Suppression of microtubule dynamic instability and treadmilling by deuterium oxide

Deuterium oxide (D2O) is known to promote the assembly of tubulin into microtubules in vitro, to increase the volume of mitotic spindles and the number and length of spindle microtubules, and to inhibit mitosis. Reasoning that its actions on cellular microtubules could be due to modulation of microtubule dynamics, we examined the effects of replacing H2O with D2O on microtubule dynamic instability, treadmilling, and steady-state GTPase activity. We found that replacing 50% or more of the H2O with D2O promoted microtubule polymerization and stabilized microtubules against dilution-induced disassembly. Using steady-state axoneme-seeded microtubules composed of pure tubulin and video microscopy, we found that 84% D2O decreased the catastrophe frequency by 89%, the shortening rate by 80%, the growing rate by 50%, and the dynamicity by 93%. Sixty percent D2O decreased the treadmilling rate of microtubules composed of tubulin and microtubule-associated proteins by 42%, and 89% D2O decreased the steady-state GTP hydrolysis rate by 90%. The mechanism responsible for the ability of D2O to stabilize microtubule dynamics may involve enhancement of hydrophobic interactions in the microtubule lattice and/or the substitution of deuterium bonds for hydrogen bonds.