competitive inhibition of high-affinity oryzalin binding ... · plant tubulin by the phosphoric...

Plant Physiol. (1994) 105: 309-320

Competitive Inhibition of High-Affinity Oryzalin Binding to Plant Tubulin by the Phosphoric Amide Herbicide

Amiprophos-Methyl'

Jaitra V. Murthy, Hyong-Ha Kim, Virginia R. Hanesworth, Jeffrey D. Hugdahl, and Louis C. Morejohn*

Department of Botany, University of Texas at Austin, Austin, Texas 78713

Amiprophos-methyl (APM), a phosphoric amide herbicide, was previously reported to inhibit the in vitro polymerization of isolated plant tubulin (L.C. Morejohn, D.E. Fosket [1984] Science 224: 874- 876), yet l itt le other biochemical information exists concerning this compound. To characterize further the mechanism of action of APM, its interactions with tubulin and microtubules purified from cultured cells of tobacco (Nicofiana tabacum cv Bright Yellow-2) were investigated. Low micromolar concentrations of APM depolymerized preformed, taxol-stabilized tobacco microtubules. Re- markably, at the lowest APM concentration examined, many short microtubules were redistributed into fewer but 2.7-fold longer microtubules without a substantial decrease in total polymer mass, a result consistent with an end-to-end annealing of microtubules with enhanced kinetic properties. Quasi-equilibrium binding measurements showed that tobacco tubulin binds ['4C]oryzalin with high affinity to produce a tubulin-oryzalin complex having a dissociation constant (&) = 117 nM (pH 6.9; 23'C). Also, an estimated maximum molar binding stoichiometry of 0.32 indicates pharmacological heterogeneity of tobacco dimers and may be related to structural heterogeneity of tobacco tubulin subunits. APM inhibits competitively the binding of ["Cloryzalin to tubulin with an inhibition constant (KJ = 5 PM, indicating the formation of a moderate affinity tubulin-APM complex that may interact with the ends of microtubules. APM concentrations inhibiting tobacco cell growth were within the threshold range of APM concentrations that depolymerized cellular microtubules, indicating that growth inhibition i s caused by microtubule depolymerization. APM had no apparent effect on microtubules in mouse 313 fibroblasts. Because cellular microtubules were depolymerized at APM and oryzalin concentrations below their respective Ki and Kd values, both herbicides are proposed to depolymerize microtubules by a substoichiometric endwise mechanism.

Phosphoric amide herbicides such as APM (Tokunol M) and butamiphos (Cremart) were developed as preemergence herbicides and are effective on annual grasses and broadleaf weeds (Aya et al., 1975). The uses of phosphoric amides are similar to those of the dinitroaniline herbicides, including

' Supported by grants to L.C.M. from the National Science Foun- dation (MCB-8996274) and the University Research Institute (Uni- versity of Texas at Austin). J.V.M. was supported, in part, by the Junior Fellows Program (University of Texas at Austin) and a Re- search Experience for Undergraduates Fellowship from the National Science Foundation. J.D.H. was supported by a Plant Biology Post- doctoral Fellowship from the National Science Foundation.

* Corresponding author; fax 1-512-471-3878. 309

oryzalin (Surflan) and trifluralin (Treflan) (Ashton and Crafts, 1981). The structures of oryzalin and APM are given in Figure 1. Both APM and oryzalin produce gross morphological effects in plants, the most characteristic of which is swollen root tips (Sumida and Ueda, 1976; Upadhyaya and Nooden, 1977; Ashton and Crafts, 1981). These herbicides halt plant growth by inhibiting cell division and elongation; the orientation of cellulose microfibril deposition in the cell wall becomes random, producing cells having more isodiametric shapes typical of "tumor" roots. These cellular activities are disrupted because of the depolymerization of microtubules, which usually direct the polar distribution of subcellular components (Morejohn, 199 1). These and other observations have provided ample evidence that microtubules are essential subcellular determinants of morphogenesis in plants (Lloyd, 1991).

Microtubules are filamentous polymers consisting mainly of tubulin, a protein heterodimer with similar a and 0 subunits, each having a molecular mass of about 50 kD. Dimeric tubulin polymerizes in a head-to-tail manner to form 13 linear protofilaments within the microtubule wall, and a dynamic equilibrium is maintained between microtubules and the soluble pool of tubulin dimers. Kinetic properties of microtubules, such as dynamic instability and treadmilling, have been observed mainly in animal microtubules (Gelfand and Bershadsky, 199 1). Plant cells have severa1 functionally distinct microtubule arrays that form at particular stages of the cell cycle (Lloyd, 1991), and plant microtubules have unique in vitro polymerization characteristics (Bokros et al., 1993; Hugdahl et al., 1993).

Studies of the mechanisms of action of antimicrotubule herbicides offer crucial information concerning the structure and function of plant tubulin and the regulation of plant microtubule dynamics and thus are important to our under- standing of plant cell growth and differentiation. In contrast to the vast data available regarding drugs that bind to mammalian tubulin (Wilson and Jordan, 1993), very little information exists concerning the pharmacology of plant tubulin (Morejohn, 1991). Our studies have demonstrated that plant tubulin binds both colchicine and oryzalin, although their

Abbreviations: APM, amiprophos-methyl; DAPI, 4 ',6-&amidino- 2-phenylindole; IB, isolation buffer; Kdep, microtubule depolymerization constant; NP-40, Nonidet P-40; r, molar binding ratio (mo1 oryzalin bound per mo1 tubulin); t, threshold concentration; TA, tubulin-APM; TC, tubulin-colchicine; TO, tubulin-oryzalin.

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310 Murthy et al. Plant Physiol. Vol. 105, 1994

\ 02N

Ory zal i n

APM

Figure 1. Chemical structures of oryzalin and APM.

binding characteristics are very different (Morejohn et al., 1987a; Hugdahl and Morejohn, 1993). Colchicine binds slowly and with very low affinity (Kd = 1.03 mM) to plant tubulin to form a TC complex that does not readily dissociate into unliganded. tubulin and free colchicine, thus exhibiting "tight" binding characteristics (Morejohn et al., 1984, 1987b). In contrast, oryzalin binds rapidly and with high affinity (Kd = 97 nM) to plant tubulin to form a TO complex that readily dissociates into unliganded tubulin and free oryzalin (Hugdahl and Morejohn, 1993). TC and TO are proposed to inhibit tubulin assembly by co-polymerizing with unliganded tubulin and inhibiting further tubulin polymerization (Strachen and Hess, 1983; Morejohn and Fosket, 1984a; Morejohn et al., 1987a, 198713; Hugdahl and Morejohn, 1993). However, the number of different drug-binding sites on plant tubulin is not known, and it has not been determined whether different classes of antimicrotubule herbicides bind to the same or different sites on plant tubulin (Morejohn, 1991).

Although a number of studies have used phosphoric amides as probes of microtubule function in cells of algae (Kiermayer and Fedtke, 1977; Quader and Filner, 1980; La Claire, 1987; Mizuta et al., 1989; Schibler and Huang, 1991; James et al., 1993), protoctists (Pape et al., 1991), and advanced land plants (Mita and Shibaoka, 1983; Bajer and Mole'-Bajer, 1986; Falconer et al., 1988), only one report exists concerning the in vitro interaction of phosphoric amides with plant tubulin (Morejohn and Fosket, 1984b). APM at low micromolar concentrations was shown to inhibit the taxol-induced polymerization of tubulin isolated from cultured cells of rose (Rosa sp.), with no effect on mammalian tubulin assembly. Turbidimetric measurements demonstrated that APM inhibits both the rate and extent of microtubule formation, and EM revealed fewer and shorter microtubules with increased APM concentrations. Polymer sedimentation assays confirmed that APM causes a concentration-dependent decrease in rose tubulin polymer mass (Morejohn and Fosket, 1984b). While these results showed an interaction of APM with plant tubulin, the experiments were conducted using a buffer containing 1 M SUC, which itself was found subsequently to inhibit both the rate and extent of taxol- induced plant tubulin polymerization (Bokros et al., 1993).

Furthermore, Suc stabilizes preformed microtubules, making an examination of the effects of APM on preforrned microtubules unfeasible. Finally, because Suc increases solution viscosity substantially and tubulin polymerization is altered at least in part by slowed dimer and polymer diffusion rates, it was important to reexamine the interaction of APM with plant tubulin in an assembly buffer not containing SUC.

The piimary purpose of this study was to characterize in greater detail the interaction of APM'with purified plant tubulin and microtubules in solutions not containing Suc and, when appropriate, to compare the results to those obtained in parallel experiments with oryzalin. We used tobacco BY-2 cells (Nicotiana tabacum cv Bright Yellow-2), widely adopted as a model plant cell line (Nagata et al., 1992), as a source of tubulin amd for experiments on cellular microtubules.

MATERIALS AND METHODS

Materials

Analytical grade APM [N-isopropyl O-methyl O-(2-nitro- p-tolyl) phosphoramidothioate; Tokunol M] was kindly provided by Dr. Carl Fedtke (Bayer AG, Leverkusen, Germany). Aphidicolin and monoclonal antibody DMlB were from Amersham. Fluorescein isothiocyanate-conjugated rabbit anti-mouse antibody was from Pierce. Sorbitol, BSA, NP-40, DAPI, and N-propyl gallate were from Sigma. Mowiol was obtained from Calbiochem. A11 other materials were obtained from soiirces as described previously (Bokros et al., 1993; Hugdahl and Morejohn, 1993).

Suspension Culture of Tobacco Cells

Suspeinsion cultures of tobacco (Nicotiana tilbacum, cv Bright Yellow-2) were grown as previously described (Nagata et al., 1992; Bokros et al., 1993), except that 2 :mL of cells were trainsferred to 100 mL of medium at 7-d intervals.

Protein lilectrophoresis

One-dimensional SDS-PAGE of pure tobacco tubulin was done according to the method of Studier (1973), and gels were stained with silver.

Ligand-Binding Methods

Quasi-Equilibrium Dialysis Measurements

The binding of [14C]oryzalin to tobacco tubulin was performed with equilibrium dialysis, which provides quasi-equilibrium measurements as described previously (Hugdahl and Morejohn, 1993). A11 ligand-binding experiments were performed in a tubulin IB consisting of 50 mM Pipes-KOH (pH 6.9), 1 IIIM EGTA, 0.5 mM MgS04, and 1 mM DTT, supplemented ,with 0.1 mM GTP, 2% (v/v) DMSO, 50 &mL Na- p-tosyl-r-arginine methyl ester, and 5 wg/mL each of pepsta- tin A, leupeptin hemisulfate, and aprotinin. Details of the technica'l limitations of [14C]oryzalin binding, iricluding its low aqueous solubility and low specific radioactivity, have been published (Hugdahl and Morejohn, 1993).

Most known and suspected antimicrotubule herbicides have relatively low aqueous solubilities and may bind to the



Amiprophos-Methyl Binding to Tobacco Tubulin 311

surfaces of containers and be lost from solution (Fedtke, 1982;Hugdahl and Morejohn, 1993). Pilot experiments were car-ried out to determine the solubility characteristics of APM inIB solutions containing 2% (v/v) DMSO. Because a radiola-beled form of APM was not available, the solubility of APMwas monitored spectrophotometrically. Maximum absorptionof APM in IB occurs at 235 nm (e = 1.2 X 10"), and a linearrelationship between A and APM concentration was foundat <200 MM. The lower limit of detection of APM with A was50 MM. Various concentrations of APM were incubated for 3h at 23°C in the dialysis chamber to be used for ligandbinding, and APM concentrations were measured at A235- Itwas determined that at APM concentrations >110 MM consid-erable APM bound to the dialysis chamber and was lost fromsolution. Thus, all ligand-binding and depolymerization ex-periments were conducted with APM concentrations wellbelow 110 MM.

Gel-Filtration Chromatography

To test the reversibility of APM binding to tobacco tubulin,two 0.5-mL tubulin samples were preincubated at 23°C for1 h in the presence or absence of APM and loaded ontoseparate, identically prepared gel-filtration columns of Seph-adex G-10 (bed volume = 1.5 mL) equilibrated with IBcontaining the supplements indicated above. Samples werechromatographed at a flow rate of 0.8 mL/min at 23°C. Afterthe void volume was discarded, fractions of the eluted proteinwere collected and assayed for protein, and the first fourfractions containing tubulin were retained and recombinedto give 3 MM tubulin in each sample.

Indirect Immunofluorescence Microscopy

Microtubules in tobacco cells were stained according to themethod of Simmonds et al. (1985), with several modifica-tions. Cells were fixed for 1 h at room temperature in 4%(w/v) paraformaldehyde in IB containing 0.4 M sorbitol, 5mM EGTA, and the protease inhibitors listed above. Cellswere washed three times for 5 min, and cell walls werepermeabilized by a 1-h digestion at room temperature in 2%(w/v) cellulysin and 0.2% (w/v) pectolyase in the samebuffer. Cells were washed three times in a buffer consistingof 10 mM Tris-HCl, pH 7.4, 5 mM EGTA, and 0.15 M NaCl(TEN) supplemented with protease inhibitors as describedabove. Cells were settled onto poly-L-Lys-coated slides andextracted in TEN containing 1% (v/v) NP-40. Cells wereblocked for 15 min with 2% (w/v) BSA in 0.05% (v/v) NP-40 in TEN and incubated in a humidified chamber for 1 h atroom temperature with a 1:500 (v/v) dilution of mousemonoclonal antibody against /3-tubulin (DM1B) (Blose et al.,1984) (Amersham) in the same buffer. Cells were washedthree times for 5 min with TEN and incubated with a 1:100(v/v) dilution of fluorescein isothiocyanate-conjugated, rabbitanti-mouse antibody (Pierce). Cells were washed and DNAwas stained simultaneously with three rinses in TEN contain-ing 0.2 Mg/mL DAPI (Sigma). Cells were mounted undercoverslips with Mowiol (Calbiochem) medium containing 2%(w/v) N-propyl gallate (Sigma) to prevent fading (Osbornand Weber, 1982). Observations and photographs were madeon an Olympus BH-2 fluorescence microscope.

For staining of microtubules in mouse 3T3 fibroblasts, cellswere grown and processed as described previously (Hugdahland Morejohn, 1993). Fibroblast cultures were obtained fromDr. Ben A. Murray (Department of Developmental and CellBiology, University of California, Irvine).

Miscellaneous Techniques

All other materials and methods used for protein isolationand purification, protein determination, EM, microtubulelength measurements, and tubulin polymer sedimentationanalysis were the same as described previously (Bokros et al.,1993; Hugdahl et al., 1993; Hugdahl and Morejohn, 1993).

RESULTS

Depolymerization of Taxol-Stabilized TobaccoMicrotubules by APM

Tubulin was isolated from stationary phase cells of tobacco(N. tabacum cv Bright Yellow-2) by DEAE-Sephadex A50chromatography (Morejohn and Fosket, 1982; Bokros et al.,1993). Microtubules were polymerized by gradual tempera-ture ramping from 0 to 25°C in the presence of taxol andwere purified by sedimentation through a Sue cushion (Bok-ros et al., 1993). The inset in Figure 2 shows silver-stainedSDS-PAGE analysis (Shadier, 1973) of microtubules andreveals electrophoreric purity of tobacco a- and /3-tubulinpolypeptides.

The effect of APM on the mass of taxol-stabilized tobaccomicrotubules was quantified by polymer sedimentation as-

120

100

80•o c0) O

o0.

20

1 2

Q|——,———^———,———^——0.1 1 10 100

Drug Concentration (/j,M)

Figure 2. APM- and oryzalin-induced depolymerization of puretaxol-stabilized tobacco microtubules. The amount of tubulin poly-mer obtained at each concentration of APM (•) and oryzalin (O) isexpressed as a percentage of the control microtubule sample havingno herbicide. The dashed lines and arrows designate half-maximaldepolymerization of taxol-stabilized tobacco microtubules by 15^M APM (filled arrowhead) and 5.6 HM oryzalin (open arrowhead).The inset shows a silver-stained, 9% polyacrylamide SDS gel (Stu-dier, 1973) containing 10 /ig of purified tobacco tubulin (lane 1)and molecular mass markers (lane 2) of 45, 66, 97, and 116 kD.




says (Hugdahl and Morejohn, 1993), and the results were compared to those obtained in parallel experiments with oryzalin. Taxol-stabilized microtubules were resuspended to 5 p~ tubulin in buffer containing different concentrations of APM (0.5-20 p ~ ) or oryzalin (0.5-25 p ~ ) at 23OC, and after 30 min of depolymerization samples were sedimented and protein assays were performed on tubulin polymer pellets. A plot of the polymer yield versus the herbicide concentration is given in Figure 2. The data show that both APM and oryzalin produced concentration-dependent decreases in the yield of tobacco tubulin polymer, with oryzalin causing more effective depolymerization than APM. The estimated herbicide concentrations producing half-maximal depolymerization (Kdep) were 5.6 and 15 p~ for oryzalin and APM, respectively .

The effects of APM on the ultrastructure of tobacco tubulin polymers were examined. Taxol-stabilized tobacco microtubules were resuspended to 5 p~ tubulin in buffer containing different concentrations of APM (4-15 p ~ ) , and after 30 min at 23OC samples were negatively stained and examined by EM. Figure 3A shows that in the control sample numerous short microtubules with a mean length of 1.4 f 0.7 pm and some small polymorphic polymers were assembled, as reported previously for taxol-induced tobacco tubulin polymerization (Bokros et al., 1993). At the lowest concentration of APM tested (4 p ~ ) , small polymorphic polymers were absent, and fewer but r2.7-fold longer microtubules (3.8 f 1.6 pm) were observed. This result indicated that a redistribution of microtubule number and length occurred at an APM concentration at which 290% of polymer was estimated to be assembled (Fig. 2). Figure 3B shows long microtubules with a mean length of 4.8 f 3.2 pm after treatment with 6 PM APM, and Figure 3C also shows long microtubules (4.6 +. 1.9 pm) present after treatment with 15 p~ APM. The summary of microtubule length measurements at different APM concentrations in Table I shows that a11 APM-treated samples had microtubules significantly longer than the control.

Another notable structural feature of APM-treated microtubules is the intermittent lattice discontinuities that appear to be sites of end-to-end microtubule annealing (Fig. 3B, arrowheads). Because a low APM concentration produced fewer but significantly longer microtubules without a large reduction in total polymer mass (Fig. 2; Table I), long microtubules probably arose from an end-to-end annealing of short microtubules, a phenomenon described previously with animal microtubules in vitro (Rothwell et al., 1986). Apparently, annealing of tobacco microtubules occurred as a result of an APM-induced enhancement of dynamics at microtubule ends.

High-Affinity Oryzalin Binding to Tobacco Tubulin

The primary objective of this study was the documentation of interactions between APM and plant tubulin and microtubules. Although the unavailability of radiolabeled APM precluded direct binding measurements, information concerning APM may be deduced by examining the effects of APM on the binding of oryzalin to tobacco tubulin. Thus, we began with a characterization of ['4C]oryzalin binding to tobacco tubulin.

Soluble tobacco tubulin was prepared from microtubules as described previously (Bokros et al., 1993; Hugdahl and Morejohn, 1993), and the dependence on oryzalin c'oncentra- tion for its binding to tubulin was examined. Pule tobacco tubulin (9: p ~ ) was incubated with different concentrations of ['4C]oryzalin (0.1-3 FM), and binding was assayed at 23OC with equilibrium dialysis, which provides quasi-equilibrium measurements (Hugdahl and Morejohn, 1993). Figure 4A displays a plot of the amount of bound oryzalin versus free oryzalin at each ligand concentration and shows saturable formation of TO complex. An initial estimate of the K d was made with Langmuir binding isotherm analysis (Klotz, 1982; Hulme artd Birdsall, 1992). The inset in Figure 4A shows a semilogarithmic plot of the concentration of free oryzalin versus thc r of oryzalin and tubulin and provides a sigmoidal curve, with estimated half-maximal binding to tubulin (inflection point) at 100 nM free oryzalin.

Estimation of the Kd according to the method of Scatchard (1949) was made by plotting r/[free oryzalin] (PM-') versus r. The plot in Figure 4B provides a line corresponding to a single affinity-class of oryzalin-binding sites (correlation coefficient = 0.96) with Kd = 117 nM and a maximum estimated molar binding stoichiometry ( r ) of 0.32. The results show that tobacco tubulin binds oryzalin with high affinity Imt that it has a relatively low oryzalin-binding capacity, with only one- third of the available dimers binding oryzalin. Decay of the oryzalin-binding site was tested by preincubating pure tubulin (1 p ~ ) at 23OC for different times prior to performing equilibrium dialysis ['4C]oryzalin binding. A plot of r versus time showed no decay of oryzalin binding for preincubation periods UIJ to 6 h (data not shown), as observed previously with maize tubulin (Hugdahl and Morejohn, 1993).

Competition of APM with Oryzalin for Binding to Tobacco Kubulin

Because APM has a relatively low solubility ir( aqueous solutions (Fedtke, 1982) and may bind to the dialysi:; chamber as does oryzalin (Hugdahl and Morejohn, 1993), pilot studies were conducted on its solubility in the buffer used for ligand- binding experiments. It was determined that an APM concentration 5110 p~ remains constant for periods up to 3 h, as described in "Materials and Methods."

The concentration-dependent effects of APM on the binding of ['4C:]oryzalin to pure tobacco tubulin were examined with equilibrium dialysis. Tobacco tubulin (2 PM) was dialyzed for 3 h at 23OC with different concentrations of ['4C]oryzalin (0.2-2 p ~ ) in the absence or presence of different concentrations of APM (20-60 p ~ ) , and quasi-eyuilibrium binding measurements were analyzed on a Lineweaver-Burk plot. Figure 5 displays the plot revealing an APM concentration-dependent inhibition of oryzalin binding to tobacco tubulin. The convergence of the four derived lines on the y intercept nndicates a pattern of competitive inhibition of oryzalin binding by APM. A replot of the slopi?s of the Lineweaver-Burk plot provides an apparent K, of 5 p~ (Fig. 5, inset). The analysis demonstrated that APM ancl oryzalin bind to the same site on the tobacco tubulin dimer, suggesting that plant tubulin binds APM to form a TA complex.




t^^^^^~

Figure 3. Electron micrographs of negatively stained taxol-stabilized microtubules after treatment with ARM. Samples arethose treated with 2% (v/v) DMSO (control) (A), 6 MM ARM (B), and 15 ^M ARM (C). Arrowheads indicate discontinuitiesin microtubule lattice. Bar = 0.5 nm.




0.0

Table 1. Effects of APM on preformed, taxo/-stabilized tobacco microtubule length

samdes were used to measure microtubule lennth. Electron microscope negatives of negatively stained polymer

APM Concentration Microtubule Length

PM

O 4.0 6.0

10.0 12.5 15.0

pm f SD (n)"

1.4 f 0.7 (50) 3.8 f 1.6 (33) 4.8 f 3.2 (73) 3.5 f 2.0 (31) 3.8 f 2.0 (61) 4.6 & 1.9 (72)

a The number of microtubules measured is aiven in Darentheses.

Readily Reversible APM Binding to Tobacco Tubulin

The binding results above provided circumstantial evidence of a TA complex, but it was not clear whether the putative TA was a tight complex like the TC complex (Morejohn et al., 1987b), or whether it dissociated readily into unliganded tubulin and free APM in the manner previously described for the TO complex (Hugdahl and Morejohn, 1993). A functional assay of tubulin polymerization was used to examine the reversibility of TA complex formation. Tobacco tubulin (5 p ~ ) was divided into two samples (0.5 mL), 2% (v/v) DMSO was added to one sample (control), and 5 p~ APM in 2% (v/v) DMSO was added to the other sample (experimental). Both samples were incubated at 23OC for 1 h and

0.5

zi 0.4 3.

7 0.3

h

v - 3 O 9 h 6 0.2 Y

0.1

subjected to Sephadex G10 gel-filtration chromatography, which was accomplished within 2 min as described in "Ma- terials ancl Methods." Each chromatographed sample was subdivided into two 100-pL aliquots of 3 PM tubulin. One aliquot each of the control sample (control A) and of the test sample (experimental A) was supplemented with 2% (v/v) DMSO alone, whereas the other aliquot of the control sample (control B) and of the test sample (experimental B) was supplemented with 3 p~ APM in 2% (v/v) DMSO. Taxo1 (3 PM) was added to a11 samples and incubated at rooni temperature for 1 h. Tubulin polymer was sedimented through a 20% (w/v) Suc cushion by centrifugation at 30,OOOg for 30 min at 25°C (Bokros et al., 1993). Protein assays were performed on supernatant and polymer pellet fractions, and the results are summarized in Table 11.

Taxol-induced polymerization of tubulin was inhibited by APM only when APM was added after chromatography (control B and experimental B). Preincubation of tubulin with APM had no effect (experimental A and control A), jndicating that TA complex dissociated during the gel filtration, which yielded free tubulin. Thus, APM binding to tobacco tubulin is readily reversible.

Depolymerization of Microtubules in Tobacco Cells by APM and Oryzalin

To understand better the mechanism of the antimicrotubule action of APM, it was important to obtain information also concerning the APM sensitivity of microtubules in tobacco cells. The effects of APM on microtubules in tobacco

2.0 h

r

5

E 1.0

v - ar E

O Y

\ L

B \ =117nM

'I \

0.0 0.2 0.4 r

Figure 4. Oryzalin concentration-dependent binding to pure tobacco tubwlin. A, Saturation binding of oryzalin to purc? tobacco tubulin. The mean of triplicate binding assays at each concentration of oryzalin (0.1-3 *M) was used to determine the concentrations of bound oryzalin ([Orybound]) and free oryzalin ([Oryf,,,,]). The line was drawn using the hyperbolic: curve-fitting equation y = (a . xP)/(b + xp). The inset shows the Langmuir binding isotherm of TO formation at 23°C: (Klotz, 1982; Hulme and Birdsall, 1992). Values of r are plotted versus the concentration of free oryzalin ([Oryf,,,]), antl the arrow denotes the estimated concentration of oryzalin (0.1 PM) thait coincides with the inflection point of t h e sigmoidal curve. The line was drawn using the sigmoidal curve fitting equation y = [(a . xp)/(b + xp)] + c. B, Apparent K,j for oryzalin binding to pure tobacco tubulin. Binding data were analyzed by plotting r/[Ot,,,] (~LM-') versus r according to Scatchard (1 949). The slope of the linear regression (correlation coefficieni = 0.96) represents the negative reciproca1 of the apparent Kd of 11 7 nM, and extrapolation of the line to the x intercept provides an estimated maximum molar bindinl; stoichiometry ( r ) of 0.32.




150

100

50

Anti-Tubulin DAPI

0 2 4 6 81/[Ory](/zM"i)

Figure 5. Competitive inhibition by ARM of oryzalin binding topure tobacco tubulin. A, Lineweaver-Burk plot of quasi-equilibriumbinding data from [14C]oryzalin binding to pure tobacco tubulin inthe absence (•) and presence of 20 ^M (O), 40 HM (•), and 60 ^M(D) ARM. All values are the means from triplicate assays. The insetshows a replot of the slopes of the Lineweaver-Burk plot versusthe ARM concentration and affords K, = 5.0 MM (correlation coef-ficient = 0.99).

cells were examined by indirect immunofluorescence micros-copy, and the results were compared to those obtained withoryzalin. Different concentrations of APM and oryzalin(0.01-1 JJM) were added to exponentially growing suspensioncultures of tobacco, and both control and herbicide-treatedsamples were taken at different times (1-14 h) and fixed formicroscopy. Microtubules were visualized with indirect im-munofluorescence microscopy (Simmonds et al., 1985), andchromatin was stained with DAPI in the same cells.

Figure 6A shows abundant microtubules in a typical controlinterphase cell, with bright staining of microtubules aroundthe nucleus (Fig. 6B). All microtubule arrays in cells treatedwith 10 and 100 nM APM were present, even after 14 h oftreatment, although preprophase bands appeared wider andmitotic spindles were shorter than in control cells. At theselow APM concentrations, phragmoplasts formed, normalchromosome distributions during all stages of mitosis wereseen, and no polyploid or micronuclei were observed, evenat later times.

In cultures treated with 1 fiM APM, microtubules in many

Table II. Effect of gel filtration chromatography on the dissociationof TA comp /ex

Tubulin PreincubatedSample with APM

Control AControl BExperimental AExperimental B

NoNoYesYes

Supplementedwith APM

NoYesNoYes

TubulinPolymerized

%

950

970

Figure 6. Depolymerization of microtubules in tobacco cells byAPM and oryzalin. Microtubules were visualized with anti-tubulinantibody and indirect immunofluorescence microscopy, and DMAwas stained with DAPI in the same cells of control (A and B), 1 JIMAPM-treated (C and D), and 100 nM oryzalin-treated (E and F)tobacco cultures. Bars = 15 ̂ m.

cells were depolymerized within 1 h, and very few cellsretained a normal-appearing microtubule cytoskeleton. After2 h of treatment, most cells had only short residual microtu-bules. At later times many cells accumulated in prometaphaseand displayed chromosomes arranged in a "ball metaphase,"indicating incomplete chromosome congression to the meta-phase plane. Figure 6C shows an interphase cell (top) and amitotic cell (bottom) after treatment with 1 HM APM. Theinterphase cell has some short residual microtubules sur-rounding the nucleus (Fig. 6D), and the mitotic cell haspunctate staining corresponding to short tufts of kinetochoremicrotubules attached to chromosomes arranged in a ballprometaphase (Fig. 6D). No normal metaphase, anaphase, ortelophase cells were seen, and mitotic inhibition resulted inthe formation of partial phragmoplasts, with d cells havingrestitution nuclei or a few aneuploid micronuclei.

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Figure 7. Inhibition of tobacco cell mitosis by ARM. DNA is stainedwith DAPI in control cells (A) and 5 MM APM-treated tobacco cells(B). Control cell nuclei and chromosomes are labeled according tostage in the cell cycle or mitosis as follows: GI, S/G2, metaphase(m), and telophase/cytokinesis (t/c). APM-treated cells are labeledas prometaphase (pm), scattered chromosomes (sc), and restitutionnuclei (rn). Bar = 15 ̂ m.

arrangements and nuclear morphology seen in DAPI-stainedpreparations of control cells (Fig. 7A) and cells treated with5 /iM APM (Fig. 7B). In APM-treated cells chromosomes hadno kinetochore microtubules, and scattered chromosomesindicated a complete inhibition of prometaphase chromo-some congression. No normal metaphase, anaphase, or telo-phase figures were observed, and at later treatment timeschromosomes decondensed to form irregularly shaped resti-tution nuclei or various numbers of aneuploid micronuclei.In summary, the above results show that plant mitosis isinhibited by APM concentrations that cause the loss of mi-crotubules. The threshold concentration (t) range of APMcausing microtubule depolymerization and mitotic inhibitionis 0.1 JUM < f < 1 /XM.

Tobacco cells treated with 10 nM oryzalin had shorterspindles than control cells, but other microtubule arraysappeared normal, and cells divided normally. In cells treatedwith 50 nM oryzalin, many interphase microtubules andmitotic spindles were absent or shorter than in control cells.Although preprophase bands appeared wider, phragmoplastsformed and cells divided apparently normally without gen-erating micronuclei. In cells treated with >100 nM oryzalin,most interphase microtubules were depolymerized within 1h. Figure 6E shows a few remaining microtubules in aninterphase cell in the vicinity of the nucleus (Fig. 6F) aftertreatment with 100 nM oryzalin. Chromosomes in 100 nMoryzalin-treated cells accumulated at prometaphase, onlyfragments of phragmoplasts developed, and GI cells usuallyformed various numbers of irregularly sized micronuclei.Thus, as seen with APM above, oryzalin concentrationscausing the loss of microtubules in cells also inhibit mitosis.The threshold oryzalin concentration range for microtubuledepolymerization and mitotic inhibition is 50 nM < t <100 nM.

Effects of APM on Growth of Tobacco CellsThe effects of APM on tobacco cell growth in suspension

culture were examined. Day seven stationary phase cells (2

mL) were transferred to medium (100 mL) in siliconized flaskscontaining different concentrations of APM (50 and 500 nM)and were grown as described previously (Bokros et al., 1993).Both the control and APM-containing flasks contained a finalconcentration of 1% (v/v) DMSO. At 1-d intervals triplicatesamples (0.5 mL) of cells were sedimented by brief centrifu-gation in tared microfuge tubes, and fresh weights weredetermined. The results presented in Figure 8 show thatfollowing a 2-d lag phase, control cells grew rapidly for 4 dand entered stationary phase by d 6. Tobacco cells grownwith 50 nM APM showed growth kinetics similar to those ofthe control culture. However, the fresh weights of thesecultures were slightly greater, in general, than the controlsduring the stationary phase, suggesting a promotion ofgrowth with 50 nM APM. Cells grown with 500 nM APMexhibited slow increases in fresh weight, attaining only 58%of the control fresh weight by d 9 (Fig. 8). The data demon-strate tobacco cell growth to be inhibited near the thresholdAPM concentration, causing the loss of microtubules andinhibiting mitosis, as described above. We are investigatinghow tobacco cell growth may have been enhanced by lowAPM concentrations.

Resistance of Mammalian Cell Microtubules to APM

Although there have been no comprehensive studies re-ported regarding the effects of phosphoric amides on micro-tubules in mammalian cells, we previously observed little orno effect of APM on microtubules in cultured PtK2 cells, afibroblast line derived from the kidney of the marsupial ratkangaroo (Morejohn and Fosket, 1984b). To assess the poten-tial antimicrotubule action of APM in cells of an advancedmammal, mouse 3T3 fibroblasts were treated with 100 JIMAPM (0.5% [v/v] DMSO) in culture medium at 37°C. Cul-tures were treated also with 0.5% (v/v) DMSO alone (nega-tive control) or 400 nM colchicine (positive control). At

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Figure 8. Effects of APM on tobacco cell growth. Cells were grownin the absence (•) or presence of 50 nM (O) or 500 nM (•) APM.All fresh weight values represent the means ± so from triplicatesamples. www.plantphysiol.orgon January 9, 2020 - Published by Downloaded from




different times (0.5, 1, 2, and 4 h) cells were fixed andmicrotubules were visualized by indirect immunofluores-cence microscopy (Hugdahl and Morejohn, 1993).

Fibroblasts treated with APM had abundant, normal-ap-pearing microtubules during interphase (Fig. 9A) and brightlystaining spindles during mitosis, even after 4 h of treatment.Both the arrangement and abundance of microtubules inAPM-rreated fibroblasts were indistinguishable from thoseobserved in DMSO-treated fibroblasts (Hugdahl and More-John, 1993). However, after a 1-h treatment with 400 nMcolchicine, virtually all interphase and mitotic microtubuleswere lost, and only centrioles within centrosomes and shortmicrotubules emerging from centrosomes were stained. Fig-ure 9B shows some punctate staining and no microtubulesafter a 4-h treatment with 400 nM colchicine. These resultsconfirm that APM has no obvious effect on microtubules inanimal cells, extending this observation to include cells froman advanced mammal.

DISCUSSION

Oryzalin-binding experiments in the present study confirmour previous observations of a high-affinity interaction oforyzalin and plant tubulin (Hugdahl and Morejohn, 1993).Oryzalin was found to bind rapidly, reversibly, and in a pH-dependent manner to tubulin from maize (Zea mays L. cvBlack Mexican Sweet) to form a TO complex having a JCd =95 HM and a maximum r = 0.5. Because unpolymerized maizetubulin has a much higher oryzalin-binding capacity thanpolymerized tubulin (r = 0.003), the primary effect of oryzalinis to bind dimer to form a TO complex. Oryzalin and/or TOcomplex may inhibit microtubule polymerization and depo-lymerize microtubules through its substoichiometric co-po-lymerization with unliganded tubulin (Hugdahl and More-john, 1993). In the current work, oryzalin bound to tobaccotubulin to form a TO complex having Kd = 117 nM and r =0.32. The competitive inhibition by APM of oryzalin bindingto tobacco tubulin (Kt = 5 MM) indicates that APM and oryzalinbind to the same site on plant tubulin. We presume that APM

Figure 9. Effects of APM on microtubules in mouse 3T3 fibroblasts.Microtubules in drug-treated cells were visualized with indirectimmunofluorescence microscopy using anti-tubulin antibody. A,Cells treated with 100 MM APM in 0.5% (v/v) DMSO. B, Cells treatedwith 0.4 fiM colchicine. Bar = 15 ̂ m.

binding also proceeds via a bimolecular interaction betweentubulin and APM to form a moderate-affinity TA complex,as we have proposed previously for tubulin and oryzalin.Thus far, we have found no evidence for tight binding in theTO or putative TA complexes, and all evidence indicates thatboth complexes are readily dissociated (Hugdahl and More-john, 1993). This reversibility of ligand binding is unlike theinteraction of colchicine and tubulin, since TC complex doesnot readily dissociate into free colchicine and unligandedtubulin. Tight binding of TC has been documented withtubulins from higher plants (Morejohn et al., 1984, 1987b)and animals (Wilson and Jordan, 1993). Therefore, the effectsof TO and putative TA on plant microtubule dynamics couldbe expected to be different from those caused by TC on plantand animal microtubules.

Polymer sedimentation analysis demonstrated that APMdepolymerizes preformed, taxol-stabilized tobacco microtu-bules in vitro. The estimated herbicide Kdep values were 5.6and 15 MM for oryzalin and APM, respectively. Relativelyhigh concentrations of APM were necessary for depolymeri-zation in vitro, because taxol has strong tubulin polymeriza-tion-promoting and microtubule-stabilizing effects (More-john and Fosket, 1982; Bokros et al., 1993). Furthermore,taxol lowers the critical concentration of the tobacco tubulindimer from approximately 8 to 1.3 HM (Bokros et al., 1993),leaving very little free tubulin available for APM binding.Taxol was used in these studies because it afforded muchmore polymer from the obtainable tubulin (Bokros et al.,1993). Taxol binds to the polymer and not the dimer (Parnessand Horwitz, 1981), and it slows the rate of dimer exchangein microtubules (Kumar, 1981; Caplow and Zeeberg, 1982;Wilson et al., 1985). Thus, much lower concentrations ofAPM and oryzalin are effective in depolymerizing microtu-bules in cells than in vitro.

At the lowest APM concentration (4 /UM) examined, anapparent reduction in the number of taxol-stabilized tobaccomicrotubules was observed concomitantly with a >2.7-foldincrease in the mean microtubule length. Because there wasnot a large reduction in microtubule mass at this APMconcentration (Fig. 2), the most likely explanation for polymerlength redistribution is that many short microtubules an-nealed end-to-end to form fewer but longer microtubules.Endwise annealing of animal microtubules in vitro was de-scribed previously by Rothwell et al. (1986). Further evidencefor tobacco microtubule annealing was seen in the form ofstructural discontinuities along polymer walls, where theends of different microtubules appeared to have joined. Be-cause a given preparation of taxol-stabilized plant microtu-bules contains polymers with different numbers of protofi-laments (Bokros et al., 1993), structural discontinuities maypersist primarily where two microtubules having differentnumbers of protofilaments are joined. Interestingly, we re-cently found that during taxol-induced polymerization ofmaize tubulin, oryzalin caused the formation of fewer butlonger maize microtubules (Hugdahl and Morejohn, 1993).This result is quite different from those obtained previouslyduring the polymerization of plant tubulin in the presence of1 M Sue; both APM and oryzalin produced a concentration-dependent decrease in plant microtubule length (Morejohnand Fosket, 1984b; Morejohn et al., 1987a). This may have www.plantphysiol.orgon January 9, 2020 - Published by Downloaded from




occurred as a result of the high viscosity and concomitantly lower rates of diffusion of herbicide, dimer, and polymer.

Our in vitro observations of APM-induced microtubule length redistribution indicate that the primary molecular mechanism of APM, and possibly oryzalin, is different from those of antimicrotubule drugs active against mammalian microtubules and mitosis (Wilson and Jordan, 1993). Studies on microtubule-associated protein-stabilized mammalian microtubules undergoing treadmilling have shown that the addition of low concentrations of colchicine or vinblastine results in drug incorporation into the net assembly ends of microtubules and a dramatic decrease in the addition of unliganded tubulin (Jordan and Wilson, 1990; Skoufias and Wilson, 1992). Kinetic stabilization of assembly ends occurs with little or no change in the total mass of polymer or in the lengths of microtubules (Jordan and Wilson, 1990; Skoufias and Wilson, 1992). Although we did not examine directly the kinetics at ends of APM-treated tobacco microtubules, we found that the addition of a low concentration of APM to taxol-stabilized tobacco microtubules had no great effect on polymer m a s but produced an apparent annealing of microtubules. Taxol-stabilized microtubules have slow treadmilling rates because taxo1 decreases the magnitude of the dissociation rate constants at both microtubule ends (Kumar, 1981; Caplow and Zeeberg, 1982; Wilson et al., 1985), and they have concomitantly low annealing rates (Rothwell et al., 1986). Because the rate of microtubule annealing is greater, with microtubules having more dynamic ends (Rothwell et al., 1986), annealing of tobacco microtubules probably results from an APM-induced increase in the kinetic activities of taxol-stabilized microtubule ends. An interesting possibility is that, if APM increases the magnitude of dissociation rate constants of both ends of taxol-stabilized microtubules, the net effect might be to increase the treadmilling rate through taxol-stabilized microtubules. Studies of the rates of tubulin exchange at plant microtubule ends will be necessary to test these possibilities.

The observations of herbicide effects on microtubules in tobacco cells also indicate that APM and oryzalin interact with plant microtubules differently from microtubules in mammalian cells. In mammalian cells, low concentrations of antimicrotubule drugs such as vinblastine and nocodazole change the organization of the mitotic spindle and block mitosis in metaphase without causing substantial depolymerization of spindle microtubules and, thus, act primarily by kinetic stabilization of spindle microtubules (Jordan et al., 1992). Although we found that tobacco cells treated with low concentrations of APM (10 and 100 nM) or oryzalin (10 nM) had shorter spindles than control cells, cultures were not arrested in metaphase. Mitotic inhibition in tobacco cells occurred within the threshold range of APM concentrations causing the loss of microtubules. This result further suggests that low concentrations of APM destabilize the kinetics of microtubule ends rather than stabilize them. It is interesting to speculate that the promotion of tobacco cell growth by 50 nM APM results from an enhancement of microtubule kinetics, which produces an acceleration of mitosis or cytokinesis. We are studying this possibility with populations of synchro- nized cells.

Although our work demonstrates that APM and oryzalin

bind to tlhe same site on plant tubulin, the location of the APM/oryzalin-bindmg site on tubulin is not known. Infor- mation concerning the chemical nature of the APM/oryzalin- binding site may be deduced, at least in part, from the different solubility characteristics and binding a1 finities of APM and oryzalin. In the buffer used for our ligand-binding studies, the maximum solubilities of APM and oryzalin were 110 and 3 PM, respectively, showing an approximate 37-fold higher solubility of APM than oryzalin (Hugdahl m d More- john, 1993). A comparison of the derived values of K, (5 PM)

for APM and Kd (117 nM) for oryzalin binding to tobacco tubulin irtdicates a 43-fold lower affinity of tobacco tubulin for APM than for oryzalin. Both APM and oryzaliri are much more hydrophobic than colchicine and have much higher affinities for plant tubulin than does colchicine (Kd = 1.03 mM) (Morejohn et al., 1987b). Apparently, APM artd oryzalin bind to a1 relatively hydrophobic site on the plant tubulin dimer, artd the similar magnitudes of these solubility and affinity dlifferences suggest that binding affinity increases with increased hydrophobicity. It will be interesting in future studies to determine whether colchicine binds to the APM/ oryzalin site or to a different site on plant tubulin. Because we foundl no decay of the oryzalin-binding site after preincubation at 23OC for up to 6 h, the relatively low molar binding stoichiometries for tobacco (Y = 0.32) and maize (Y = 0.5) tubulins indicate pharmacological heterogeneity among plant tubulin dimers. Such heterogeneity may be derived from differences between the a or p subunit isotypes encoded by small inultigene families (Fosket and Morejohn 1992), an idea that should be addressed.

Althou,ph tubulin is generally considered to be a highly conserved protein, there are numerous differenlzes in the pnmary amino acid sequences of plant and mammalian tubulins, wnth most nonconservative substitutions in a-tubulin (Morejohm, 1991; Fosket and Morejohn, 1992). Because plant a-tubulins also have unusual electrophoretic, immunological and peptide-mapping characteristics (Morejohn and Fosket, 1982, 19136; Morejohn et al., 1984; Fosket and Morejohn, 1992), the structure of a-tubulin alone could account for the different m d kingdom-specific pharmacological properties of plant tubulins. We speculate that different plant a-tubulin isotypes are pharmacologically distinct from one another.

Neither APM nor oryzalin is an effective antimicrotubule drug in animal systems. Studies using pure boyrine brain tubulin have reported the absence of oryzalin binding (Morejohn et al., 1987a; Hugdahl and Morejohn, 1993) and no effect of APM or oryzalin on microtubule assembly in vitro (Morejohn and Fosket, 1984b; Morejohn et al., 1987a). Oryzalin has no effect on microtubules in cultured cells of frog (Xenopus laevis) (Bajer and Mole’-Bajer, 1986) or of mouse (Hhgdahl and Morejohn, 1993). APM was reported to have no effect on cellular microtubules in cells of frog (Bajer and Mole’-Bajer, 1986) or of a marsupial i(Morejohn and Fosket, 1984b). Our current results with ntouse 3T3 fibroblasts show that APM has no discernible effect on microtubules in cells of an advanced mammal. Taken to- gether, these observations demonstrate that animd tubulins do not bind APM or oryzalin and that microtubules in plants and animals are pharmacologically distinct.

In suminary, a consideration of the present ligan d-binding




data and cellular observations provides information concerning the mechanism of antimicrotubule herbicide action. The determined value of Kd for oryzalin binding is the herbicide concentration saturating half of the available tobacco tubulin dimers and the determined Ki value for APM competitive inhibition of oryzalin binding is the APM concentration at which half of the oryzalin-binding sites are occupied by APM (Hulme and Birdsall, 1992; Hugdahl and Morejohn, 1993). Thus, the value of Ki for APM is predicted to be equal to or greater than the undetermined Kd for APM binding to tobacco tubulin. Because the threshold concentrations of both APM (0.1 PM < t I 1 PM) and oryzalin (50 nM < t I 100 nM) causing microtubule depolymerization in tobacco cells were lower than their values of Ki (5 PM) and Kd (117 nM), respectively, both herbicides must act to depolymerize microtubules in cells by similar substoichiometric binding mechanisms.

ACKNOWLEDGMENTS

We are grateful to Dr. Carl Fedtke (Bayer AG) for supplying APM and Dr. Ben A. Murray (University of Califomia, Irvine) for kindly providing the mouse 3T3 fibroblasts. We also thank Dr. Brooke S. Kirby for critica1 reading of the manuscript. EM was performed in the Cell Research Institute, University of Texas, Austin.

Received November 30, 1993; accepted January 27, 1994. Copyright Clearance Center: 0032-0889/94/105/0309/12.

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