Temperature-dependent reversible assembly of taxol-treated microtubules.
Taxol is a plant alkaloid that binds to and strongly stabilizes microtubules. Taxol-treated microtubules resist depolymerization under a variety of conditions that readily disassemble untreated microtubules. We report here that taxol-treated microtubules can be induced to disassemble by a combination of depolymerizating conditions. Reversible cycles of disassembly and reassembly were carried out using taxol-containing microtubules from calf brain and sea urchin eggs by shifting temperature in the presence of millimolar levels of Ca2+. Microtubules depolymerized completely, yielding dimers and ring-shaped oligomers as revealed by negative stain electron microscopy and Bio-Gel A-15m chromatography, and reassembled into well-formed microtubule polymer structures. Microtubule-associated proteins (MAPs), including species previously identified only by taxol-based purification such as MAP 1B and kinesin, were found to copurify with tubulin through reversible assembly cycles. To determine whether taxol remained bound to tubulin subunits, we subjected depolymerized taxol-treated microtubule protein to Sephadex G-25 chromatography, and the fractions were assayed for taxol content by reverse-phase HPLC. Taxol was found to be dissociated from the depolymerized microtubules. Protein treated in this way was found to be competent to reassemble, but now required conditions comparable with those for protein that had never been exposed to taxol. Thus, the binding of taxol to tubulin can be reversed. This has implications for the mechanism of taxol action and for the purification of microtubules from a wide variety of sources for use in self-assembly experiments.
D000470:Alkaloids / Q000494:pharmacology*; D000818:Animals; D001921:Brain / Q000648:ultrastructure; D002118:Calcium / Q000494:pharmacology; D002417:Cattle; D002458:Cell Fractionation; D005260:Female; D007700:Kinetics; D046911:Macromolecular Substances; D008854:Microscopy, Electron; D008868:Microtubule Proteins / Q000378:metabolism*; D008870:Microtubules / Q000187:drug effects / Q000648:ultrastructure*; D010063:Ovum / Q000648:ultrastructure; D017239:Paclitaxel; D012617:Sea Urchins; D013696:Temperature
D000470:Alkaloids; D046911:Macromolecular Substances; D008868:Microtubule Proteins; D017239:Paclitaxel; D002118:Calcium