Evidence for two growth steps in microtubule polymerization

Abstract

For microtubule assembly, the data reported here support an initial nucleation phase followed by a growth or elongation phase. The nucleation phase was not detected kinetically. Evidence for this step was given by the existence of the critical concentration and the dependence of the number of microtubules on oligomer concentration. Kinetic evidence indicated the existence of two consecutive steps in the growth phase of microtubules. The fast process increased and the slow one decreased with the concentration of microtubule protein. Similar kinetics were found upon recombination of tubulin oligomer and dimer which had been resolved by agarose chromatography. The fast process increased with oligomer and decreased with dimer concentration while the slow one depended positively on dimer concentration. Microtubules were formed when the oligomeric fraction only was employed. In contrast, under identical conditions, no microtubule formation was detected turbidimetrically or by electron microscopy from dimer alone. When dimer caused elongation of seed tubules, there was only one growth step with a rate constant of the same order of magnitude as the slow process for the other experiments.