The biochemistry of compounds with anti-microtubule activity in plant cells (original) (raw)
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Taxol-induced rose microtubule polymerization in vitro and its inhibition by colchicine
The Journal of Cell Biology, 1984
Tubulin was isolated from cultured cells of rose (Rosa, sp.cv. Paul's scarlet) by DEAE-Sephadex A50 chromatography, and the taxol-induced polymerization of microtubules in vitro was characterized at 24 degrees C by turbidity development, sedimentation analysis, and electron microscopy. Numerous, short microtubules were formed in the presence of taxol, and maximum levels of turbidity and polymer yield were obtained at approximately 2:1 molar ratios of taxol to tubulin. The critical concentration of rose tubulin for polymerization in saturating taxol was estimated to be 0.21 mg/ml. Colchicine inhibited the taxol-induced polymerization of tubulin as shown by sedimentation assays; however, much higher concentrations of colchicine were required for the inhibition of taxol-induced rose tubulin assembly than for inhibition of taxol-induced mammalian brain tubulin assembly. On the basis of the relative sensitivity of rose tubulin assembly to taxol and its insensitivity to colchicine, we...
Characterization of the reversible taxol-induced polymerization of plant tubulin into microtubules
Biochemistry, 1993
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, 141-1471, 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 297,4264281, most of the tubulin polymerizes at 25 OC, with critical dimer concentrations (C,) 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 OC temperature jump, 42% of polymer is polymorphic, presumably due to aberrant nucleation events. Taxol-induced assembly at 2 OC 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 195% 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/Ca2+-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.
Effects of taxol on microtubule arrays in cultured higher plant cells
Cell Motility and the Cytoskeleton, 1986
Treatment with 10 pm taxol disrupted mitotic and cytoplasmic arrays of microtubules (MT) in cultured cells of two higher plants, Viciu hajusturn (vetch) and Zinnia elegans. When treated for 1, 24, and 48 h, cells in both cultures showed similar effects. After 1 h, multipolar arrays of MT were noted in prophase, large aster-like arrays of MT appeared in metaphase, and extra MT shared poles with otherwise normal-appearing metaphase and anaphase configurations. After 24 and 48 h, some phragmoplasts were multipartite or misplaced. In interphase cells, micronuclei and multinucleate cells were evidence of irregular mitosis and cytokinesis. Cytoplasmic MT in elongated cells were oriented parallel to, instead of at right angles to the long axis of the cell. Some interphase cells lost asymmetry while maintaining organized arrays of MT. Taxol appears to disrupt mitotic and cytoplasmic arrays of MT, seemingly overriding the mechanism(s) regulating MT polymerization and orientation.
Protoplasma, 1998
The herbicide carbetamide [(R)-l-(ethylcarbamoyl) ethylphenylcarbamate], in the 0.4 to 0.8 mM range, efficiently induced multipolar mitoses in Allium cepa L. The frequency of multipolar anaphases rose earlier and reached higher values when both concentration and time of treatment increased, up to a maximum of 90% after 1 h of treatment. To identify the physiological target, the kinetics of induction of multipolar mitoses were followed during recovery from very short treatments (5, 10, and 15 min). Tubulin immunodetection showed that phenylcmbamate immediately disrupts the cohesion between the different bundles of microtubule minus ends which converge at the pole. The spindle was rendered multipolar about three times more efficiently in metaphase than in anaphase. The observations do not support any effect of the herbicide on the tubulin polymerization-depolymerization cycle, and suggest that the minus ends of the microtubules remained stabilized in carbetamide. Thus, the density of kinetochore microtubnles and their lengths were unmodified in the individual chromosomes which became detached from both spindle poles in response to the herbicide. Extra microtubule-organizing centres for the assembly of both preprophase band and phragmoplast (the tubulin arrays which characterize the microtubular cycle responsible for cytokinesis in plant cells) were also rapidly induced.
Cell Biology International, 2008
To clarify the mechanism of isopropyl-N-phenyl carbamate (IPC) action on higher plant cells the sensitivity of microtubules (cortical network and mitotic arrays) and microtubule organizing centers to IPC treatment (30 mM) in IPC-resistant and sensitive Nicotiana sylvestris lines was studied. It was clearly demonstrated that IPC does not depolymerize plant MTs but causes the MTOC damage in cells, which results in MTOC fragmentation, splitting of the spindle poles and in abnormal division spindle formation. It was also found that IPC-resistance of mutant N. sylvestris line correlates not with tubulin resistance to IPC action but possibly with resistance of one of the proteins involved in MTOC composition. Ó
Pesticide Biochemistry and Physiology, 2006
Mitotic disrupter herbicides are best known for their macroscopic eVect on root tip swelling and their microscopic eVect on the progression of chromosomes through mitosis. However, irregularities with the phragmoplast microtubules and cell plate formation occur at lower herbicide concentrations than these more familiar eVects. Instead of the relatively straight cell plates found in control tissue, cell plates after mitotic disrupter treatment are often branched and grow irregularly throughout the cytoplasm. Sometimes these abnormal plates adhere to one wall and in most cases do not eVectively divide the potential daughter cytoplasms. To determine the chemical composition of these abnormal cell plates, thin sections of treated onion root tips were probed with a battery of antibodies and cytochemical probes. Abnormal cell plates are greatly enriched in callose compared to control cell plates and accumulate very low levels of cellulose. The development of these wildly undulated and excessively branched or heavily thickened cell plates indicates the importance of microtubules in forming a proper cell plate and perhaps the necessity of stable microtubule arrays for the addition of cellulose to these structures. Because the abnormal plates occur at herbicide concentrations below that required for induction of mitotic arrest or root tip swelling, this eVect may be the primary phytotoxic eVect of these herbicides.
Mutation Research/Genetic Toxicology and Environmental Mutagenesis, 2014
Microtubules (MT) are formed by the assembly of ␣and -tubulins and MT-associated proteins. We characterized the effects of pharmaceutical formulations containing the microtubule disruptors thiabendazole (TBZ) and griseofulvin (GF) on the mitotic machinery of plant (A. cepa) meristematic cells. GF concentrations between 10 and 250 g/ml were tested. GF induced mitotic index inhibition and genotoxic effects, including chromosome fragments, bridges, lagged chromosomes, C-metaphases, tripolar cell division, disorganized anaphases and nuclear abnormalities in interphase cells. Efects on the mitotic machinery were studied by direct immunofluorescence with -tubulin labeling and by DNA counterstaining with 4 ,6-diamidino-2-phenylindole (DAPI). Exposure of meristematic root cells to TBZ or GF, 100 g/ml, caused microtubular damage which led to abnormal MT arrays. Our results suggest that GF induces abnormalities in spindle symmetry/polarity, while TBZ causes chromosome missegregation, polyploidy, and lack of cytokinesis.
Microtubule Components of the Plant Cell Cytoskeleton
Plant physiology, 1994
Corresponding author; fax 1-612-625-5754. Abbreviations: MAP, microtubule-associated protein; MTOC, microtubule organizing center; PPB, preprophase band. Copyright Clearance Center: 0032-0889/94/l04/0001/06. LITERATURE CITED Asada T, Sonobe S, Shibaoka H j1991) Microtubule translocation in the cytokinetic apparatus of cultured tobacco cells. Nature 350: 238-241 Brown RC, Lemmon BE (1992) Polar organizers in monoplastidic mitosis of hepatics (Bryophyta). Cell Motil Cytoskel22 72-77 Caplow M (1992) Microtubule dynamics. Curr Opin Cell Biol 4 Carpenter JL, Kopczak SD, Snustad DP, Silflow CD (1993) Semiconstitutive expression of an Arabidopsis thaliana a-tubulin gene. Plant Mo1 Biol21: 937-942 Carpenter JL, Ploense SE, Snustad DP, Silflow CD (1992) Preferential expression of an a-tubulin gene of Arabidopsis in pollen. Plant Cell4 557-571 Chu B, Snustad DP, Carter JB (1993) Alteration of 0-tubulin gene expression during low-temperature exposure in leaves of Arabidopsis thaliana. Plant Physiol 103 371-377 Cleary AL, Gunning BES, Wasteneys GO, Hepler PK (1992) Microtubule and F-actin dynamics at the division site in living Tradescanfia stamen hair cells. J Cell Sci 103 977-988 Colasanti J, Cho S-O, Wick S, Sundaresan V (1993) Localization of the functionalp34'd'2 homologue of maize in dividing cells of the root tip and stomatal complex: association with the predicted division site in premitotic cells. Plant Cell5 1101-1 11 1 Cyr RJ, Palevitz BA (1989) Microtubule-binding proteins from carrot. Planta 177: 245-260 Fosket DE, Morejohn LC (1992) Structural and functional organization of tubulin. Annu Rev Plant Physiol Plant Mo1 Biol 43: Fulton C, Simpson PA (1976) Selective synthesis and utilization of flagellar tubulin. The multi-tubulin hypothesis. In R Goldman, T Pollard, J Rosenbaum, eds, Cell Motility. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp 987-1005 Giddinge TH Jr, Staehelin LA (1991) Microtubule-mediated control of microfibril deposition: a re-examination of the hypothesis. In
Planta, 1987
The inhibition of the polymerization of tubulin from cultured cells of rose (Rosa. sp. cv. Paul's scarlet) by colchicine and the binding of colchicine to tubulin were examined in vitro and compared with data obtained in parallel experiments with bovine brain tubulin. Turbidimetric measurements of taxol-induced polymerization of rose microtubules were found to be sensitive and semiquantitative at low tubulin concentrations, and to conform to some of the characteristics of a nucleation and condensation-polymerization mechanism for assembly of filamentous helical polymers. Colchicine inhibited the rapid phase of polymerization at 24 ~ C with an apparent inhibition constant (Ki) of 1.4.10-4 M for rose tubulin and an apparent Ki= 8.8" 10-v M for brain tubulin. The binding of [3H]colchicine to rose tubulin to form tubulin-colchicine complex was mildly temperature-dependent and slow, taking 2-3 h to reach equilibrium at 24 ~ C, and was not affected by vinblastine sulfate. The binding of [3H]colchicine to rose tubulin was saturable and Scatchard analysis indicated a single class of low-affinity binding sites having an apparent affinity constant (K) of 9.7.102 M-1 and an estimated molar binding stoichiometry (r) of 0.47 at 24 ~ C. The values for brain tubulin were K=2.46" 106 M-1 and r=0.45 at 37 ~ C. The binding of [3H]colchicine to rose tubulin was inhibited by excess unlabeled colchicine, but not by podophyllotoxin or tropolone. The data demonstrate divergence of the colchicine-binding sites on plant and animal tubulins and indicate that the relative resistance of plant microtubule polymerization to