The specific aims of this project are to elucidate the mechanisms by which the active sliding between tubules of the 9+2 axoneme are controlled and coordinated to produce the propagated bending waves that are characteristic of cilia and sperm flagella, and to clarify how the dynein arms interact with the axonemal structures and with ATP to produce this active sliding. The invertebrate sperm flagellum will continue to serve as model material for studying the interaction of the dynein cross bridge with its sites on the adjacent B-tubule, with particular attention to the possibly different roles of the individual two-headed domains on each cross bridge, as well as the less well studied isoenzymes of the inner dynein arm. Recent discovery of two vanadate-mediated photocleavages that are highly specific for dynein ATPases will provide a valuable approach to these problems. Studies using Tetrahymena cilia, which possess a three heavy chain, three- headed dynein molecule that, unlike sea urchin dynein, can bind to reconstituted brain microtubules, will explore the nature of the structural and ATP-sensitive binding sites through recombination experiments with dynein-depleted sperm flagella as well as with reconstituted brain microtubules. Newly developed techniques of micromanipulation will be used to investigate the interaction between externally imposed vibration and flagellar waveform, as well as to test the hypothesis that the coordination of tubule- sliding within the cylinder of axonemal doublet microtubules is determined by the plane of the central tubules. An attempt will be made to identify the axonemal polypeptides that mediate the control of flagellar beating via cAMP-mediated kinase and phosphatase activities, using radiolabelling techniques. The possibility that one of the peptides associated with outer arm dynein links the axoneme to the sperm flagellar membrane will be further investigated for its potential relationship to the transport of vesicles along microtubules in nerve axons. The work of this project has direct relevance to the consequences of axonemal defects in the cilia and sperm flagella in man that lead to severe respiratory problems associated with the """"""""immotile cilia syndrome"""""""", and to male infertility, and to other cellular processes involving microtubule-based motility, such as the movement of chromosomes during cell division and organelle transport in axons.
Showing the most recent 10 out of 18 publications
