Intraflagellar transport process in primary cilium maintenance

项目总结 初级纤毛是一种非移动的天线状传感器，它伸入细胞外空间，用于检测广泛的 一系列信号，包括控制细胞增殖的信号。初级纤毛装配中存在的问题和 结构/长度维持，纤毛感觉蛋白未能分布到纤毛的特定位置， 与多种医学疾病和发育异常(称为纤毛疾病)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 纤毛依靠马达驱动的鞭毛内运输(IFT)列车沿轴丝微管复合体行进 (MTCS)。了解初级纤毛的IFT过程的细节是理解 初级纤毛中感觉蛋白的适当分布。理解IFT的职能作用 初级纤毛的组装和维持过程是相关纤毛疾病研究的基础。这个 这项提案的目的是确定顺行IFT列车如何从底部沿着轴丝移动 到达纤毛尖端，然后过渡到逆行列车，移回原生纤毛，以及它们是如何 活动有助于维持初级纤毛轴丝的结构/长度。的长期目标是 这一建议是为了发展对功能角色和相关生理角色的机械性理解 原生纤毛在组织动态平衡和纤毛病变中的过程。我们的中心假设是行为和 初级纤毛和活动鞭毛中的IFT序列构筑不同。我们将在三年内测试我们的中心假设 具体目标：1)通过使用相关研究确定IFT列车如何沿初级纤毛轴突行进 光学显微镜(LM)和细胞电子显微镜(EM)；2)确定连续IFT的贡献 连续切片电子断层扫描和三维成像对原发性纤毛维持的研究 分析；3)确定了纤毛尖端IFT序列的结构单元和构象变化。 电子断层扫描和三维图像分析。我们将推行结合3D细胞的创新战略 利用3D-EM标本制备和数据分析技术的培养方法和光学显微镜 用可抑制的IFT克服研究上皮细胞原代纤毛的实验困难。建议数 研究具有重要意义，因为它将提供关于IFT列车沿 初级纤毛，明确IFT在维持初级纤毛结构中的作用，并提供第一个结构水平 初级纤毛尖端促进IFT顺行逆行的分子环境的描述 过渡，货物释放，外胚体萌发和其他重要的分子事件对生物的生理功能 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 对于初级纤毛在发育、器官发生、组织中的作用机制的研究没有参考文献 动态平衡和伤口修复。

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.