The kinetics of microtubule assembly. Evidence for a two-stage nucleation mechanism

Abstract

A model describing the nucleation and assembly of purified tubulin has been developed. The novel feature of this model is a two stage nucleation process to allow the explicit inclusion of the two-dimensional nature of the early stages of microtubule assembly. In actin assembly the small starting nucleus has only one site for subunit addition as the two-stranded helix is formed. In contrast, microtubule assembly begins with the formation of a small two-dimensional section of microtubule wall. The model we propose is a modification of the work of Wegner and Engel (Wegner, A., and Engel, J. (1975) Biophys. Chem. 3, 215-225) wherein we add a second stage of nucleation to directly account for lateral growth, i.e. the addition of a small number of subunits to the side of an existing sheet structure. Subsequent elongation of the sheets is treated in the usual way. The experimental system used to test this model was the Mg2+/glycerol induced assembly of purified tubulin. The computer simulation of the polymerization time courses gave a fairly good fit to experimental kinetics for our model, where the primary nucleus comprises two protofilaments, of four and three subunits, and lateral growth requires a three-subunit nucleus to initiate a new protofilament.