Characterization of the reversible taxol-induced polymerization of plant tubulin into microtubules

Abstract

Taxol has been reported to induce the polymerization of plant tubulin into microtubules, albeit weakly when compared to that of mammalian tubulin [Morejohn, L.C., & Fosket, D.E. (1984) J. Cell Biol. 99, 141-147], suggesting that taxol, a product of plant secondary metabolism, may interact poorly with plant microtubules. To test this idea in detail, we have investigated critical parameters affecting taxol-dependent microtubule polymerization and stability using tubulins from model cell lines of maize [Zea mays cv. Black Mexican Sweet (BMS)] and tobacco [Nicotiana tabacum cv. Bright Yellow 2 (BY-2)]. When plant tubulin dimer is isolated by using a modified version of the original method [Morejohn, L.C., & Fosket, D.E. (1982) Nature 297, 426-428], most of the tubulin polymerizes at 25 degrees C, with critical dimer concentrations (Cc) of 0.06 mg/mL for BMS tubulin and 0.13 mg/mL for BY-2 tubulin. When taxol-induced assembly is initiated with a 0-25 degrees C temperature jump, 42% of polymer is polymorphic, presumably due to aberrant nucleation events. Taxol-induced assembly at 2 degrees C minimizes the formation of polymorphic structures and is much more rapid than that of purified bovine brain tubulin, indicating a functional difference in the polymerization domains of these diverse tubulins. Temperature ramping during taxol-induced polymerization affords > or = 95% assembly of plant tubulin into polymer consisting of 86% microtubules, which may be completely depolymerized by a combined treatment with low temperature and Ca2+. We report for the first time that plant tubulin may be subjected to numerous cycles of efficient taxol-induced polymerization and cold/Ca(2+)-induced depolymerization with little loss of polymerization competence. Gel filtration chromatography at low temperature may be used to separate taxol from soluble plant tubulin dimer, which retains its characteristic polymerization and herbicide-binding properties. Our results demonstrate that despite its origin from plants, taxol is a potent drug for the reversible polymerization of plant microtubules.

Rapid and Reversible High-Affinity Binding of the Dinitroaniline Herbicide Oryzalin to Tubulin from Zea mays L

“…Taxol was kindly provided by Dr. Nancita Lomax, Drug Synthesis and Chemistry Branch, Division of Cancer Treatment, National Cancer Institute (Bethesda, MD (Bokros et al, 1993).…”

“…Suspension cultures of maize (Zea mays cv Black Me:ucan Sweet) were grown in the dark at 28OC on an orbital shaker as described previously (Bokros et al, 1993). At 7-d inteivals, cells were subcultured by 10-fold dilution into fresh medlium.…”

“…Maize tubulin was isolated from stationary phase (d 7-8) cells using DEAE-Sephadex A50 chromatography accoding to the method of Morejohn and Fosket (1982) with modifications as described by Bokros et al (1993). Ammoinium sulfate precipitates of DEAE-tubulin were resuspended i n an IB consisting of 50 m~ Pipes-KOH (pH 6.9), 1 m~ EGTA, 0.5 m~ MgS04, and 1 m~ DTT and supplemented with 0.1 m~ GTP, 50 p g mL-' Na-p-tosyl-L-Arg methyl ester, and 5 pg mL-' each of pepstatin A, leupeptin hemisulfate, and aprotinin.…”

“…For certain experiments, maize DEAE-tubulin was purified to homogeneity by a single cycle of taxol-induced microtubule polymerization and cold/Ca2+-induced depolymerization (Bokros et al, 1993). Samples were thawed in an ice bath and clarified of particulates by centrifugation for 1 h at lO0,OOOg (2OC) in a Beckman TL-100 ultracentrifuge (I'LA-100 rotor).…”

“…Ligand-binding and polymerization experiments were performed with a buffer containing 1 M SUC, a tubulin-stabilizing component known to prevent the normal decay of colchicine binding to tubulin (Frigon and Lee, 1972). However, we have found recently that SUC inhibits both the rate and extent of taxol-inducedplant microtubule polymerization (Bokros et al, 1993). Because SUC increases solution viscosity substantially, polymerization is inhibited, at least in part, by slowed diffusion rates of tubulin dimer and polymer, and the interaction of oryzalin and tubulin may be altered by SUC as well.…”