There are two objectives of this proposal: (a) to study the chemistry and structure of ciliary and flagellar microtubules; and (b) to investigate the mechanisms that control ciliary and flagellar motility. Microtubules are involved in vital cell processes including cell divisions, cytoplasmic motility and transport, cell-shape changes, secretion, and ciliary and flagellar motility. Normal microtubule structure and function is often seriously affected by pharmacological drugs, environmental toxins and disease. Our understanding of mocrotubule function will benefit from studies on flagella, the best characterized of the microtubule systems, for it is with flagella that the functional roles of microtubules can most unambiguously be determined. This proposal rests on my recently published findings that flagellar microtubules can be fractrionated into special filaments or protofilaments which are composed of highly insoluble, filamentous proteins chemically distinct from tubulin and which have properties in common with the class of cytoplasmic filaments known as """"""""intermediate filaments."""""""" These studies will be continued as follows, using echinoderm and molluscan cilia an sperm flagella: (1) The relevant filamentous proteins will be purified by standard procedures. Some physical-chemical properties of these proteins will be determined and compared with those of flagellar tubulin and vertebrate and invertebrate intermediate filament proteins. (2) Antibodies will be prepared against the purified proteins and used to further characterize these flagellar proteins. (3) The protein composition and localization in the microtubule wall of the unique (proto-)filaments and associated dynein-components will be studied by combining biochemical, immunological and electron microscope techniques. (4) Finally, in separate studies I will investigate the effects of solution variables (e.g., Ca+, pH, temperature) on the coiling behavior of flagellar microtubules and protofilament ribbons using my recently installed image-intensification-video-microscope. The observed coiling effects will be correlated with the above structural and biochemical data.
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