A reassessment of the factors affecting microtubule assembly and disassembly in vitro

Current models of microtubule assembly from pure tubulin involve a nucleati on phase followed by microtubule elongation at a constant polymer number. B oth the rate of microtubule nucleation and elongation are thought to be tig htly influenced by the free GTP-tubulin concentration, in a law of mass act ion-dependent manner. However, these basic hypotheses have remained largely untested due to a lack of data reporting actual measurements of the microt ubule length and number concentration during microtubule assembly. Here, we performed simultaneous measurements of the polymeric tubulin conce ntration, of the free GTP-tubulin concentration, and of the microtubule len gth and number concentration in both polymerizing and depolymerizing condit ions. In agreement with previous work we find that the microtubule nucleati on rate is strongly dependent on the initial GTP-tubulin concentration. But we find that microtubule nucleation persists during microtubule elongation . At any given initial tubulin-GTP concentration, the microtubule nucleatio n rate remains constant during polymer assembly, despite the wide variation in free GTP-tubulin concentration. We also find a remarkable constancy of the rate of microtubule elongation during assembly. Apparently, the rate of microtubule elongation is intrinsic to the polymers, insensitive to large variations of the free GTP-tubulin concentration. Finally we observe that w hen, following assembly, microtubules depolymerize below the free GTP-tubul in critical concentration, the rate-limiting factor for disassembly is the frequency of microtubule catastrophe. At all time-points during disassembly , the microtubule catastrophe frequency is independent of the free GTP-tubu lin concentration but, as the microtubule nucleation rate, is strongly depe ndent on the initial free GTP-tubulin concentration. We conclude that the d ynamics of both microtubule assembly and disassembly depend largely on fact ors other than the free GTP-tubulin concentration. We propose that intrinsi c structural factors and endogenous regulators, whose concentration varies with the initial conditions, are also major determinants of these dynamics. (C) 2000 Academic Press.