Characterization of the reversible taxol-induced polymerization of plant tubulin into microtubules.
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.
Bokros
C L
CL
Department of Botany, University of Texas, Austin 78713.
Hugdahl
J D
JD
Hanesworth
V R
VR
Murthy
J V
JV
Morejohn
L C
LC
D002118:Calcium / Q000494:pharmacology; D002478:Cells, Cultured; D066298:In Vitro Techniques; D008854:Microscopy, Electron; D008870:Microtubules / Q000737:chemistry*; D017239:Paclitaxel / Q000378:metabolism / Q000494:pharmacology*; D010944:Plants / Q000648:ultrastructure*; D011108:Polymers; D011485:Protein Binding; D013696:Temperature; D014404:Tubulin / Q000737:chemistry* / Q000378:metabolism
D011108:Polymers; D014404:Tubulin; D017239:Paclitaxel; D002118:Calcium