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.

项目摘要： 微管（MTS）及其在真核细胞中起着重要作用的结构。最著名的动态 来自A/B-微管蛋白异二聚体组装的聚合物，在许多细胞过程中绝对需要MT， 包括有丝分裂。这些活动中的许多活动取决于动态MT行为，但是有关键的细胞功能 这需要稳定的微管。神经元中稳定的单线MTS充当轴突运输的轨道，稳定的双线 轴突中的mts在纤毛中产生力，在中心三粒和基体中发现稳定的三重态mts 分别组织中心体和纤毛。尽管它们的重要性，但我们几乎不知道基于MT的稳定 结构组装，维护和拆卸。因为相同的小管蛋白二聚体组装动态 和大多数生物中的稳定MT，包括纤毛四氢菌Hythyphila，不同的MT行为 归因于相关的蛋白质和蛋白质修饰。在Doublet和Triplet MT中，有些相关 蛋白质在空心MT内发现；这些微管内蛋白（MIP）是我们工作的重点。 MIP最初是使用各种形式的电子显微镜发现的，它是未知的结构 轴突双管微管内部的组成。提出了MIPS来减轻变形和压力 在双重纤毛跳动引起的双打MT上。睫状跳动将细胞外流体移动到一个方向，这是 许多基本过程所必需的，例如清除气道粘液，促进鸡蛋的运动 在输卵管中，并在大脑中产生脑脊液流动。在结构上类似于机动性 纤毛，鞭毛是精子运动所必需的。缺陷破坏纤毛纤毛会导致广泛的人类 病理学，包括两性睫状运动障碍（PCD），脑积水和不育症。 了解睫状缺陷如何导致运动问题和疾病受到限制。以前，我们确定了 纤毛纤毛中的rib72a和rib72b作为正常纤毛殴打所需的MIP。比较蛋白质组学 从野生型和rib72a-，rib72b- null细胞中分离出的轴突的分析，确定了其他MIP，例如 FAP115和钙质样蛋白，其组装在突变体中有缺陷。我们进一步描述了 FAP115，表明它对于正常的细胞运动和轴突稳定性至关重要。同时，通过比较 四膜虫，衣原体Reinhardtii和Bos Taurus的双双脑MT结构 这些进化上遥远的生物中MIP的保护和多样性，揭示了必不可少的和不同的 功能。该项目的长期目标是使用生物物理，遗传和高级显微镜工具进行 更好地了解纤毛运动的功能和组装机制。为此，我们计划确定 轴突双线和基部三胞胎MT中的四心hymena MIP 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 拟议的工作将大大提高我们对纤毛组装机制和功能机制的理解 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。