Dynamics of Antarctic fish microtubules at low temperatures

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Abstract
The tubulins of Antarctic fishes, purified from brain tissue and depleted of microtubule-associated proteins (MAPs), polymerized efficiently in vitro to yield microtubules at near-physiological supraphysiological temperatures (5, 10 and 20.degree. C). The dynamics of the microtubules at these temperatures were examined through the use of labeled guanosine 5''-triphosphate (GTP) as marker for the incorporation retention, and loss of tubulin dimers. Following attainment of a steady state in microtubule mass at 20.degree. C, the rate of incorporation of [3H]GTP (i.e., tubulin dimers) during pulses of constant duration decreased asymptotically toward a constant, nonzero value as the interval prior to label addition to the microtubule solution increased. Concomitant with the decreasing rate of label incorporation, the average length of the microtubules increased, and the number concentration of microtubules decreased. Thus, redistribution of microtubule lengths (probably via dynamic instability and/or microtubule annealing) appears to be responsible for the time-dependent decrease in the rate of tubulin uptake. When the microtubules had attained both a steady state in mass and a constant length distribution, linear incoporation of labeled tubulin dimers over time occurred at rates of 1.45 s-1 at 5.degree. C, 0.48 s-1 at 10.degree. C, and 0.18 s-1 at 20.degree. C. Thus, the microtubules displayed greater rates of subunit flux, or treadmilling, at lower, near-physiological temperatures. At each temperature, most of the incorporated label was retained by the microtubules during a subsequent chase with excess unlabeled GTP. In contrast, when microtubules were asembled de novo in the presence of [.alpha.-32P]GTP at 5.degree.C and then exposed to a pulse of [3H]GTP, the 32P label was lost over time during a subsequent chase with unlabeled GTP, whereas the 3H label was retained. Together, these results indicate that the microtubules of Antarctic fishes exhibit, at low temperatures, behaviors consistent both with subunit treadmilling and with dynamic instability and/or microtubule annealing.