End-to-end joining of taxol-stabilized GDP-containing microtubules

By the use of the drug taxol, microtubules were assembled from tubulin that had GDP at its exchangeable nucleotide binding site. By means of dilution experiments and measurements of exchange of labeled subunits, it was determined that the rate of interchange of subunits between these microtubules and the solution is very slow: the upper limit of the dissociation rate constant was measured to be 0.2 subunit s-1 end-1. When they were broken into short pieces by gentle shearing, these microtubules were found to undergo a rapid subsequent spontaneous increase in length. This increase was attributed to end-to-end joining (also called annealing), because dynamic instability and other mechanisms involving either nucleotide hydrolysis or subunit interchange at the ends could be ruled out. To characterize the process, a diffusion-controlled joining mechanism was hypothesized, and a length-independent bimolecular rate constant, gamma, was defined. Length distributions were measured at a series of times after the initial shearing. By means of a novel iterative calculation, the best-fitting value of gamma was determined from the time-dependent changes in the length distributions. Fitting was carried out at each of three concentrations of microtubules. The resulting values show that end-to-end joining of microtubules is remarkably efficient and that gamma is concentration-dependent.