Is microtubule assembly a biphasic process? A fluorimetric study using 4',6-diamidino-2-phenylindole as a probe.

Abstract

The kinetics of microtubule assembly followed by turbidimetry usually describe an exponential process but hyperbolic or biphasic assembly curves have also been reported. A combination of turbidimetry, 4',6-diamidino-2-phenylindole (DAPI) fluorescence, electron microscopy, rapid sedimentation and wavelength dependence of scattered light have been used here to investigate these apparently biphasic kinetics of microtubule assembly. Experimental conditions have been developed under which the increase in DAPI fluorescence is a quantitative measurement of polymer formation which, in contrast to turbidity, does not depend on the size of the microtubules. Monophasic assembly curves were obtained when the increase in DAPI fluorescence was measured, whereas under the same conditions the turbidity change was a biphasic process. Biphasic turbidity curves are observed at low (less than 0.5 mM) magnesium ion concentration; the data are consistent with a rapid polymerization of tubulin into short polymers, followed by extensive length redistribution. The morphology of the early intermediates in microtubule assembly appears dependent on the concentration of Mg2+ ions.