This work seeks to understand the internal mechanisms responsible for the movements of flagella and cilia. These mechanisms include the dynein- microtubule interactions responsible for active sliding, the oscillatory mechanism, and control mechanisms that determine a particular type of bending pattern and its parameters. Because of the importance of cilia and flagella in normal respiratory and reproductive functions (as evidenced by the pathology of """"""""immotile cilia syndromes""""""""), an understanding of the functioning of these organelles is important in its own right. In addition, simple flagella provide a particularly accessible and highly organized system that is a major source of detailed information about microtubule-mediated motility, providing insights that increase our understanding of other systems such as mitosis and axonal transport, as well as systems involving the other motor enzymes, kinesin and myosin. This work will utilize simple flagella such as those of sea urchin and tunicate spermatozoa. Most of the experimental work will involve ATP- reactivated movement of Triton-demembranated spermatozoa. Moving sperm flagella are photographed with high spatial and temporal resolution. The films are analyzed by computerized image analysis methods to obtain quantitative descriptions of movement under various conditions. When necessary, direct measurements of microtubule sliding can be made using 40 nm gold beads as markers of positions on the exposed outer doublet microtubules of demembranated flagella. Interpretation of results will be assisted by using programs for computer simulation of flagellar motility. Major questions that will be addressed include: What distribution of active sliding is required to initiate a new bend at the base of a flagellum? What triggers the initiation of active sliding in a newly formed bend? What determines the relatively constant bend angle of propagating bends? What differences between the processes of bend initiation and bend propagation make it possible to inhibit them separately? What are the relationships between specific alterations in the dynein ATPase cycle and the characteristics of flagellar bending? How do the outer arm dyneins modify the bending pattern? How do changes in phosphorylation of flagellar proteins determine whether bend propagation occurs in addition to bend initiation? What protein kinases are responsible for these phosphorylations?
