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: 10798049
• 负责人: Haixin Sui
• 金额: $ 7.98万
• 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/10798049
• 关键词: 3-Dimensional; Architecture; Back; Behavior; Cell Proliferation; Cilia; Complex; Correlative Study; Cryo-electron tomography; Data Analyses; Defect; Development; Disease; Electron Microscopy; Environment; Epithelial Cells; Evaluation; Event; Extracellular Space; Failure; Flagella; Foundations; Goals; Homeostasis; Image Analysis; Knowledge; Length; Link; Location; Maintenance; Malignant Neoplasms; Medical; Methods; Microtubules; Molecular; Molecular Conformation; Motor; Organogenesis; Physiological; Physiological Processes; Preparation; Process; Proteins; Public Health; Research; Role; Sensory; Signal Transduction; Signaling Molecule; Specimen; Structure; Techniques; Telomere Length Maintenance; Testing; Three-Dimensional Image; Tissues; Training; Transport Process; Travel; anterograde transport; base; cell motility; ciliopathy; developmental disease; electron tomography; innovation; light microscopy; sensor; three dimensional cell culture; trafficking; 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.

项目摘要 初级纤毛是非运动的触角状传感器，其突出到细胞外空间中以检测广泛的细胞外信号。 信号范围，包括用于细胞增殖控制的信号。初级纤毛组装中存在的问题， 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。 纤毛骨架（轴丝）的组装和维持以及初级感觉蛋白的分布 纤毛依赖于马达驱动的鞭毛内运输（IFT）列车，该列车沿着轴丝微管复合体行进 （MtCs）。了解初级纤毛中IFT过程的细节是理解IFT过程的重要基础。 初级纤毛中感觉蛋白的适当分布。理解IFT的职能作用 初级纤毛组装和维持过程是相关纤毛病研究的基础。的 这个建议的目的是确定顺行的IFT序列是如何从基底沿着轴丝运动的 到达纤毛尖端，然后过渡到逆行列车，在初级纤毛中向后移动，以及它们的 活动有助于维持初级纤毛轴丝的结构/长度。的长期目标 这一建议是为了发展对功能作用和相关生理学的机械理解， 组织稳态和纤毛病中初级纤毛的过程。我们的中心假设是， 初级纤毛中IFT序列的结构不同于运动鞭毛中IFT序列的结构。我们将在三个方面检验我们的中心假设 具体目的：1）通过相关研究确定IFT序列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如何贡献 通过连续切片电子断层扫描（SSET）和三维（3D）成像， 3）用冷冻法确定纤毛顶端IFT链的结构单元和构象变化。 电子断层扫描和3D图像分析。我们将采取创新战略，将3D细胞 培养方法和光学显微镜与3D-EM标本制备和数据分析技术， 克服了实验上的困难，研究了具有可重复IFT的上皮细胞的初级纤毛。拟议 研究是重要的，因为它将提供关键知识的过程中，IFT列车移动沿着 初级纤毛，定义IFT在初级纤毛结构维持中的作用，并提供第一个结构水平 描述初级纤毛尖端的分子环境，促进IFT的顺行-逆行 转换，货物释放，外泌体出芽和其他重要的分子事件的生理功能， 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， 对研究初级纤毛在发育、器官发生、组织中的功能机制没有参考价值 体内平衡和伤口修复。