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

通过超分辨率显微镜阐明初级纤毛如何调节 Hedgehog 信号传导

• 批准号: 10152612
• 负责人: Xiaoyu Shi
• 金额: $ 24.9万
• 依托单位: UNIVERSITY OF CALIFORNIA-IRVINE
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-05-18 至 2023-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10152612
• 关键词: 3-Dimensional; Active Biological Transport; Adult; Algorithms; Basal cell carcinoma; Binding Sites; Cells; Cellular Structures; Cilia; Data; Defect; Disease; Docking; Embryo; Foundations; Frequencies; Goals; Heterogeneity; Homeostasis; Image; Knock-out; Label; Malignant Neoplasms; Maps; Measures; Mediating; Mediator of activation protein; Membrane; Microscopy; Modeling; Molecular; Movement; Mutation; Nature; Optics; Pattern; Prevention; Preventive screening; Proteins; Receptor Signaling; Regulation; Reporting; Resolution; Role; Signal Transduction; Signaling Molecule; Structure; Techniques; Technology; Therapeutic; Therapeutic Intervention; Time; Tissues; Uncertainty; base; congenital heart disorder; developmental disease; insight; mutant; nanometer resolution; novel; novel strategies; novel therapeutics; particle; preventive intervention; protein protein interaction; reconstruction; smoothened signaling pathway; spatial relationship; spatiotemporal; structural biology; trafficking

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

抽象的 Hedgehog 信号是胚胎模式和成体组织稳态的关键调节因子。最后， Hedgehog 信号传导缺陷可能导致发育性疾病、先天性心脏病和癌症，例如 如基底细胞癌。迫切需要了解其背后的分子机制 Hedgehog 信号通路的调节及其潜在的预防和治疗价值。据了解， 脊椎动物 Hedgehog 信号传导依赖于 Hedgehog 信号受体的纤毛运输，其中 smoothened (SMO) 是 Hedgehog 信号传导的核心正介体。如果突变发生在任一intrafla- 胶质转运蛋白 (IFT) 或纤毛过渡区的 SMO 活动可能会受到严重干扰。然而， IFT颗粒和过渡区如何调节SMO运输的分子机制目前尚未确定 已知。确定分子调节机制是本申请的目的。我们的初步 随机光学重建显微镜（STORM）获得的所有数据显示了共定位 过渡区蛋白与 SMO 和 IFT88，表明过渡区蛋白和 IFT 颗粒相互作用 与SMO。根据之前的研究和我们自己的主要数据，我的中心假设是转变 区域作为 Hedgehog 信号受体的检查点，IFT 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颖的控制机制 纤毛贩运和不同纤毛货物之间的交叉相互作用。它可能允许新的应用程序 操纵刺猬信号的方法，并为治疗由刺猬引起的疾病奠定了基础 Hedgehog 信号传导缺陷。为了完成这个项目，我计划将 SMO 分子和 IFT 粒子映射到 过渡区使用多色 3D STORM。它将揭示SMO分子之间的空间关系 这些纤毛成分的分辨率约为 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，提供约 5 nm 的精度 所研究蛋白质之间的距离，表明蛋白质-蛋白质相互作用。与 静态结构研究，我还计划检测SMO分子、IFT粒子和过渡态之间的相互作用 使用单粒子跟踪和光转换成像动态地分析区域蛋白质。拟议项目 不仅为 Hedgehog 信号调节的分子机制提供新的见解，而且 提出一套基于显微镜的技术和算法，可广泛应用于细胞领域 信号传导和结构生物学。此外，预计结果将产生广泛影响，因为 该项目将确定的监管成分将为预治疗提供新机制和新药物筛选。 通风和治疗干预。 1