Hedgehog signaling at the cell's antenna: Smoothened and the primary cilium

摘要 在脊椎动物中，刺猬信号是一种细胞间通讯的手段，它依赖于静止的细胞。 称为初级纤毛的附属物。在我们之前的工作中，我们发现脊椎动物刺猬的信号会移动 平滑到初级纤毛，这种运动对于平滑的活动是必要的，并且某些癌症 依赖它们的纤毛进行结构性的活性刺猬途径活动。 尽管有这些关于刺猬信号的见解，但如何平滑纤毛运动到纤毛是如何调节的，以及如何纤毛的 平滑激活了下游通路，但尚不清楚。在此续订申请中，我们将研究 Hedgehog受体Patched1控制光滑活性的分子机制(目标1)，如何 平滑在纤毛内激活(目标2)，以及平滑如何激活其下游效应器 转录因子GLI2(目标3)。 我们发现睫状膜有一种独特的脂类成分，而特定的睫状脂是 是激活哺乳动物刺猬信号的必要条件和充分条件。因此，前两个目标集中在一定程度上， 关于Patched1如何调节睫状脂以及睫状脂如何控制平滑的活动。这些实验 将有助于揭示脂质是如何控制信号的，并可能确定阻断刺猬的新治疗策略 途径相关的癌症形成。Smoothed如何与GLI2通信仍是个未知数。我们已经创造了 一种生物化学上易于处理的敲入Gli2等位基因，将使我们能够揭示纤毛调节机制 GLI2活性在胚胎发育和肿瘤发生中的作用 因此，拟议的实验使用了哺乳动物遗传学、细胞生物学、成像和 生物化学方法揭示刺猬信号转导途径如何利用纤毛传递 发展和疾病方面的信息。

ABSTRACT Hedgehog signaling is a means of intercellular communication that, in vertebrates, relies on immotile cellular appendages called primary cilia. In our previous work, we discovered that vertebrate Hedgehog signals move Smoothened to primary cilia, that this movement is necessary for Smoothened activity, and that certain cancers depend on their cilia for constitutively active Hedgehog pathway activity. Despite these insights into Hedgehog signaling, how Smoothened movement to cilia is regulated and how ciliary Smoothened activates the downstream pathway remain unclear. In this renewal application, we examine the molecular mechanisms by which the Hedgehog receptor, Patched1, controls Smoothened activity (Aim 1), how Smoothened is activated within the cilium (Aim 2), and how Smoothened activates its downstream effector, the transcription factor GLI2 (Aim 3). We have discovered that the ciliary membrane has a unique lipid composition, and that specific ciliary lipids are necessary and sufficient to activate mammalian Hedgehog signaling. Therefore, the first two aims focus, in part, on how Patched1 regulates ciliary lipids and how ciliary lipids control Smoothened activity. These experiments will help reveal how lipids control signaling, and may identify new therapeutic strategies for blocking Hedgehog pathway-related cancer formation. How Smoothened communicates to GLI2 remains unknown. We have created a biochemically tractable knock-in Gli2 allele that will allow us to uncover mechanisms by which cilia regulate GLI2 activity in embryogenesis and oncogenesis. Thus, the proposed experiments use a combination of mammalian genetic, cell biological, imaging and biochemical approaches to reveal how the Hedgehog signal transduction pathway uses cilia to transmit information, both in development and disease.