MIPS (Microtubule Inner Proteins) function in cilia and basal bodies

项目概要： 微管（MT）及其形成的结构在真核细胞中起着重要作用。最著名的动态 由a/b-微管蛋白异二聚体组装的聚合物，MT在许多细胞过程中是绝对需要的， 包括有丝分裂。这些活动中的许多依赖于动态MT行为，但也有关键的细胞功能 需要稳定的微管神经元中稳定的单线态MT充当轴突运输的轨道， 轴丝中的MT在纤毛中产生力，并且在中心粒和基体中发现稳定的三联体MT， 分别组织中心体和纤毛。尽管它们很重要，但我们对基于MT的 结构被组装、维护和拆卸。因为同样的微管蛋白二聚体 和稳定的MT在大多数生物体中，包括纤毛四膜虫thermophila，不同的MT行为 归因于相关蛋白和蛋白修饰。在双重和三重MT中，一些相关的 在中空的MT内发现了蛋白质;这些微管内蛋白（MIP）是我们工作的重点。 最初使用各种形式的电子显微镜发现，MIP表现为未知的结构， 轴丝双线微管内的组成。提出了MIPs来减轻变形和应力 纤毛跳动引起的双峰MT。纤毛跳动使细胞外液向单一方向移动， 这是许多基本过程所必需的，例如清除气道中的粘液，促进卵子的运动， 并在大脑中产生脑脊髓液流动。在结构上类似于 纤毛，鞭毛是精子运动所必需的。破坏运动纤毛的缺陷引起广泛的人类疾病， 病理学，包括原发性纤毛运动障碍（PCD）、脑积水和两性不育。 对纤毛缺陷如何导致运动问题和疾病的理解是有限的。此前，我们发现 Rib 72 A和Rib 72 B在四膜虫纤毛中作为正常纤毛跳动所需的MIP。比较蛋白质组学 对从野生型和rib 72 A-、rib 72 B-缺失细胞分离的轴丝的分析鉴定了另外的MIP，例如 Fap 115和钙磷蛋白样蛋白，其组装在突变体中是有缺陷的。我们进一步表征 Fap 115，并表明它是必不可少的正常细胞运动和轴丝的稳定性。同时，通过比较 四膜虫、莱茵衣藻和普通牛的双重MT结构，我们发现两者 保守性和多样性的MIP在这些进化上遥远的生物，揭示了基本的和不同的 功能协调发展的该项目的长期目标是使用生物物理，遗传和先进的显微镜工具， 更好地了解运动纤毛的功能和组装机制。为此，我们计划确定 四膜虫MIPs在轴丝双联体和基体三联体MT，映射蛋白质相互作用，驱动 MIP定位和组装，并阐明MIP如何有助于基体和纤毛功能。我们 这项工作将大大促进我们对纤毛组装和功能机制的理解 并将有助于揭示这些过程中的功能障碍如何导致人类纤毛病变。

Project Summary: Microtubules (MTs) and the structures they form play essential roles in eukaryotic cells. Best known as dynamic polymers assembled from a/b-tubulin heterodimers, MTs are absolutely required in numerous cellular processes, including mitosis. Many of these activities depend on dynamic MT behavior, but there are critical cellular functions that require stable microtubules. Stable singlet MTs in neurons act as tracks for axonal transport, stable doublet MTs in axonemes generate force in cilia, and stable triplet MTs are found in centrioles and basal bodies that organize centrosomes and cilia, respectively. Despite their importance, we know little of how stable MT-based structures are assembled, maintained, and disassembled. Because the same tubulin dimers assemble dynamic and stable MTs in most organisms, including the ciliate Tetrahymena thermophila, the different MT behaviors are attributed to associated proteins and protein modifications. In doublet and triplet MTs, some associated proteins are found inside the hollow MT; these microtubule inner proteins (MIPs) are the focus of our work. Originally discovered using various forms of electron microscopy, MIPs appeared as structures of unknown composition inside axonemal doublet microtubules. MIPs are proposed to mitigate the deformation and stress on doublet MTs caused by ciliary beating. Ciliary beating moves extracellular fluid in a single direction, which is necessary for many essential processes, such as clearing mucus from airways, facilitating the movement of eggs in the fallopian tube, and generating cerebrospinal fluid flow in the brain. Structurally analogous to the motile cilium, the flagellum is required for sperm motility. Defects disrupting motile cilia cause a wide range of human pathologies, including primary ciliary dyskinesia (PCD), hydrocephalus, and infertility in both sexes. Understanding of how ciliary defects lead to motility problems and disease is limited. Previously, we identified Rib72A and Rib72B in Tetrahymena cilia as MIPs required for normal cilia beating. Comparative proteomic analyses of axonemes isolated from wild type and rib72A-, rib72B- null cells identified additional MIPs, such as Fap115 and Calciphosin-like protein, whose assembly is defective in the mutants. We further characterized Fap115 and showed it to be essential for normal cell motility and axoneme stability. Meanwhile, by comparing the doublet MT structures of Tetrahymena, Chlamydomonas reinhardtii, and Bos taurus, we find both conservation and diversity of MIPs in these evolutionarily distant organisms, revealing essential and divergent functions. The long-term goal of this project is to use biophysical, genetic, and advanced microscopy tools to better understand the function and assembly mechanisms of motile cilia. To do this, we plan to identify Tetrahymena MIPs in both axonemal doublet and basal body triplet MTs, to map protein interactions that drive MIP localization and assembly, and to illuminate how MIPs contribute to basal body and cilia function. Our proposed work will significantly advance our understanding of the mechanisms of cilia assembly and function and will help reveal how dysfunction in these processes contributes to human ciliopathies.