Flagellar Motility and Assembly

鞭毛运动和组装

• 批准号: 10152610
• 负责人: George B Witman
• 金额: $ 63.61万
• 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 2024-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10152610
• 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； 纤毛蛋白缺陷会导致人类疾病，称为“纤毛病”。 研究将利用这些细胞器的结构、组装和功能。 衣藻和小鼠作为模型生物体，并将专注于高度相关的过程和蛋白质 遗传、生化和细胞生物学方法的结合。 调查将集中在对于了解情况特别重要的三个相关领域。 首先，实验将确定纤毛和纤毛病的功能和具体位置。 纤毛内含有未表征的纤毛蛋白 纤毛含有超过 650 种蛋白质，其中少于 一半的轴丝微管往往特别丰富。 未表征的蛋白质，因此最初的工作将对其进行检查。 鞭毛内运输（IFT）机制，即大型多亚基“列车”从基部的运动 从纤毛到纤毛尖端，然后返回到细胞体，这些序列由包括以下在内的复合物组成。 IFT-A 和 IFT-B，运载纤毛列车组装和维护所需的货物。 细胞体内的形成涉及将 IFT-A 和 IFT-B 募集到纤毛基部，装载货物 到综合体上，将​​电机连接到综合体上，并将完整的列车注入到 当这个过程有缺陷时，纤毛组装就会失败，但人们对其中的各个步骤知之甚少。 研究将使用高分辨率结构照明显微镜和突变体来训练。 在各个步骤中阻止形成，以确定这些步骤的顺序以及单个蛋白质的作用 相关研究将探讨 IFT-A 在纤毛组装中的具体功能。 此外，还将进行单颗粒冷冻电子显微镜以确定 IFT-A 和 IFT-B，这对于理解这些综合体在火车中的排列方式非常重要。 将位于过渡区，即基底体和睫状轴丝之间的特殊区域。 过渡区作为一个屏障，与 IFT 相结合，对于建立和维持 然而，过渡区在很大程度上仍然是一个存在缺陷的“黑匣子”。 将研究过渡区蛋白，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 型连接的组成，该连接连接 过渡区微管到上覆膜，对于过渡区的屏障至关重要 这些结果将填补我们对纤毛和鞭毛的认识的重大空白，并提供新的见解。 特定纤毛蛋白的缺陷会导致人类疾病。

项目成果

TIM, a targeted insertional mutagenesis method utilizing CRISPR/Cas9 in Chlamydomonas reinhardtii.

TIM，一种在莱茵衣藻中利用 CRISPR/Cas9 的靶向插入诱变方法。

• 发表时间: 2020
• 影响因子: 3.7
• 作者: Picariello, Tyler;Hou, Yuqing;Kubo, Tomohiro;McNeill, Nathan A;Yanagisawa, Haru;Oda, Toshiyuki;Witman, George B
• 通讯作者: Witman, George B

The N-terminus of IFT46 mediates intraflagellar transport of outer arm dynein and its cargo-adaptor ODA16.

IFT46 的 N 末端介导外臂动力蛋白及其货物适配器 ODA16 的鞭毛内运输。

• 发表时间: 2017-09-01
• 影响因子: 3.3
• 作者: Hou, Yuqing;Witman, George B
• 通讯作者: Witman, George B

Proteome of the central apparatus of a ciliary axoneme.

纤毛轴丝中央装置的蛋白质组。

• 发表时间: 2019-06-03
• 作者: Zhao, Lei;Hou, Yuqing;Picariello, Tyler;Craige, Branch;Witman, George B
• 通讯作者: Witman, George B

Characterization of a new oda3 allele, oda3-6, defective in assembly of the outer dynein arm-docking complex in Chlamydomonas reinhardtii.

莱茵衣藻外动力蛋白臂对接复合体组装缺陷的新 oda3 等位基因 oda3-6 的表征。

• 发表时间: 2017
• 影响因子: 3.7
• 作者: Brown, Jason M;Mosley, Matthew;Montes;Hou, Yuqing;Yang, Fan;Scarbrough, Chasity;Witman, George B;Wirschell, Maureen
• 通讯作者: Wirschell, Maureen

The unity and diversity of the ciliary central apparatus.

睫状体中央装置的统一性和多样性。

• 发表时间: 2020-02-17
• 作者: Zhao, Lei;Hou, Yuqing;McNeill, Nathan A;Witman, George B
• 通讯作者: Witman, George B