Flagellar Motility and Assembly

鞭毛运动和组装

• 批准号: 9277068
• 负责人: George B Witman
• 金额: $ 51.96万
• 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/9277068
• 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; Injection of therapeutic agent; 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; Recruitment Activity; 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

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.

纤毛和鞭毛基本上是相同的细胞器，在人类健康中具有重要作用;因此， 睫状蛋白的缺陷引起称为"纤毛病"的人类疾病。这项研究的长期目标是 就是了解这些细胞器的结构、组装和功能。研究将利用 衣原体和小鼠作为模式生物，并将集中在过程和蛋白质，是高度 在纤毛生物中是保守的。遗传学、生物化学和细胞生物学方法的结合 会被带走调查将集中在三个相关领域，这三个领域对于了解 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 在纤毛内的未表征的纤毛蛋白。纤毛含有超过650种蛋白质， 其中一半已经得到很好的描述。中央的一对轴丝微管倾向于特别富含 第二个重点将是基本的蛋白质，因此最初的努力将检查它。 鞭毛内运输（IFT）的机制，这是大的，多亚基“火车”从基地的运动 从纤毛到纤毛尖端，然后回到细胞体。这些列车由复杂的组成，包括 IFT-A和IFT-B携带纤毛组装和维持所需的货物。火车 在细胞体中的形成包括将IFT-A和IFT-B募集到纤毛的基部，装载货物， 到复合体上，将发动机连接到复合体上，并将完成的列车注入复合体中。 纤毛当这个过程有缺陷时，纤毛组装失败，但对纤毛组装的各个步骤知之甚少。 这个过程研究将使用高分辨率的结构照明显微镜和突变体在哪个火车 在不同的步骤中阻止蛋白质的形成，以确定这些步骤的顺序和单个蛋白质的作用 在这个过程的关键部分。相关研究将探讨IFT-A在纤毛组装中的具体功能。在 此外，还将进行单颗粒冷冻电子显微镜检查，以确定IFT-A的结构， IFT-B，这将是重要的了解这些复合体是如何安排在火车上。第三焦点 将在过渡区，一个专门的区域之间的基体和纤毛轴丝。的 过渡区作为一个屏障，与IFT一致，对于建立和维持 纤毛蛋白质含量。然而，过渡区在很大程度上仍然是一个"黑匣子"。基因突变体 过渡区蛋白将被研究，以了解更多关于这些蛋白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-链接的组成， 过渡区微管与上覆膜的连接，对过渡区的屏障至关重要 功能这一结果将填补我们对纤毛和鞭毛的认识的主要空白，并为了解其原因提供新的见解。 特定纤毛蛋白的缺陷导致人类疾病。