CILIARY MOTION: MICROTUBULE SLIDING AND THE DYNEINS

睫状体运动受损和随之而来的液体清除不足是相关的 Kartagener综合征患者纤毛中动力蛋白ATP酶缺陷 或囊性纤维化。 基于微管的滑丝机制 纤毛、鞭毛和精子尾含有两种ATP酶，21 S和13 S 动力蛋白，占据外排和内排臂位置 分别 这项建议继续研究基本的 纤毛运动的机制，表达的动力蛋白ATP酶和 滑丝机构 该提案的总体目标是 测试13 S和21 S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。 体现 通过研究比较分析， 两个ATP酶 这些问题以前没有得到解决，因为 缺乏适当的分析来区分 每个ATP酶 该提案分为两个主题，但每个主题都是 在概念上与另一个相关，并依赖于功能的重新绑定， 分离的酶提取的双峰微管。 两个一般 问题是：1）13 S和21 S的区别是什么？ ATP酶与微管相互作用，2）微管的功能是什么？ 纤毛运动中的13 S和21 S ATP酶？ 这两个问题将由 结合用于蛋白质-蛋白质相互作用的浊度测定（350 nm） 用基本的酶学程序，体外再活化和电子 显微镜 初步数据表明，外臂和内臂动力蛋白 有着根本不同的表达方式。 实验将 涉及进一步纯化、分级分离和表征 这两种动力蛋白都来自四膜虫纤毛，初步比较了 两种动力蛋白的酶学和再结合性质。 功能 两种酶的体外特性的表达将通过以下方法进行评估： 在重新结合任一种或两种ATP酶后，提取的轴丝重新激活。 13 S ATP酶的活性可能代表调节中介 负责耦合主要滑动与传播弯曲， 这两种功能可以通过改变13 S的基底来分离 ATP酶 这些研究不仅将提供关于 纤毛中的13 S和21 S动力蛋白，但它们也会立即 细胞质动力蛋白研究的意义， 与13 S动力蛋白相比， 21 S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.