Question

1) Comparisons of microtubule behavior between species point to differences that raise questions about the biological importance of dynamic instability. Notothenioid fish, for example, which live in the Southern Ocean at a constant temperature of –1.8°C, have remarkably stable microtubules compared with warm-blooded vertebrates such as the cow. This is an essential modification for notothenioid fish because normal microtubules disassemble completely into αβ-tubulin dimers at 0°C. Measurements on individual microtubules in solutions of pure tubulin show that notothenioid fish microtubules grow at a much slower rate, shrink at a much slower rate, and only rarely switch from growth to shrinkage (catastrophe) or from shrinkage to growth (rescue) (Table 1).

A. The amino acid sequences of the α- and β-tubulin subunits from notothenioid fish differ from those of the cow at positions and in ways that might reasonably be expected to stabilize the microtubule, in accord with the data in Table 16–1. Would you expect these changes to strengthen the interactions between the α- and β-tubulin subunits in the αβ-dimer, between adjacent dimers in the protofilament, or between tubulin subunits in adjacent protofilaments? Explain your reasoning.

B. Dynamic instability is thought to play a fundamental role in the rapid microtubule rearrangements that occur in cells. How do you suppose cells in these notothenioid fishes manage to alter their microtubule architecture quickly enough to accomplish essential cell functions? Or do you suppose that these cells exist with a stable microtubule cytoskeleton that only slowly rearranges itself?

TABLE 16 domestic cow (Problem 16-79) Microtubules -1 Properties of individual microtubules in notothenioid fish and the Growth Shrinkage Catastrophe Rescue frequency min-1 frequency rate (um/min) (um/min) rate (min 0.008 0.52 Notothenioid fish 0.27 0.8 0.0004 Domestic cow 2.18 61.2 Multiple individual microtubules were observed by video microscopy near the body temperature for each species: 5°C for fish and 37°C for cow. Average growth rates were calculated for growing microtubules, and average shrinkage rates were calculated for shrinking microtubules Changes from growth to shrinkage (catastrophe) and from shrinkage to growth (rescue) were averaged over the observation period and expressed as frequency of events per minute.

Answer 1

jbc.org

Cold adaptation of microtubule assembly and dynamics structural interpretation of primary sequence changes present in the α-and β-tubulins of antarctic fishes

H William Detrich, Sandra K Parker, Robley C Williams, Eva Nogales, Kenneth H Downing

Journal of Biological Chemistry 275 (47), 37038-37047, 2000

The microtubules of Antarctic fishes, unlike those of homeotherms, assemble at very low temperatures (− 1.8 C). The adaptations that enhance assembly of these microtubules are intrinsic to the tubulin dimer and reduce its critical concentration for polymerization at 0 C to∼ 0.9 mg/ml (Williams, RC, Jr., Correia, JJ, and DeVries, AL (1985) Biochemistry 24, 2790–2798). Here we demonstrate that microtubules formed by pure brain tubulins of Antarctic fishes exhibit slow dynamics at both low (5 C) and high (25 C) temperatures; the rates of polymer growth and shortening and the frequencies of interconversion between these states are small relative to those observed for mammalian microtubules (37 C). To investigate the contribution of tubulin primary sequence variation to the functional properties of the microtubules of Antarctic fishes, we have sequenced brain cDNAs that encode 9 α-tubulins and 4 β-tubulins from the yellowbelly rockcod Notothenia coriiceps and 4 α-tubulins and 2 β-tubulins from the ocellated icefish Chionodraco rastrospinosus. The tubulins of these fishes were found to contain small sets of unique or rare residue substitutions that mapped to the lateral, interprotofilament surfaces or to the interiors of the α-and β-polypeptides; longitudinal interaction surfaces are not altered in the fish tubulins. Four changes (A278T and S287T in α; S280G and A285S in β) were present in the S7-H9 interprotofilament “M” loops of some monomers and would be expected to increase the flexibility of these regions. A fifth lateral substitution specific to the α-chain (M302L or M302F) may increase the hydrophobicity of the interprotofilament interaction. Two hydrophobic substitutions (α: S187A in helix H5 and β: Y202F in sheet S6) may act to stabilize the monomers in conformations favorable to polymerization. We propose that cold adaptation of microtubule assembly in Antarctic fishes has occurred in part by evolutionary restructuring of the lateral surfaces and the cores of the tubulin monomers.