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

项目总结： 微管及其形成的结构在真核细胞中起着至关重要的作用。最著名的是动态 由a/b-微管蛋白异二聚体组装而成的聚合物，MTS在许多细胞过程中是必不可少的， 包括有丝分裂。其中许多活动依赖于MT的动态行为，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MTs作为轴突运输的轨迹，稳定的二重体 轴丝中的MT在纤毛中产生力量，在中心粒和基底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 对结构进行组装、维护和拆卸。因为相同的微管蛋白二聚体可以动态组装 在大多数生物中，包括嗜热四膜虫在内的大多数生物中都有稳定的MT，不同的MT行为 归因于相关蛋白质和蛋白质修饰。在二联体和三联体MT中，一些相关 在中空的MT中发现了蛋白质；这些微管内部蛋白质(MIP)是我们工作的重点。 MIP最初是用各种形式的电子显微镜发现的，看起来像是未知的结构 轴丝双线微管内的成分。为了减轻变形和应力，提出了MIPS 关于纤毛搏动引起的双重MTS。纤毛跳动使细胞外液单向运动，这是 对许多基本过程都是必需的，如清除呼吸道粘液，促进卵子的移动 在输卵管中，并在大脑中产生脑脊液流动。在结构上类似于运动的 纤毛，鞭毛是精子运动所必需的。扰乱活动纤毛的缺陷导致广泛的人类 病理，包括原发性睫状体运动障碍(PCD)，脑积水，以及男女不育。 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 纤毛四膜虫中的Rib72A和Rib72B作为正常纤毛跳动所需的分子印迹。比较蛋白质组学 从野生型和rb72a、r72B缺失细胞中分离的轴丝分析发现了其他的MIP，如 Fap115和类Calciphosin蛋白，其组装在突变体中存在缺陷。我们进一步描述了 Fap115，表明它对正常的细胞运动和轴丝稳定是必不可少的。同时，通过比较 四膜虫、莱茵衣藻和金牛的双重MT结构，我们发现两者 这些进化上遥远的生物体中分子印迹蛋白的保守性和多样性，揭示了本质和分歧 功能。该项目的长期目标是使用生物物理、遗传学和先进的显微镜工具来 更好地了解活动纤毛的功能和组装机制。为了做到这一点，我们计划确定 轴突二联体和基体三联体MTS中的四膜虫MIP，以绘制驱动 MIP的定位和组装，并阐明MIP对机体和纤毛功能的贡献。我们的 提出的工作将极大地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是如何导致人类纤毛疾病的。

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.