9617183 Horwath Recently, proteinaceous antifreezes, once thought to be an adaptation restricted to polar fishes and overwintering terrestrial insect, have been found among much broader phylogenetcally diverse groups of both plant and animal species. Yet, despite the now numerous examples of their role in survival of subzero temperatures, very little is known about the cellular mechanisms regulating production. This study will use a newly developed cell culture system derived from the beetle, Tenebrio molitor, to begin for the first time a more direct assessment of environmental, hormonal, and cellular mechanisms influencing production, storage and secretion of a specific insect antifreeze protein. This is possible because this in vitro system allows one to track and quantitate specific antifreezes from their biosynthesis, throughout their trafficking within the cell, through to secretion into the blood, using sensitive and specific biochemical, immunological, and computer imaging tools. In addition, molecular studies that have recently resulted in cloning of a specific insect antifreeze gene, will be expanded to obtain more molecular characterization of this gene and other possible homologues. Thus, through a combination of in vitro and molecular approaches, this project will do much to unravel the cellular and genetic mechanisms contributing to antifreeze activity and thus effective cold hardening, and should lead to practical cryobiological applications.
