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.

纤毛和鞭毛是在人类健康中具有重要作用的基本相同的细胞细胞器。因此， 睫状蛋白的缺陷引起人类疾病，称为“纤毛病”。这项研究的长期目标 要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型生物，将集中于高度的过程和蛋白质 在纤毛生物中保守。遗传，生化和细胞生物学方法的结合 将被带走。调查将集中在三个相关领域，对于理解 纤毛和纤毛病的基本生物学。首先，实验将确定功能和特定位置 在未表征的睫状蛋白的纤毛内。纤毛含有超过650种蛋白质，其中少于 一半的特征是。轴突微管的中央对易于富含 未表征的蛋白质，因此最初的努力将研究它。第二个重点将放在基本上 flagallar运输（IFT）的机制，这是从底座的大型多工厂“火车”的运动 纤毛到睫状尖端，然后回到细胞体。这些火车由包括 IFT-A和IFT-B，载有纤毛的组装和维护所需的cargos。火车 细胞体中的形成涉及将IFT-A和IFT-B募集到纤毛的底部，货物的装载 在复合物上，电动机附着在复合物上，并将完整的火车注入 纤毛。当此过程有缺陷时，睫状组件会失败，但对单个步骤中的步骤几乎不知道 这个过程。研究将使用高分辨率的结构化弹性显微镜和突变体，其中训练 形成在各个步骤中被捕，以确定这些步骤的顺序和单个蛋白质的作用 在过程的关键部分中。相关研究将探索IFT-A在纤毛组装中的特定功能。在 另外，将执行单粒子冷冻电子显微镜，以确定IFT-A和 IFT-B，这对于理解这些复合物如何在火车中排列至关重要。第三个重点 将位于过渡区，这是基本体和睫状轴突之间的专门区域。 过渡区充当与IFT协同的障碍，对于建立和维护 纤毛的蛋白质含量。但是，过渡区仍然主要是一个“黑匣子”。有缺陷的突变体 将研究过渡区蛋白质，以更多地了解这些蛋白质在过渡中的特定作用 区域功能和组装，并确定高度组成的Y链接的组成，连接 向上膜的过渡区微管对过渡区的屏障至关重要 功能。结果将填补我们对纤毛和鞭毛的了解，并提供有关为什么 特定睫状蛋白的缺陷引起人类疾病。