Intraflagellar transport process in primary cilium maintenance

初级纤毛维持中的鞭毛内运输过程

• 批准号: 10626341
• 负责人: Haixin Sui
• 金额: $ 4.92万
• 依托单位: WADSWORTH CENTER
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-01 至 2025-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10626341
• 关键词: 3-Dimensional; Architecture; Back; Behavior; Cell Proliferation; Cells; Chlamydomonas; Cilia; Complex; Correlative Study; Cryo-electron tomography; Cryoelectron Microscopy; Data Analyses; Defect; Development; Disease; Electron Microscopy; Electrons; Environment; Epithelial Cells; Evaluation; Event; Experimental Designs; Extracellular Space; Failure; Flagella; Formulation; Foundations; Freezing; Frequencies; Funding; Goals; Homeostasis; Image Analysis; Individual; Kinesin; Knowledge; Lead; Length; Light; Link; Location; Maintenance; Malignant Neoplasms; Measures; Medical; Methods; Microscopic; Microtubules; Modeling; Molecular; Molecular Conformation; Motor; Organogenesis; Peripheral; Physiological; Physiological Processes; Preparation; Process; Proteins; Public Health; Regulation; Research; Role; Sensory; Signal Transduction; Signaling Molecule; Specimen; Structure; Techniques; Telomere Length Maintenance; Testing; Three-Dimensional Image; Time; Tissues; Tomogram; Training; Transport Process; Travel; anterograde transport; base; cell motility; ciliopathy; cilium motility; developmental disease; electron tomography; innovation; insight; light microscopy; sensor; three dimensional cell culture; three dimensional structure; trafficking; wasting; wound healing

PROJECT SUMMARY Primary cilia are non-motile antenna-like sensors that protrude into extracellular space for detecting a wide range of signals, including signals for cell proliferation control. Problems in primary cilium assembly and structure/length maintenance, and failures to distribute ciliary sensory proteins to specific locations of the cilium, are linked to a wide variety of medical disorders and developmental abnormalities (termed ciliopathies). Assembly and maintenance of the ciliary framework (axoneme) and distribution of sensory proteins in the primary cilium rely on motor-driven intraflagellar transport (IFT) trains that travel along axonemal microtubule complexes (MtCs). Knowing details about IFT processes in the primary cilium is an essential foundation for understanding the proper distribution of sensory proteins in the primary cilium. Understanding the functional roles of IFT processes in primary cilium assembly and maintenance is fundamental for studies of related ciliopathies. The objectives of this proposal are to determine how anterograde IFT trains move from the base along the axoneme to reach the cilium tip, and then transition into the retrograde trains to move back in primary cilia, and how their activities contribute to maintenance of the structure/length of the primary cilium axoneme. The long-term goal of this proposal is to develop a mechanistic understanding of the functional roles and associated physiological processes of primary cilia in tissue homeostasis and ciliopathies. Our central hypothesis is that the behavior and architecture of IFT trains in primary cilia differ from that in motile flagella. We will test our central hypothesis in three specific aims: 1) Determine how IFT trains travel along the primary cilium axoneme by a correlative study using both light microscopy (LM) and cellular electron microscopy (EM); 2) Determine how continuous IFT contributes to primary cilium maintenance by serial section electron tomography (SSET) and three-dimensional (3D) image analysis; and 3) Define the structural units and conformational changes of the IFT trains at the cilium tip by cryo- electron tomography and 3D image analysis. We will pursue an innovative strategy that combines 3D cell culturing methods and light microscopy with techniques of 3D-EM specimen preparation and data analysis to overcome experimental difficulties for studying primary cilia of epithelial cells with inhibitable IFT. The proposed research is significant because it will provide critical knowledge about the process of IFT trains moving along the primary cilia, define the roles of IFT in primary-cilium structure maintenance, and offer the first structural-level description of the molecular environment at the primary cilium tip that facilitates the IFT anterograde-retrograde transition, cargo release, ectosome budding, and other important molecular events for physiological functions of primary cilia. The impact of the proposed studies is broad, as the anticipated knowledge will become a previously unavailable reference for studies of primary cilium functional mechanisms in development, organogenesis, tissue homeostasis and wound repairing.

项目概要 初级纤毛是不可移动的类似天线的传感器，伸入细胞外空间以检测广泛的 一系列信号，包括细胞增殖控制信号。初级纤毛组装问题和 结构/长度维持，以及未能将纤毛感觉蛋白分配到纤毛的特定位置， 与多种医学疾病和发育异常（称为纤毛病）有关。 纤毛框架（轴丝）的组装和维持以及初级感觉蛋白的分布 纤毛依赖于电机驱动的鞭毛内运输（IFT）列车，沿着轴丝微管复合体行进 （MTC）。了解初级纤毛中 IFT 过程的细节是理解的重要基础 初级纤毛中感觉蛋白的正确分布。了解 IFT 的职能角色 初级纤毛组装和维持过程是相关纤毛病研究的基础。这 该提案的目标是确定顺行 IFT 列车如何沿着轴丝从底座移动 到达纤毛尖端，然后过渡到逆行列车以移回初级纤毛，以及它们如何 活动有助于维持初级纤毛轴丝的结构/长度。长期目标是 该提案旨在对功能作用和相关生理学产生机械性的理解 初级纤毛在组织稳态和纤毛病中的过程。我们的中心假设是行为和 初级纤毛中的 IFT 序列结构与运动鞭毛中的结构不同。我们将在三个方面检验我们的中心假设 具体目标： 1) 通过相关研究确定 IFT 列车如何沿着初级纤毛轴丝行进 光学显微镜 (LM) 和细胞电子显微镜 (EM)； 2) 确定连续 IFT 如何发挥作用 通过串行截面电子断层扫描 (SSET) 和三维 (3D) 图像进行初级纤毛维护 分析; 3) 通过冷冻定义纤毛尖端 IFT 序列的结构单元和构象变化 电子断层扫描和 3D 图像分析。我们将追求结合 3D 细胞的创新战略 培养方法和光学显微镜，采用 3D-EM 样本制备和数据分析技术 克服了研究具有可抑制 IFT 的上皮细胞初级纤毛的实验困难。拟议的 这项研究意义重大，因为它将提供有关 IFT 列车沿线行驶过程的关键知识。 初级纤毛，定义了 IFT 在初级纤毛结构维护中的作用，并提供了第一个结构水平 初级纤毛尖端促进 IFT 顺行-逆行的分子环境的描述 转变、货物释放、胞外体出芽和其他重要的生理功能分子事件 初级纤毛。拟议研究的影响是广泛的，因为预期的知识将成为以前的知识 对于发育、器官发生、组织中的初级纤毛功能机制的研究没有参考资料 体内平衡和伤口修复。