Elucidating How Primary Cilia Regulate Hedgehog Signaling by Super-Resolution Microscopy

摘要 刺猬信号是胚胎模式和成年组织动态平衡的关键调节因子。因此， Hedgehog信号的缺陷可能导致发育疾病、先天性心脏病和癌症等 作为基底细胞癌。迫切需要了解其背后的分子机制。 Hedgehog信号通路的调控及其潜在的预防和治疗价值。这是众所周知的- 脊椎动物Hedgehog信号依赖于Hedgehog信号受体的纤毛运输，其中 Smooth(SMO)是Hedgehog信号的中枢正向调节因子。如果在任何一个肺内发生突变- 凝胶转运体(IFTS)，或在睫状移行区，SMO的活动可能会严重中断。然而， 目前，IFT颗粒和过渡区调控SMO转运的分子机制尚不清楚。 为人所知。确定分子调控机制是这一应用的目标。我们的初选是- 随机光学重建显微镜(STORM)获得的基本数据表明， 过渡区蛋白与SMO和IFT88结合，提示过渡区蛋白与IFT颗粒相互作用 和SMO一起。基于之前的研究和我们自己的主要数据，我的中心假设是 区域作为Hedgehog信号受体的检查点，IFTS帮助Hedgehog信号受体 穿过过渡区。这种过渡区检查点模型代表了一种新的控制机制 纤毛运输和不同纤毛之间的交叉相互作用。它可能会允许新的AP- 处理刺猬信号的方法，为治疗由刺猬引起的疾病奠定基础 刺猬信号中的缺陷。为了接近这个项目，我计划将SMO分子和IFT粒子映射到 过渡区采用多色3D风暴。它将揭示SMO分子之间的空间关系 这些纤毛成分的分辨率为~15 nm。将开发算法以减少不确定性 由结构异质性和免疫染色引起的空间缓和，提供~5 nm的精度 研究蛋白质之间的距离，表明蛋白质之间的相互作用。与 静态结构研究，我还计划检测SMO分子、IFT粒子和跃迁之间的相互作用 利用单粒子跟踪和光转化成像技术动态研究区带蛋白质。拟议中的项目 这不仅为了解刺猬信号调控的分子机制提供了新的思路，也为进一步研究刺猬信号转导机制提供了新的思路。 Vance是一套基于显微镜的技术和算法，可广泛应用于细胞领域 信号和结构生物学。此外，预计结果将产生广泛影响，因为这一规定-- 该项目将确定的刺激成分将提供新的机制和新药筛选 预防性和治疗性干预。 1

Abstract Hedgehog signals are the key regulators of embryonic patterning and adult tissue homeostasis. Consequently, defects in Hedgehog signaling can cause developmental diseases, congenital heart disease, and cancers such as basal cell carcinoma. There is an urgent need to understand the molecular mechanisms that underlie the modulation of Hedgehog signaling pathway for its potential preventative and therapeutic value. It is known ver- tebrate Hedgehog signaling relies on the ciliary trafficking of Hedgehog signaling receptors, among which smoothened (SMO) is the central positive mediator of Hedgehog signaling. If mutations occur in either intrafla- gellar transporters (IFTs), or in the ciliary transition zone, SMO activities can be severely disrupted. However, the molecular mechanism of how IFT particles and transition zone regulate trafficking of SMO is currently un- known. Determination of the molecular regulation mechanisms is the objective of this application. Our prelimi- nary data acquired by Stochastic Optical Reconstruction Microscopy (STORM) showed the colocalization of transition zone proteins with SMO and IFT88, suggesting that transition zone proteins and IFT particles interact with SMO. Based on previous studies and our own primary data, my central hypothesis is that the transition zone serves as a checkpoint for Hedgehog signaling receptors, and IFTs help Hedgehog signaling receptors cross the transition zone. This transition zone checkpoint model represents a novel mechanism for the control of cilium trafficking and the cross-interaction between different ciliary cargos. It could potentially allow new ap- proaches to manipulate Hedgehog signals, and underlie the foundation for treatments of diseases caused by defects in Hedgehog signaling. To approach to the project, I plan to map SMO molecules and IFT particles in the transition zone using multicolor 3D STORM. It will reveal the spatial relationship between SMO molecules and these ciliary components at a resolution of ~15 nm. Algorithms will be developed to reduce the uncertainty of the spatial easements caused by structural heterogeneity and immunostaining, providing a ~ 5nm precision of the distance between investigated proteins, indicating protein-protein interaction. Equally important as the static structural study, I also plan to detect the interactions among SMO molecules, IFT particles, and transition zone proteins dynamically using single-particle tracking and photoconversion imaging. The proposed project will not only offer new insights into the molecular mechanisms of Hedgehog signaling regulation, but also ad- vance a suite of microscopy-based technologies and algorithms that can be broadly applied to the fields of cell signaling and structural biology. Furthermore, the results are expected to have broad impact, because the reg- ulatory components to be identified by this project will provide new mechanisms and new drug screen for pre- ventive and therapeutic interventions. 1