最近的发现基础上的，即离子梯度和膜脂是关键 HH信号的关键初始步骤的调节者。这些研究现在提供了一个平台来调查 膜胆固醇是连接上游途径事件和SMO活性的候选“信使”，它结合和 激活SMO。这些努力还揭示了SMO的其他监管因素，我们将确定这些因素 生化方面的。2)一旦被激活，SMO如何与控制GLI的转录因子进行通讯 HH途径靶基因的表达？我们将确定SMO长期寻找的直接下游效应器 在HH途径中，并检验纤毛主要作为集中注意力的“汇合处”的假设 共同激活通路组件。3)这些调节影响是如何在纤毛内起作用的？这些 实验将测试我们通过生物化学确定的调节因子的生理意义，以及 定量比较并选择性地操纵纤毛和“细胞体”间隔中的信号转导。 每个项目还提供了研究相关机制的垫脚石，这些机制在许多情况下支配着7TM蛋白 其他纤毛信号级联反应。部署的技术和从HH级联中学到的原则将因此 可广泛应用于其他睫状神经通路。在机械层面上定义这些级联对于 为一系列破坏性疾病开发诊断和治疗工具；包括先天性 神经、心血管和排泄系统的缺陷、恶性肿瘤和功能障碍。

PROJECT SUMMARY / ABSTRACT: The primary cilium is a tiny, immotile antenna-shaped protrusion found on nearly every cell in the human body. This organelle is essential for organ development and function, serving as a hub for several critical cell signaling pathways. Perturbing cilia instigates a slew of ciliopathies that include developmental, sensory, metabolic, and regenerative disorders. While the biological roles of the cilium are known, it is unclear at a biochemical level how or why the cilium is needed for its constituent cascades to operate. This prevents an understanding of how the cilium serves any of its biological functions. It also stymies our ability to control ciliary signaling therapeutically. The goal of the work proposed here is to understand the biochemical and biophysical principles governing ciliary signal transduction. Rather than relying on conventional genetic and cell biological approaches in the field, we are harnessing non-traditional biochemical and physiologic tools from other areas of biology; this includes reconstitution systems and live-cell sensors for key transduction events. This proposal uses the Hedgehog (Hh) pathway, a model ciliary cascade and fundamental regulator of embryogenesis and stem cell biology, to address these outstanding questions. At the ciliary membrane, the seven-transmembrane (7TM) protein Smoothened (SMO) is the pivotal molecule controlling essentially all the Hh pathway’s biological activities. The following three projects will together uncover the molecules and mechanisms by which SMO and related ciliary 7TM proteins signal: 1) What are biochemical mechanisms that regulate SMO activation? This builds on the recent discovery that ion gradients and membrane lipids are key regulators of the crucial initial steps of Hh signaling. These studies now provide a platform to investigate how membrane cholesterol, a candidate “messenger” linking upstream pathway events to SMO activity, binds to and activates SMO. These efforts have also revealed additional SMO regulatory factors, which we will identify biochemically. 2) How does SMO, once activated, communicate to GLI transcription factors that control expression of Hh pathway target genes? We will pinpoint SMO’s long-sought immediate downstream effector in the Hh pathway and test the hypothesis that the cilium serves primarily as a “meeting place” to concentrate activated pathway components together. 3) How do these regulatory influences operate within the cilium? These experiments will test the physiological significance of the regulatory factors we identify biochemically, as well as quantitatively compare and selectively manipulate signal transduction in the ciliary and “cell body” compartments. Each project also provides stepping stones to investigate related mechanisms that govern 7TM proteins in many other ciliary signaling cascades. The techniques deployed and principles learned from the Hh cascade will thus be broadly applicable to other ciliary pathways. Defining these cascades at a mechanistic level is essential for the development of diagnostic and therapeutic tools for a range of devastating disorders; this includes congenital defects, malignancies, and dysfunction of the nervous, cardiovascular, and excretory systems.