The proposed research constitutes the first effort to investigate the mechanism by which hair bundles on tentacles of sea anemones self-repair. Whereas certain avian species are known to respond to trauma to their hair bundles by replacing entire hair cells, no organism is yet known to repair existing hair bundles. Preliminary data detailed in this proposal suggest that repair is accomplished by proteins secreted by the anemone in response to trauma to its hair bundles. Research plans include fractionation of repair proteins (exclusion chromatography and ion- exchange chromatography). Fractions will be tested for biological activity (i.e., the ability to shorten the time course of recovery in test animals). Biologically active fractions will be subjected to SDS-PAGE gels. Specific, purified repair proteins will be biotinylated. Provided such proteins retain biological activity, they will be used in combination with avidin-fluorochromes or with avidin-colloidal gold to localize binding sites for repair proteins on the anemone at the light (confocal) and electron (field emission scanning) microscopic levels. Any repair proteins losing biological activity as a result of biotinylation will be used as antigens to generate polyclonal antibodies. The antibodies will be used in immunocytochemistry. Video-enhanced differential interference contrast (DIC) microscopy will be used to investigate the repair process on hair bundles of living specimens. Field emission scanning microscopy will be used to characterize the effects of repair proteins on linkages (including tip links) interconnecting stereocilia of the hair bundle. Long range plans for this project including testing the usefulness of anemone repair proteins as possible therapeutic agents for damaged hair bundles in vertebrates.
