Magnesium ion effects on microtubule nucleation in vitro

Abstract

Much interest has currently been attached to the length distribution of microtubules polymerized in vitro and the related question of their possible ‘dynamic instability’. Fundamental to this question is the mechanism of microtubule nucleation, which controls the rates of assembly and disassembly of microtubule protein in vitro. These kinetics are affected by a number of factors, including both the guanine nucleotides, GTP and GDP, and magnesium ion. Mg 2+ exerts complex effects, as indicated by the existence of an optimal Mg 2+ concentration for the maximum assembly rate of microtubule protein, and we investigate these effects in this report. At [Mg 2+] > 0.5 mM, the characteristic lag-phase is substantially increased and the rate of assembly is greatly reduced without affecting the critical concentration significantly. We show that increasing [Mg 2+] has two effects on the assembly process: nucleation is less efficient and the intrinsic rate constant for the elongation reaction is reduced. Lowering [Mg 2+] (less than 0.5 mM) also inhibits nucleation. These effects of varying [Mg 2+] can be explained predominantly in terms of enhanced stability of the microtubule-associated protein-containing oligomeric species present in the microtubule protein preparation. [Mg 2+] is thus found to be a further important factor in microtubule nucleation, and hence, in determining length distributions in assembling microtubules.