Structure, Function, and Expression of Tubulins, Globins, and Microtubule-Dependent Motors from Cold-Adapted Antarctic Fishes
P.I. William Detrich
As the Southern Ocean cooled during the past 25 million years, the fishes of Antarctic coastal waters evolved biochemical and physiological adaptations that maintain essential cellular processes such as cytoskeletal function and gene transcription. Their microtubules, for example, assemble and function at body temperatures (-1.8 to +1 oC) well below those of homeotherms and temperate poikilotherms. The long range goals of the proposed research are to determine, at the molecular level, the adaptations that enhance the assembly of microtubules, the function of kinesin motors, and the expression of globin and tubulin genes. The specific objectives are three: 1) to determine the primary sequence changes and posttranslational modifications that contribute to the efficient polymerization of Antarctic fish tubulins at low temperatures; 2) to evaluate the biochemical adaptations required for efficient function of the brain kinesin motor of Antarctic fishes at low temperatures; and 3) to characterize the structure, organization, and promoter-driven expression of globin and tubulin genes from an Antarctic rockcod (Notothenia coriiceps) and a temperate congener (N. angustata). Brain tubulins from Antarctic fishes differ from those of temperate and warm-blooded vertebrates both in unusual primary sequence substitutions (located primarily in lateral loops and the cores of tubulin monomers) and in posttranslational C-terminal glutamylation. Potential primary sequence adaptations of the Antarctic fish tubulins will be tested directly by production of wild-type and site directed tubulin mutants for functional analysis in vitro. The capacity of mutated and wild-type fish tubulins to form "cold-stable" microtubules will be determined by measurement of their critical concentrations for assembly and by analysis of their dynamics by video-enhanced microscopy. Three unusual substitutions in the kinesin motor domain of Chionodraco rastrospinosus may enhance mechanochemical activity at low temperature by modifying the binding of ATP and/or the velocity of the motor. To test the functional significance of these changes, the fish residues will be converted individually, and in concert, to those found in mammalian brain kinesin. Reciprocal substitutions will be introduced into the framework of the mammalian kinesin motor domain. After production in Escherichia coli and purification, the functional performance of the mutant motor domains will be evaluated by measurement of the temperature dependence of their ATPase and motility activities. Molecular adaptation of gene expression in N. coriiceps will be analyzed using an a-globin/b-globin gene pair and an a-tubulin gene cluster. Structural features of N. coriiceps globin and tubulin gene regulatory sequences (promoters and enhancers) that support efficient expression will be assessed by transient transfection assay of promoter/luciferase reporter plasmid constructs in inducible erythrocytic and neuronal model cell systems followed by assay of luciferase reporter activity. Together, these studies should reveal the molecular adaptations of Antarctic fishes that maintain efficient cytoskeletal assembly, mechanochemical motor function, and gene expression at low temperatures. In the broadest sense, this research program should advance the molecular understanding of the poikilothermic mode of life.
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