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A mitotic inhibitor for chromosomal studies in slime moulds

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A mitotic inhibitor for chromosomal studies in slime moulds
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  FEMS Microbiology Letters 5 (1979) 25-27 © Copyright Federation of European Microbiological Societies Published by Elsevier/North-Holland Biomedical Press 25 A MITOTIC INHIBITOR FOR CHROMOSOMAL STUDIES IN SLIME MOULDS P. CAPPUCCINELLI, MARIA FIGHETTI and S. RUBINO Institute of Microbiology Medical School University of Sassari Viale Mancini 5 0 7100 Sassari Sardinia Italy Received 24 August 1978 1. Introduction Chromosomal studies in slime moulds and particu- larly in Dictyostelium discoideum cannot be per- formed with the same ease as in other systems due to many limiting factors. Among other things, the DNA content ofD. discoideum is one of the lowest of any eukaryotic organism [1], chromosomes are small and not easily studied [2], and metaphase blocking of cell division is difficult to obtain, due to the high resis- tance to antimitotic drugs shown by this organism [3,4]. Despite these limiting factors, chromosomal studies have been recently conducted using high con- centrations of the mitotic inhibitor colchicine [4], or the release from starvation conditions [5] to increase the mitotic index. However, the mitotic indices obtainable in this way are extremely low if compared with mammalian systems. We now report the use of a synthetic antimitotic inhibitor, nocodazole, for chromosomal studies in slime moulds which enables large numbers of mitoti- cally arrested cells to be obtained. 2 Materials and Methods Nocodazole or methyl [5-(2-thienylcarbonyl)- 1H- benzimidazol-2-yl)]-carbamate batch No. 191076 was obtained from Janssen Pharmaceutica. Stock solutions were prepared by dissolving 5 mg/ml of nocodazole in DMSO and stored at -30°C until use. 2.1. Growth and metaphase arrest of amoebae Myxamoebae ofD. discoideum strain Ax2 were grown axenically at 22°C either in HL5 medium con- taining 86 mM glucose or in medium with no added sugar as described [6]. D. discoideum NC-4 strain and D. mucoroides were grown non-axenically on SM agar plates with A. aerogenes or E. coli as associated bacteria [7]. Ax2 cells in exponential growth were incubated with the desired concentration of nocodazole (1-10/ag/ml) in HL5 medium and shaken in a rotating shaker (120 rev./min) until use. Non-axenic amoebae were incu- bated with the inhibitor in 0.05 M phosphate buffer pH 6.5 containing washed E. coli (109 cells/ml) at 22°C. 2.2. Chromosomal examination Amoebae were fixed and stained with Gurr's Giemsa stain (improved R66) as described elsewhere [2,4]. Mitotic index was determined by examining all nuclei in a series of microscopic fields randomly chosen using 1000x magnification. No less than 1000 nuclei per sample were examined. Nuclei in all stages of mitosis were scored as mitotic. Photographs were taken using a Leitz Ortolux microscope equipped with a Leica DBP camera on Agfaortho 25 film and printed on Ilford Ilfobrom B111 photographic paper, number 4. 3 Results Cells grown in the presence or absence of glucose showed the same sensitivity to the microtubule inhib- itor nocodazole. However, in most of the experiments, glucose grown cells were preferred for their shorter doubling time (7.5 h). When D. discoideum amoebae  26 5o- T T 40 x Ill e~ z_ 30 o_ I- o. . I/j O-- 1 3 ~ 7 NOCODAZOLE pg/ml) Fig. 1. Effect of nocodazole on the mitotic index of Dictyo stelium discoideum Ax2 amoebae were incubated at various concentrations of inhibitor for 4 =) and 7 *) h. Results are expressed as mean +- S.D. vertical range bars) of five different experiment s. R 50 40 30 20 I:t? T 1 3 l ,/ TIME hr) Fig. 2. Effect of nocodazole 5 pg/ml) upon the mitotic index of ictyostelium discoideum amoebae at various incubation times. Results are expressed as mean -+ S.D. vertical range bars) of four different experiments. Fig. 3. Typical appearance of ictyostelium discoideum chromosomes at different stages of metaphase: a) low-magnification score of a microscopic field. Almost all of the nuclei are in mitosis; b) a multinucleated cell showing three metaphase-arrested nuclei; e,d) late metaphase figures; e,f) intermediate metaphase showing pairing and connections between chromosomes; g,h) early metaphase showing chromosomal condensation. Glucose-growing Ax2 amoebae were incubated for 5-7 h at 22°C in 5 pg/ ml of nocodazole. Bar marker represents 10 pm in a,b and 1 pm in c,d,e,f,g,h.  are incubated with various concentrations of noco- dazole in HL5 glucose medium and the mitotic index determined after 7 h, the indices thus obtained are proportional to the inhibitor concentration. The amoebae demonstrate a maximum mitotic index at 7/~g/ml of the inhibitor. The same pattern can be seen after 4 h of incubation. In this case, the mitotic index is much lower, reaching a maximum of 23 as compared to 49 of the 7-h group (Fig. 1). The extent of metaphase arrest varied slightly depending on the sample of nocodazole used. How- ever, maximum mitotic indices approximating 50 were always obtained with inhibitor concentrations between 5 and 7/ag/ml. Fig. 2 shows the effect of 5/ag/ml of nocodazole on the mitotic index ofD. discoideum amoebae in HL5 medium at various times. The maximum is reached at 7 h; incubation periods of longer duration did not increase the mitotic index, but rather resulted in a very rapid deterioration of chromosomal struc- ture. When Giemsa-stained slides were scored at low magnification (Fig. 3a) many mitotic nuclei could be seen; multinucleated cells, which in Ax2 strain axeni- cally grown represent a 20-30 of the total cell population [8], also presented most of the nuclei in mitosis (Fig. 3b). Chromosomes were usually well- preserved and all the stages ofmetaphase could be seen (Fig. 3c,d,e,f,g,h). Frequently, chromosomal pairing or connections between chromosomes were evident (Fig. 3f, g). In the haploid Ax2 strain chromo- somal number was 7. In the diploid strain NC-4 the number was usually 14. 4 Discussion This paper presents a method for obtaining large numbers of metaphase arrested D. discoideum cells for chromosomal studies using a new mitotic inhibi- tor, nocodazole, that is extremely active in slime moulds, usually poorly affected by other classical microtubule inhibitors such as colchicine or vinka alkaloids. The specific effect of nocodazole is to inhibit tubulin polymerization and, therefore, to dis- rupt the mitotic apparatus in a dose-dependent way [9,10]. it acts specifically on tubulin where apparently it shares the same binding site with colchi- cine [11]. However, in D. discoideum colchicine is much less active than nocodazole regarding metaphase arrest and the cohcentrations at which inhibitory effects can be seen are extremely high if compared 27 with mammalian systems [3]. This colchicine effect seems to be due to intrinsic properties ofD. discoi- deum tubulin, rather than to permeability phenomena. Dictyostelium tubulin is in some respects, e.g. the ability to copolymerize with pig and sheep tubulin and the immunological cross reactivity with mamma- lian tubulin [ 12], similar to that of mammalian cells, but its colchicine-binding activity is far less [ 13]. The low colchicine-binding activity may account for the low mitotic indices that can be seen using this drug even at high concentrations [4]. The metaphase- arrested cells which can be reproducibly obtained with 5-7/ag/ml for 7-8 h incubation with nocoda- zole are about 50 . This figure is much higher than either the 11 reached with colchicine treatment for 14-16 h [4] or the 6 than can be seen by releasing amoebae from starvation on filters [5]. Chromosomal patterns ofD. discoideum as revealed after nocodazole treatment are identical to those ob- tained with other methods. For these reasons the mi- totic inhibitor nocodazole is likely to be the drug of choice for chromosomal studies in slime moulds. References [ 1 ] Ashworth, J.M. and Watts, D.J. (1970) Biochem. J. 119, 175-182. [2] Brody, T. and Williams, K.L. (1974) J. Gen. Microbiol. 82,371-383. [3] CappuccineUi, P. and Ashworth, J.M. (1976) Exp. Cell Res. 103,387-393. [4] Zada-Hames, I.M. (1977) J. Gen. Microbiol. 99,201- 208. [5 ] Robson, G.E. and Williams K.L. (1977) J. Gen. Micro- biol. 99,191-200. [6] Watts, D.J. and Asworth, J.M. (1970) Biochem. J. 119, 171-174. [7] Sussman,M. (1966) in Methods in Cell Physiology (Pres- cott, D., Ed.), Vol. 2, pp. 397-410, Academic Press, New York. [8] Zada-Hames, I.M. and Ashworth, J.M. (1977) in Devel- opment and Differentiation in the Cellular Slime Moulds (Cappuccinelli, P. and Ashworth, J.M., Eds.), pp. 69-78, Elsevier/North Holland, Amsterdam. [9] De Brabander, M., Van de Veire, R., Aerts, F.E.M., Borges, M. and Janssen, P.A.J. (1976) Cancer Res. 36, 905-910. [10] De Brabander, R., Van de Veire, R., Aerts, F., Guens, G. and Hoebeke, J. (1976) J. Natl. Cancer Inst. 56, 319-325. [ 11 ] Hoebeke, J., Van Nijen, G. and De Brabander, M. (1976) Biochem. Biophys. Res.Commun. 69, 319-324. [12] CappuccineUi, P., Martinotti, M.G. and Hames, B.D. (1978) FEBS Lett., in press. [13] CappuccineUi, P. and Hames, B.D. (1978) Biochem. J. 169,499-504.
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