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.
