Dynein molecules interact with ciliary and flagellar microtubules to generate the ciliary beat, with the concomitant hydrolysis of ATP. An understanding of their mode of action may facilitate the analysis of such clinical disorders as infertility, emphysema, and cystic fibrosis, and may pertain to the mechanics of the mitotic spindle and to microtubule-mediated shape changes during embryology. Methods have been developed in these laboratories for the isolation and purification of dyneins from the outer and inner arms, and for visualizing these molecules in situ and in vitro using the quick-freeze deep-etch technique. The long-term objective is to understand the dynein force-generating cycle in the fashion now possible for the myosin cycle.
Five specific aims are described. 1. Outer-arm dynein will be dissociated into subunits and fractionated by high-performance liquid chromatography. By combining deep-etching with studies on ATPase activity, microtubule-binding ability, monoclonal-antibody binding, and the like, it should be possible to generate a functional map of the protein's domains. 2. Outer-arm dynein will be studied as it decorates microtubules and responds to ATP, the goal being to analyze the location and the modification of its ATP and tubulin binding sites. These studies will hopefully complement the studies on dissociated dynein. 3. A functional map of the inner-arm dyneins will be generated, and their role in ciliary mechanics will be assessed. 4. Unusual dyneins or dynein-like proteins from various sources will be studied to establish homologies and look for functional similarities with """"""""standard"""""""" dyneins. 5. Motile detergent-extracted cells will be studied to learn how dyneins change their configuration, and their nucleotide phosphate associations, during the in situ beat cycle.
