Cilia are important motile cell organelles of organisms including man. This study continues a comprehensive attack on the structure and function of cilia, centering on ultrastructural correlates of ciliary motion, including analysis of the mechanism of movement and its regulation. This approach has been influential in the development of the sliding microtubule model of ciliary motility, now widely accepted. Despite the success of this model, fundamental questions remain regarding the basic interaction responsible for sliding and the hierarchy of regulatory processes between the sliding event and actual ciliary beat. One major aim of this proposal is to strengthen the hypothesis that a unique cycle of dynein arm activity is responsible for microtubule sliding in motile cilia, and to specify the structural bases of this cycle further. The subunit structure of the arm will be studied using negative stain, freeze etch and rotary shadow electron microscopy. Structure will be correlated with polypeptide composition, location of microtubule attachment points and ATPase activity.
A second aims i s to use such structural information to probe possible cooperative and/or asynchronous arm activity in the axoneme. Asynchronous arm activity is embodied in several axonemal switching hypotheses relating sliding of specific microtubules to defined beat positions. These hypotheses will be tested by direct readout of arm configuration along single doublets and from doublet to doublet in cilia treated with various ATP analogs or arrested in specific stroke positions, for example after treatment with Ca2+ or calmodulin (CaM)-directed drugs. The possibility that CaM or CaM-binding proteins are part of the dynein arm in some cilia will be explored. This information should lead to further understanding of normal ciliary activity and of ciliary malfunction in respiratory disease.
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