Abstract
Methods have been developed for differentially inhibiting microtubule nucleation and elongation in vitro. By use of polyanions, assembly-competent tubulin solutions of several milligrams/milliliter can be prepared which do not exhibit appreciable spontaneous assembly during the time-course of an experiment. Microtubule elongation can be initiated by the addition of known numbers of microtubule fragments. A detailed analysis of the resulting process demonstrates that: (a) rings are not obligatory intermediates in the nucleation sequence, and neither rings nor protofilament sheets are obligatory intermediates in the elongation reaction. (b) The end of an elongating microtubule often has a short region of open protofilament sheet or "C-microtubule" similar to that observed in vivo. (c) The development of turbidity follows a simple exponential approach to an equilibrium value. (d) The final equilibrium values are independent of the number of added nucleating fragments, while the initial growth rates and half-times to reach equilibrium are dependent on the number of added nuclei. (e) The final lengths of the microtubules at equilibrium are inversely proportional to the number of added fragments. (f) The equilibrium constants are independent of microtubule length. (g) The number of assembly and disassembly sites per microtubule is not a function of microtubule length. (h) The forward rate constants, the final polymer concentrations, and growth rates of microtubules are dependent upon the concentration of polyanion present. These results are strongly supportive of the idea that microtubule assembly is a "condensation-polymerization" and provide basic information on the kinetics and length distributions of the elongation in vitro.