CILIARY MOTION: MICROTUBULE SLIDING AND THE DYNEINS

纤毛运动受损和随之而来的液体清除不足有关 Karagener综合征患者纤毛动力蛋白ATPase缺陷的研究 或者囊性纤维症。基于微管的滑动细丝机制 纤毛、鞭毛和精尾含有两个ATPase，即21S和13S Dyneins，占据外排和内排手臂位置 分别进行了分析。这项建议继续研究基本的 动力蛋白ATPase表达的纤毛运动机制 滑丝机构。该提案的总体目标是 测试13s和21s动力蛋白相互作用的想法 现象学上或生物化学上产生调节运动的。体现 通过研究是对化学成分和功能的比较分析 两个ATPase。这些问题以前没有得到解决，因为 缺乏适当的分析方法来区分 每个ATPase。该提案分为两个主题，尽管每个主题都是 在概念上相互关联，并依赖于 从分离的酶中提取二重微管。两位将军 问题是：1)13和21有什么不同 ATPase与微管相互作用，以及2)ATPase的功能是什么 纤毛运动中的13S和21S ATPase？这两个问题都将由 结合比浊法(350 Nm)测定蛋白质-蛋白质相互作用 通过基本的酶学程序、体外再激活和电子 显微镜。初步数据显示，外臂和内臂动力蛋白 有着根本不同的表达方式。实验将是 进一步的提纯、分级和表征 纤毛四膜虫的两种动力蛋白，初步比较了 这两种动力蛋白的酶学性质和再结合性质。功能界别 这两种酶的体外性质的表达将通过 在重新结合其中一个或两个ATPase后，提取的轴丝重新激活。 13S-ATPase的活性可能代表调节中介 负责将主滑动与传播弯曲相结合，因为 可以通过改变13S的衬底来分离这两种功能 ATPase。这些研究不仅将提供有关 纤毛的13S和21S动力蛋白，但他们也将立即 可能的细胞质动力蛋白研究的意义 这些特征可能被证明更像13S动力蛋白，而不是更像。 21s动力蛋白。

Impaired ciliary motion and consequent lack of fluid clearance are related to defective dynein ATPases in cilia of patients having Kartagener syndrome or cystic fibrosis. The microtubule-based sliding filament mechanism of cilia, flagella and spermtails incorporates two ATPases, the 21S and 13S dyneins, which occupy the outer row and inner row arm positions respectively. This proposal continues studies of the fundamental mechanisms of ciliary motion as expressed by the dynein ATPases and the sliding filament mechanism. The general objective of the proposal is to test the idea that the 13S and 21S dyneins interact either phenomenologically or biochemically to produce regulated motion. Embodied by the study is a comparative analysis of the chemistry and function of the two ATPases. These problems have not been addressed previously because of the lack of appropriate assays to distinguish between the contributions of each ATPase. The proposal is separated into two topics, although each is related conceptually to the other and depends on functional rebinding of the isolated enzymes to extracted doublet microtubules. Two general questions are asked: 1) What are the differences by which the 13S and 21S ATPases interact with microtubules, and 2) What are the functions of the 13S and 21S ATPases in ciliary motion? Both questions will be pursued by combining a turbidimetric assay (350 nm) for protein-protein interactions with basic enzymological procedures, in vitro reactivation, and electron microscopy. Preliminary data suggest that the outer and inner arm dyneins have fundamentally different modes of expression. Experiments will be directed to further purification, fractionation and characterization of both dyneins from Tetrahymena cilia, preliminary to comparisons of the enzymological and rebinding properties of both dyneins. The functional expression of the in vitro properties of both enzymes will be asseessed by reactivation of extracted axonemes after rebinding either or both ATPases. Activity of the 13S ATPase may represent the regulatory intermediary responsible for coupling primary sliding with propagated bending because the two functions can be uncoupled by altering the substrate for the 13S ATPase. These studies not only will provide fundamental information on the 13S and 21S dyneins from cilia but they also will have immediate implications for studies of putative cytoplasmic dyneins whose primary characteristics may prove to resemble 13S dynein more than they resemble 21S dynein.