INT. J. RADIAT. BIOL., 1984, VOL. 46, NO. 4, 435-442

Effect of y-irradiation on microtubule assembly in vitro

VERA GAL and DIVNA TRAJKOVICt

Institute of Biophysics, School of Medicine, University of Belgrade, andt Department of Biochemistry, Institute for Biological Research,Belgrade, Yugoslavia

(Received 3 March 1983; revision received 21 February 1984;accepted 24 April 1984)

Microtubular protein was exposed to y-radiation from 500 to 000 Gy. Withinthat dose range its polymerization ability was decreased by 20-60 per cent whensamples were irradiated in assembled state, and by 40-75 per cent when irradiatedin unassembled state. Microtubules assembled from irradiated subunits wereshorter and of more uniform lengths than control microtubules. For the dose of1000 Gy the mean length and its standard deviation were reduced to about one-half of the values of the control.

Indexing terms: microtubular protein, polymerization ability, microtubuleassembly.

1. IntroductionSeveral authors have investigated the effect of ionizing radiation on microtubular

protein in vitro (Zaremba and Irwin 1977, 1981, Coss et al., 1981). It was found thatthe ability of microtubular protein to polymerize is reduced in a dose-dependentmanner when exposed to irradiation. The loss of the ability to polymerize iscorrelated with the oxidation of sulphydryl groups (Coss et al. 1981, Zaremba andIrwin 1981). However, there is a discrepancy in the reported reduction ofpolymerization and different views are expressed as to whether the polymerized ordepolymerized state is more sensitive to irradiation (Zaremba and Irwin 1977, 1981,Coss et al. 1981).

In the present investigation, microtubular protein was exposed to -radiation in ahigher dose range (500 1000(;y) than in the above mentioned work. The mainpurpose of our study was to re-examine at higher doses the effect of irradiation on thepolymerization ability of microtubular protein in assembled and unassembled state.

2. Materials and methodsMicrotubular protein was extracted from calf brain by the temperature-

dependent assembly/disassembly method of Shelanski et al. (1973). Theassembly buffer in the first two cycles of extraction contained 01 M Mes (2-(N-morpholino)ethanesulphonic acid), 1 mM EGTA (ethylene glycol bis(2-aminoethylether) tetraacetic acid), 1 mM GTP (guanosine 5'-triphosphate), 05 mMMgCI2 , 4M glycerol and 1 mM 2-mercaptoethanol, pH 66. After two cycles ofextraction the supernatants were made 8 M in glycerol and stored in liquid nitrogen.Immediately before the use, a third cycle was carried out, but in the assembly buffer2-mercaptoethanol and glycerol were omitted. Protein determinations were carriedout by the method of owry et al. (1951).

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The polymerization was induced by transferring the sample from the ice slurryeither to a water bath or to the spectrophotometric cuvette, both maintained at 28°C.The extent of polymerization was determined by measuring the change in turbidityat 350nm in a Varian 634 spectrophotometer integrated with a heating system.

Samples for electron microscopy were applied to the grids with formvar andcarbon, stained with 2 per cent uranyl acetate and examined in a Philips 300 electronmicroscope, at 80kV.

The air-equilibrated samples were y-irradiated in a 60 Co source at a dose rate of220 Gy/min. During irradiation all samples were mounted in the same fixed positionof the cell compartment, which was kept at constant temperature.

3. ResultsIn order to study the effect of irradiation on the polymerization of microtubular

protein, it is important to keep in mind the history of the sample. The heat effect(incubation) also decreases the polymerization ability (Gaskin et al. 1974), and thishas been taken into account by the following experimental procedure. The sample inthe buffer of 01 M Mes, 0'5 mM MgCl 2, 1 mM GTP, 1 mM EGTA, pH 66 wasdivided into three aliquots. One aliquot was incubated at 28°C (control assembly),depolymerized in ice for 35 min and repolymerized at 28°C (control reassembly).The second aliquot was incubated at 28°C for 35 in, irradiated with 500, 700 or1000 Gy at 28°C, depolymerized in ice for 35min and repolymerized at 28°C(reassembly of irradiated microtubules). The third aliquot was irradiated in an iceslurry with the same dose as the microtubules, polymerized at 28°C (assembly ofirradiated free subunits), depolymerized in ice for 35 min and repolymerized at 28°C(reassembly of irradiated free subunits). Every experiment was repeated 4 or 5 times.The protein concentration was 09 mg/ml. Before and after irradiation and poly-merization aliquots were taken for electron microscopy.

Figure 1 shows typical polymerization and repolymerization curves of thecontrol sample and of a sample irradiated with 700 Gy. In the case of the controlsample, the plateau of the reassembly curve is 12 per cent below that of the assemblycurve. However, one can note that the polymerization of irradiated free subunits isless than the repolymerization of irradiated microtubules. This behaviour was foundin all experiments for all applied doses.

The decrease of polymerization with increasing doses is presented in figure 2.The extent of polymerization is expressed as the percentage of control polymeriz-ation, after 20 min of incubation, i.e. (AT350nm sample/Ar35 0nm control) x 100. Due tothe higher reproducibility of control assembly compared to reassembly, the extent ofreassembly for irradiated microtubules is also expressed relative to control assembly.If the extent of reassembly is calculated relative to the control reassembly, the uppercurve of figure 2 would be shifted 10-15 per cent upward.

The polymerization of irradiated and control microtubular protein was alsoexamined by electron microscopy. We observed that the polymerization of irradiatedsamples yields microtubules of smaller but more uniform lengths. In order toillustrate this effect electron micrographs which correspond to (re)polymerizationcurves from figure 1 are shown in figure 3. At all applied doses we found a shorteningand increased length uniformity of microtubules polymerized or repolymerizedfrom irradiated samples. However, this effect is more pronounced at higher doses.The change of the length distribution was quantitatively examined for the dose of1000 Gy. In figure 4 are presented histograms of length of control microtubules and

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Effect of radiation on microtubules 437

40TIME (min)

Figure 1. The effect of ?-irradiation at 700 Gy on assembly and reassembly of microtubularprotein. The assembly or reassembly was induced by transferring the sample from theice slurry to the spectrophotometric cuvette kept at 28°C. The change in turbidity at350nm (Ar3o.nm) was monitored. Protein concentration is 09mg/cm3. Controlassembly (); control reassembly (); irradiated free subunits assembly ();irradiated free subunits reassembly (); irradiated microtubules reassembly (A).(See §2.)

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Figure 2. The dependence of the extent of polymerization on the dose. The extent ofpolymerization is expressed as precentage of control assembly after 20 min incubation(Az3 5 0nm sample/Ar 3 50nm control) x 100. Points represent the average of 4 or 5 experi-ments and error bars the standard errors. Protein concentration is 09 mg/cm3 .

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(a) (b)

(c)

Figure 3. Negatively stained microtubules from the sample shown in figure 1,(a) corresponding to the plateau of the control assembly, (b) corresponding to theplateau of the irradiated free subunits assembly, and (c) corresponding to the plateau ofthe irradiated microtubules reassembly curve. Bar 5 m, x 4200.

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Effect of radiation on microtubules

LENGTH (ARBITRARY UNITS)

Figure 4. Histograms of length of microtubules formed from unirradiated and irradiatedsamples. Length distributions were determined by electron microscopy for (a)microtubules formed from irradiated free subunits at a dose of 1000 Gy, and (b)unirradiated microtubules. The fraction of microtubules observed within one arbitraryunit (0-61 m) length-interval is plotted on the ordinate. The total number of examinedmicrotubules as fr (a) 353 and (b) 516. The average length is for (a) 264/lm and(b) 503pm and standard deviation for (a) 1-59pm and (b) 3-72#m.

microtubules polymerized from irradiated free subunits. No change in the mor-phology of microtubules polymerized from irradiated samples has been observed, inagreement with Coss et al. (1981) and Zaremba and Irwin (1981). Electronmicrographs of microtubules before and after irradiation were indistinguishable(figures 3 (a) and 5).

In order to estimate the possible contribution of aggregates to the turbiditymeasurements, the shape of the polymerization curves (the plateau) and thereproducibility of the extent of polymerization should be taken into account. Inorder to get rid of aggregates, irradiated free subunits were centrifuged before theassembly and all other samples were centrifuged in depolymerized state immediatelybefore the reassembly. The centrifugation was performed at 100 000g and 5C for30 min. This experiment was repeated four times for the dose of 700 Gy. The meanvalue and standard error of the extent of reassembly were 74-82 + 0-87 per cent for thecontrol, 577.+ 19 per cent for the irradiated microtubules, and 396 + 2-4 per cent for

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Figure 5. Intact microtubules irradiated with 700 (v. Protein concentration 0'9mg/cm3.

Bar 5im, x4200.

the extent of assembly of irradiated free subunits. (As mentioned before, allpercentage values are expressed relative to control assembly.) Hence, theseexperiments show that the reassembly extent of the control is reduced by about 25per cent, which indicates that in the case of low doses the effect of irradiation canhardly be distinguished from the heat effect. For instance, for the dose of 300 Gy wefound that the mean value and the standard error of the reassembly extent forirradiated microtubules is 67-3 + 32 per cent whereas the assembly extent of theirradiated free subunits is 552+41 per cent.

4. DiscussionIn this work the effect of y-irradiation on the polymerization ability of

microtubular protein was investigated from 500-100 Gy. The decrease of poly-merization ability with increase in dose is presented in figure 2. These results,verified by electron microscopy, show that the polymerization is reduced to a largedegree, but even for the dose as high as 000 (Gy it is not completely inhibited. This isin agreement with experiments i vivo of Rustad (1981) which demonstrate a highradioresistance of microtubules. In these experiments, cells of sea urchin embryosexposed to 500-1000 Gy y-radiation were capable of elongating microtubules.

Our results give evidence that microtubular protein is more sensitive toirradiation in unassembled than assembled state. In all our experiments thepolymerization of irradiated free subunits was more reduced than the repolymeriz-ation of irradiated microtubules. From figure 2 it can be deduced that this differenceis about 15 per cent. Since the reassembly of unirradiated samples is reduced by9-8 + 1-8 per cent relative to the assembly, we estimate that the effect of irradiation onfree subunits is about 25 per cent higher than on microtubules. However, the

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Effect of radiation on microtubules

contribution of aggregates formed by the exposure of microtubular protein toradiation (Zaremba and Irwin 1981) should also be considered when discussing theresults shown in figure 2. When samples in depolymerized state were centrifugedafter irradiation, for the dose of 700 Gy a relative difference of about 23 per cent wasobtained between the effect of irradiation on free subunits and microtubules.Moreover, the difference in the initial turbidity between unirradiated and irradiatedsamples was about 001 and the plateau of polymerization curves of irradiatedsamples was stable within 0005 in a time interval of 10 min (see figure 1). Therefore,possible errors caused by the presence of aggregates cannot change significantly thecharacteristics of the irradiation effect presented in figure 2.

It is interesting to compare our results with those of Zaremba and Irwin (1977)and Coss et al. (1981). Although the experimental conditions are not identical,agreement and discrepancies might indicate the influence of various parameters,such as the buffer composition, the temperature of sample during irradiation, etc.Zaremba and Irwin (1977) y-irradiated air-equilibrated samples at 25°C in bufferwhich contained 1 mM CaC12 (unassembled state). Their samples irradiated with500 Gy assembled to 75 per-cent of the control, whereas those irradiated with 700 Gyassembled to 69 per cent of the control. These results are in a good agreement withour results (figure 2). These authors also found that free subunits were moresensitive than 36 S oligomers (assembled state). Coss et al. (1981) X-irradiated sealedsamples up to 300 Gy. They obtained for samples irradiated with 300 Gy an extent ofpolymerization of 60 per cent (relative to the control) for both states, assembled andunassembled. In these experiments the procedure was similar to ours (which gavethe results shown in figure 2) and their buffer contained 02 mM GTP. However,using a different procedure, Coss et al. (1981) obtained at 300 Gy extents ofpolymerization of 52'6, 10-8 and 2-6 per cent for temperatures of irradiation of 0, 22,and 37°C, respectively. In that case, the final cold disassembly before anyexperimental treatment was performed in buffer lacking GTP, and samples indepolymerized state were centrifuged after irradiation. In our experiments, whichincluded centrifugation of samples after irradiation, we obtained at 300 Gy and 0°Can extent of polymerization of 55'2 + 4'1 per cent (see § 3). This is in agreement withCoss et al. (1981) within experimental error. However, the discrepancy for theirradiation at 300 Gy at higher temperatures is large. It is possible that the differencein the composition of the buffer during the procedure is the cause for thediscrepancies, i.e. GTP is protecting the structure of microtubular protein.

We would like to emphasize the difference between the effect of radiation on(i) the structure of intact microtubules, and (ii) the decrease of polymerization abilityof the microtubular protein. The destruction of the intact microtubules is very weak(see figures 3 (a) and 5). There was no detectable difference between negativelystained irradiated and control microtubules. This is in agreement with the lengthdistribution of control and X-irradiated microtubules (20-300 Gy) found by Cosset al. (1981). They did not find a regular change in the mean length and its standarddeviation with increasing doses.

We conclude that when intact microtubules are exposed to radiation, the bondsbetween subunits are not significantly affected. However, when irradiated micro-tubules are depolymerized, the initial change of subunits leads to a conformationalstate in which the ability to polymerize is reduced. The irradiation of free subunitsprobably leads to the same conformational state (Zaremba and Irwin 1981). Fromour results it follows that a larger fraction is affected by irradiation when

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Effect of radiation on microtubules

microtubular protein is in unassembled than when it is in assembled state. Thechange in length distribution (see figure 4) indicates that irradiation has a strongereffect on elongation than nucleation.

AcknowledgmentsElectron microscopy was carried out at the Laboratory of Electron Microscopy of

the University of Belgrade, and irradiation of samples at the Laboratory of SolidState Physics and Radiation Chemistry, the Boris Kidri6 Institute of NuclearSciences, Vinca. The authors are grateful to both Institutions for their kindhospitality.

ReferencesCoss, R. A., BAMBURG, J. R., and DEWEY, W. C., 1981, Radiat. Res., 85, 99.GASKIN, F., CANTOR, C. R., and SHELANSKI, M. L., 1974, J. molec. Biol., 89, 737.LOWRY, H. Q., ROSEBROUGH, J. N., FARR, L. A., and RANDALL, J. R., 1951, J. biol. Chem.,

193, 265.RuSTAD, R. C., 1981, Radiat. Res., 88, 631.SHELANSKI, M. L., GASKIN, F., and CANTOR, C. R., 1973, Proc. natn. Acad. Sci. U.S.A.,

70, 765.ZAREMBA, T. G., and IRWIN, R. D., 1977, Radiat. Res., 71,300; 1981, Biochemistry, 20,1323.

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