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

MIPS（微管内部蛋白）在纤毛和基底体中发挥作用

• 批准号: 10655224
• 负责人: MARK WINEY
• 金额: $ 35.65万
• 依托单位: UNIVERSITY OF CALIFORNIA AT DAVIS
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-05-01 至 2027-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10655224
• 关键词: Adolescent; Affect; Axonal Transport; Behavior; Biophysics; Bos taurus; Brain; Cattle; Cell physiology; Cell surface; Cells; Centrioles; Centrosome; Chlamydomonas; Chlamydomonas reinhardtii; Cilia; Cryoelectron Microscopy; Defect; Disease; Distant; Electron Microscopy; Environment; Epilepsy; Eukaryotic Cell; Extracellular Fluid; Eye; Family; Functional disorder; Genetic; Goals; Hair; Human; Human Pathology; Hydrocephalus; Individual; Infertility; Inherited; Juvenile Myoclonic Epilepsy; Kidney; Lead; Liquid substance; Lung; Mammalian Oviducts; Maps; Mediating; Microscopy; Microtubule Triplet; Microtubules; Mitosis; Movement; Mucous body substance; Mus; Myoclonus; Neurons; Normal Cell; Null Lymphocytes; Organ; Organism; Orthologous Gene; Pathogenesis; Pathology; Pathway interactions; Phenotype; Play; Point Mutation; Polymers; Post-Translational Protein Processing; Predisposition; Primary Ciliary Dyskinesias; Process; Proteins; Proteomics; Resolution; Role; Seizures; Site; Sperm Motility; Stress; Structure; Tetrahymena; Tetrahymena thermophila; Tubulin; Vertebrates; Work; beta Tubulin; biophysical analysis; cell motility; cerebrospinal fluid flow; ciliopathy; cilium motility; comparative; dimer; egg; experimental study; improved; kinetosome; mutant; novel therapeutic intervention; protein function; protein structure; protein transport; recruit; rib bone structure; sex; tool; trafficking

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.

项目概要： 微管 (MT) 及其形成的结构在真核细胞中发挥着重要作用。最出名的是动态 由 a/b-微管蛋白异二聚体组装而成的聚合物，MT 在许多细胞过程中是绝对必需的， 包括有丝分裂。其中许多活动依赖于动态 MT 行为，但也有一些关键的细胞功能 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，稳定的双线态 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 的稳定性知之甚少 结构被组装、维护和拆卸。因为相同的微管蛋白二聚体动态组装 大多数生物体中都有稳定的 MT，包括纤毛虫嗜热四膜虫，不同的 MT 行为 归因于相关蛋白质和蛋白质修饰。在双线态和三线态 MT 中，一些相关的 中空 MT 内发现蛋白质；这些微管内部蛋白（MIP）是我们工作的重点。 MIP 最初是通过各种形式的电子显微镜发现的，其表现为未知的结构 轴丝双联微管内的组成。建议使用 MIP 来减轻变形和应力 由纤毛跳动引起的双峰 MT。纤毛跳动使细胞外液沿单一方向移动，即 对于许多基本过程来说是必需的，例如清除呼吸道粘液、促进卵的移动 在输卵管中，并在大脑中产生脑脊液流。结构上与运动类似 纤毛，鞭毛是精子活动所必需的。破坏活动纤毛的缺陷会导致广泛的人类 病理学，包括原发性纤毛运动障碍 (PCD)、脑积水和两性不孕症。 对纤毛缺陷如何导致运动问题和疾病的了解有限。此前，我们确定了 四膜虫纤毛中的 Rib72A 和 Rib72B 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 rib72A-、rib72B- null 细胞中分离出的轴丝进行分析，鉴定出其他 MIP，例如 Fap115 和钙磷蛋白样蛋白，其组装在突变体中存在缺陷。我们进一步表征 Fap115 并表明它对于正常细胞运动和轴丝稳定性至关重要。同时，通过比较 四膜虫、莱茵衣藻和牛的双联体 MT 结构，我们发现 这些进化遥远的生物体中 MIP 的保护和多样性，揭示了本质和差异 功能。该项目的长期目标是利用生物物理、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 轴丝双联体和基底体三联体 MT 中的四膜虫 MIP，以绘制驱动蛋白质相互作用的图谱 MIP 定位和组装，并阐明 MIP 如何促进基底体和纤毛功能。我们的 拟议的工作将极大地增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的功能障碍如何导致人类纤毛病。