The temperature biology of vertebrate actins and the actin-based motility of fish keratocytes.

摘要

The work described herein focuses on the evolution of vertebrate actins in species adapted to various temperatures. I began by sequencing alpha-actins from five ectotherms living across a range of environmental temperatures (-1.86°C to 45°C), and purified alpha-actin from the two species living at the ends of this temperature range to determine thermal stability in vitro , alpha-actins of the two species which display the most disparate body temperatures (a difference of 47°C), do not differ in their primary sequence and their thermal stabilities are not significantly different from each other. I conclude that vertebrate alpha-actin is sufficiently conserved to preclude thermal adaptation at the level of primary sequence. Next, these sequences allowed for a unique examination of the GC-adaptation temperature hypothesis by examining patterns of base composition of genes encoding lactate dehydrogenase-A and alpha-actin from 51 species of vertebrates whose adaptation temperatures ranged from -1.86°C to approximately 45°C. No significant positive correlation was found between any index of GC content and any index of adaptation temperature. Finally, I characterized the effect of temperature on actin-based cell motility in fish epithelial keratocytes from fish adapted to different temperatures. Keratocyte speed increases with increasing temperature; average speed is conserved at physiological temperature for temperate and tropical species. Analysis of angular velocity reveals that keratocytes take straighter paths at lower temperatures, and more circular paths or paths "doubling-back" upon themselves at higher temperatures, although this is affected by thermal acclimation. However, acclimation does not alter the effect of experimental temperature on cellular speed. These findings suggest that more than one temperature-sensitive mechanism may govern cell motility. I also investigated the role of membrane fluidity in cell motility as a putative mechanism underlying the effects of temperature. I used small particle tracking to monitor the change in diffusion coefficient at different temperatures and in the presence and absence of the detergent deoxycholate. Changes in diffusion coefficient did not correlate with changes in cell speed under any condition. These results demonstrate that membrane fluidity does not govern the rate of cell movement and cannot account for the large effects of temperature on cell speed.