Flagellar Motility and Assembly

鞭毛运动和组装

• 批准号: 9923718
• 负责人: George B Witman
• 金额: $ 66.41万
• 依托单位: UNIV OF MASSACHUSETTS MED SCH WORCESTER
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-05-01 至 2022-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9923718
• 关键词: Animal Model; Area; Back; Biochemical; Biological; Biology; Blindness; Cells; Chlamydomonas; Cilia; Complex; Cryoelectron Microscopy; Defect; Disease; Flagella; Genetic; Goals; Health; Human; Hydrocephalus; Individual; Injections; Investigation; Knowledge; Learning; Lighting; Link; Location; Maintenance; Male Infertility; Membrane; Microscopy; Microtubules; Motor; Movement; Mus; Nephronophthisis; Organelles; Organism; Pathogenicity; Polycystic Kidney Diseases; Primary Ciliary Dyskinesias; Process; Proteins; Research; Resolution; Role; Situs Inversus; Structure; Structure of ciliary processes; Syndrome; Training; base; cell motility; ciliopathy; developmental disease; experimental study; human disease; insight; kinetosome; mutant; particle; recruit

Cilia and flagella are essentially identical cell organelles that have important roles in human health; as a result, defects in ciliary proteins cause human diseases, termed “ciliopathies.” The long-term goals of this research are to understand the structure, assembly, and function of these organelles. The studies will utilize Chlamydomonas and mice as model organisms, and will concentrate on processes and proteins that are highly conserved among ciliated organisms. A combination of genetic, biochemical, and cell biological approaches will be taken. Investigations will focus on three related areas of particular importance for understanding the basic biology of cilia and ciliopathies. First, experiments will determine the functions and specific locations within the cilium of uncharacterized ciliary proteins. The cilium contains over 650 proteins, of which fewer than half have been well characterized. The central pair of axonemal microtubules is apt to be particularly rich in uncharacterized proteins, so initial efforts will examine it. A second focus will be on the fundamental mechanism of intraflagellar transport (IFT), which is the movement of large, multi-subunit “trains” from the base of the cilium to the ciliary tip and then back to the cell body. These trains are made up of complexes including IFT-A and IFT-B, which carry cargos necessary for the assembly and maintenance of the cilium. Train formation in the cell body involves recruitment of IFT-A and IFT-B to the base of the cilium, loading of cargo onto the complexes, attachment of motors to the complexes, and injection of the completed train into the cilium. When this process is defective, ciliary assembly fails, but little is known about the individual steps in this process. Studies will use high-resolution structured-illumination microscopy and mutants in which train formation is arrested at various steps to determine the order of these steps and the roles of individual proteins in key parts of the process. Related studies will explore the specific function of IFT-A in ciliary assembly. In addition, single-particle cryo-electron microscopy will be carried out to determine the structure of IFT-A and IFT-B, which will be important for understanding how these complexes are arranged in the trains. A third focus will be on the transition zone, a specialized region between the basal body and the ciliary axoneme. The transition zone acts as a barrier that, in concert with IFT, is important for establishing and maintaining the protein content of cilia. However, the transition zone is still largely a “black box.” Mutants with defects in transition zone proteins will be investigated to learn more about the specific roles of these proteins in transition zone function and assembly, and to determine the composition of the highly conserved Y-links, which connect the transition zone microtubules to the overlying membrane and are critical to the transition zone's barrier function. The results will fill major gaps in our knowledge of cilia and flagella, and provide new insight into why defects in specific ciliary proteins cause human disease.

纤毛和鞭毛本质上是相同的细胞器，对人类健康具有重要作用；因此， 纤毛蛋白的缺陷会导致人类的疾病，这种疾病被称为“纤毛疾病”。这项研究的长期目标是 是为了了解这些细胞器的结构、组装和功能。这些研究将利用 衣藻和小鼠作为模式生物，并将专注于高度 在纤毛生物中保存。遗传学、生物化学和细胞生物学方法的结合 都会被带走。调查将集中在三个相关领域，这对了解 纤毛和纤毛病的基础生物学。首先，实验将确定功能和具体位置 纤毛中的未鉴定的纤毛蛋白。纤毛含有超过650种蛋白质，其中不到 其中一半已经被很好地描述了。中央的一对轴丝微管往往特别富含 未知的蛋白质，所以最初的努力将对其进行检查。第二个重点将放在根本上 鞭毛内转运机制(IFT)，即大的、多亚单位的“列车”从底部移动 纤毛到纤毛尖端，然后回到细胞体。这些列车由复合体组成，包括 IFT-A和IFT-B，携带组装和维护纤毛所需的货物。火车 细胞体的形成包括将IFT-A和IFT-B聚集到纤毛底部，装载货物 将发动机安装到综合体上，并将完成的列车注入到 纤毛。当这个过程有缺陷时，纤毛组装就会失败，但人们对纤毛组装的各个步骤知之甚少 这一过程。研究将使用高分辨率结构照明显微镜和突变体 在不同的步骤中阻止形成，以确定这些步骤的顺序和单个蛋白质的作用 在这一过程的关键部分。相关研究将探索IFT-A在纤毛组装中的具体功能。在……里面 此外，将进行单颗粒冷冻电子显微镜来确定IFT-A和 IFT-B，这对于了解这些复合体在列车中是如何布置的将是重要的。第三个焦点 将位于过渡区，这是基底体和睫状轴丝之间的一个特殊区域。这个 过渡区充当障碍，与IFT协调一致，对于建立和维持 纤毛的蛋白质含量。然而，过渡区在很大程度上仍然是一个“黑匣子”。带有缺陷的突变体 将对过渡区蛋白质进行研究，以更多地了解这些蛋白质在过渡中的具体作用。 区域功能和组装，并确定高度保守的Y-链的组成，它们连接 过渡区对上覆膜起微管作用，是过渡区屏障的关键。 功能。这些结果将填补我们对纤毛和鞭毛知识的主要空白，并为为什么 特定纤毛蛋白的缺陷会导致人类疾病。