Taxol stabilization of mitotic spindle microtubules: analysis using calcium-induced depolymerization.

Abstract

Taxol stabilizes or promotes the assembly of microtubules. In this report we characterize the rate, extent, and reversibility of taxol stabilization of calcium-labile microtubules in isolated mitotic spindles, principally from embryos of the sand dollar Echinarachnius parma. The intense depolymerizing action of 100 microM Ca2+ was used to assess the extent of stabilization by taxol. Changes in spindle microtubule assembly were evaluated and recorded by measuring changes in spindle birefringent retardation (BR). Membrane-free mitotic spindles, isolated with a calcium-chelating, nonionic detergent buffer, were stored in an EGTA-glycerol storage buffer to prevent microtubule depolymerization. When perfused with an EGTA-buffer without glycerol, microtubules in these isolated spindles depolymerized gradually over 60-120 min; but in isolated spindles perfused with buffer that contained 100 microM Ca2+, BR decreased by 90% within 2-5 sec. In contrast, spindles that were pretreated for 3 min with 1 microM taxol, or for about 30 sec with 10 microM taxol, lost less than 10% of their initial BR when perfused with buffer containing 100 microM Ca2+. The rate and extent of microtubule stabilization by taxol depended on both the concentration and the duration of exposure to taxol. Taxol stabilization was reversible. After a 15 min preincubation with 1 microM or 10 microM taxol then washout, stability of spindle BR to 100 microM Ca2+ decreased exponentially with a time constant of 30-60 min. Thus taxol dissociates from spindle microtubules at significant rates; taxol-stabilized microtubules are not "fixed."