Suppression of microtubule dynamic instability and treadmilling by deuterium oxide

Deuterium oxide (D2O) is known to promote the assembly of tubulin into micr otubules in vitro, to increase the volume of mitotic spindles and the numbe r and length of spindle microtubules, and to inhibit mitosis. Reasoning tha t its actions on cellular microtubules could be due to modulation of microt ubule dynamics, we examined the effects of replacing H2O with D2O on microt ubule dynamic instability, treadmilling, and steady-state GTPase activity. We found that replacing 50% or more of the H2O with D2O promoted microtubul e polymerization and stabilized microtubules against dilution-induced disas sembly. Using steady-state axoneme-seeded microtubules composed of pure tub ulin 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 m echanism responsible for the ability of D2O to stabilize microtubule dynami cs may involve enhancement of hydrophobic interactions in the microtubule l attice and/or the substitution of deuterium bonds for hydrogen bonds.