Cell Differentiation 8 (1979) 243--252 © Elsevier/North-Holland Scientific Publishers Ltd.

DIFFERENTATION D I S C O I D E UM

243

WITHOUT MITOSIS IN DICTYOSTELIUM

PIERO CAPPUCCINELLI, MARIA FIGHETTI and SALVATORE RUBINO Institute of Microbiology, University of Sassari, Viale Mancini, 5, 07100 Sassari, Sardinia (Italy) Accepted January 30th, 1979 Dictyoselium discoideum Ax-2 amoebae incubated in the presence of the microtubule inhibitor nocodazole, irreversibly lost their ability to multiply. Nocodazole-treated cells remained viable and RNA and protein synthesis continued for at least 48 h. When nocodazole-treated amoebae were allowed to develop on Millipore filters or on agar slides they differentiated with some delay when compared with controls. These results show that mitosis, naturally present during the developmental cycle ofDictyostelium discoideum Ax-2, is not indispensible for differentiation. D. d i s c o i d e u m is a s i m p l e e u k a r y o t e w i t h a life cycle n a t u r a l l y divided i n t o t w o s e p a r a t e stages. During the g r o w t h phase, vegative a m o e b a e feed a n d m u l t i p l y b y m i t o t i c b y division. W h e n t h e f o o d supplies are e x h a u s t e d , cells e n t e r i n t o a s y n c h r o n o u s d e v e l o p m e n t a l c y c l e ending, as a result o f cell d i f f e r e n t i a t i o n i n t o spores, s t a l k and basal disc cells, w i t h a fruiting b o d y c o n s t r u c t i o n ( B o n n e r , 1 9 6 9 ; L o o m i s , 1 9 7 5 ) . This m i c r o r g a n i s m can, thus, be r e g a r d e d as a g o o d m o d e l f o r d e v e l o p m e n t a l studies a n d p a r t i c u l a r l y f o r investigating h o w cell cycle events are c o u p l e d w i t h d i f f e r e n t i a t i o n . T h e a i m of this i n v e s t g a t i o n was to d e t e r m i n e w h e t h e r mitosis is indisp e n s a b l e f o r cell d i f f e r e n t i a t i o n or, r a t h e r , if a c o m p l e t e d i f f e r e n t i a t i o n prog r a m m e can b e carried o u t b y cells w h e r e mitosis has b e e n p r e v e n t e d . We o b t a i n e d large a m o u n t s o f stable m i t o t i c a l l y i n h i b i t e d a m o e b a e b y using a m i c r o t u b u l e i n h i b i t o r , n o c o d a z o l e . This is also active in slime m o u l d s w h i c h are usually p o o r l y a f f e c t e d b y similar drugs ( C a p p u c c i n e l l i a n d A s h w o r t h , 1 9 7 6 ) . W h e n D. d i s c o i d e u m a m o e b a e are i n c u b a t e d for sufficient t i m e w i t h n o c o d a z o l e t h e y irreversibly lose t h e i r ability t o m u l t i p l y and can t h u s be used for such experiments. MATERIALS AND METHODS R eagen ts

Nocodazole or methyl[5-(2-thienylcarbonyl)-lH-benzoimidazol-2yl-]carb a m a t e b a t c h No. 1 9 1 0 7 6 (Aldrich C h e m i c a l Co.) was m a i n t a i n e d as the s t o c k s o l u t i o n in D M S O at a c o n c e n t r a t i o n o f 5 m g / m l . P a r t o f s t o c k was a d d e d t o t h e c u l t u r e m e d i u m t o f u r t h e r dilute t h e solution. T h e final D M S O c o n c e n t r a t i o n was n e v e r g r e a t e r t h a n 0.2%.

244

Radiochemicals [Methyl-3H]thymidine (spec. act. 35 Ci/mmol), was obtained through CEA, Gif-sur-Yvette, France. [3H]Uridine (spec. act. 41 Ci/mmol) and [14C]leucine (spec. act. 342 Ci/mol) were purchased from the Radiochemicai Centre, Amersham U.K. All other chemicals were of reagent grade. Growth D. discoideum strain Ax-2 was grown axenically at 22°C in HL-5 medium containing 86 mM glucose as described by Watts and Ashworth (1970). Cells were harvested during the exponential phase of growth at approx. 3--6 × 106 cells/ml. When growth inhibition was required, nocodazole was added directly to the medium at a concentration of between 1 and 10 ~g/ml. Development For developmental studies, amoebae were harvested from growth flasks, washed in ice-cold distilled water and spread either onto the surface of black Millipore filters (Sussman, 1966) or on agar-coated microscopic slides as described by Cappucinelli and Ashworth (1976}. Development was carried out at 22°C as a constant humidity. Fruiting b o d y construction in controls was completed in approx. 24--26 h. Cell counting and viability Direct counting was by eye using a h a e m a t o c y t o m e t e r and at least 20 fields per sample were observed. In order to count cells at various stages of development, disaggregation was obtained by washing the filters with 30 mM HEPES (pH 6.8) containing 10 mg/ml Pronase (Calbiochem) and 0.25 M BAL (2,3-dimercaptopropanol, Sigma), as described by Zada-Hames and Ashworth {1978). Cell viability was determined either by the trypan blue exclusion test or by plating clonally with Aerobacter aerogenes on SM agar plates {Sussman, 1966). Labelling conditions DNA synthesis in control and nocodazole-treated amoebae was studied both with pulse and continuous labelling techniques. In pulse labelling experiments, D. discoideum amoebae (5 × 10 s cells/ml) were incubated in HL-5 glucose medium containing 6 ~g/ml nocodazole for 21 h, washed once to remove the inhibitor, and resuspended in the medium without the inhibitor at the same cell density for an additional 27 h. For pulsing, aliquots of 0.5 ml of inhibited cells and untreated controls were incubated at 22°C with [methyl-3H]thymidine (40 uCi/ml) for 30 min. To determine the TCAprecipitable radioactivity, cells were lysed by the addition of an equal volume of 0.2 M KOH (Katz and Bourguignon, 1974) and precipitated by cold TCA (final concentration 10%}. The precipitate was collected on Sartorius nitrocellulose filters, washed, dried and counted in a toluene based scintillation fluid using a Packard Tri-Carb scintillation counter.

245

In continuous labelling experiments, amoebae were cultivated in HL-5 glucose medium in the presence of 4 pCi]ml [methyl-3H]thymidine. After 17 h, when cell densities were between 5 and 8 × 10S/ml, 5 ~g/ml of nocodazole was added. Cells were kept in the presence of the inhibitor for 24 h and then washed and resuspended in the medium containing the previous a m o u n t of radioactivity (but without nocodazole) for an additional 24 h. Radioactivity was determined as previously described on aliquots of 0.4 ml of cell suspension for the duration of the experiments. R N A and protein synthesis was studied by continuous labelling using the same experimental procedure except that [3H]uridine (4 pCi/ml) or [14C]leucine (1.2 uCi/ml) were added to cell cultures. RESULTS

Assessment of the antimitotic effect To obtain a cell population in which mitosis is prevented, D. Discoideum amoebae were treated eith the microtubule inhibitor, nocodazole. Figure 1 shows the effect of the inhibitor on growth and viability (tested by the trypan blue exclusion test). Cell growth is inhibited in a dose-dependent way; it is totally prevented for at least 60 h at a concentration of 5 ~g/ml. Cell viability, is not affected even at 7 ~g/ml; however a toxic effect is evident with higher concentrations. Chromosomal analysis shows metaphase arrested cells during the first 7--8 h of treatment; after which, extensive chromosomal breakage occurs {Cappuccinelli et al., 1978) A

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246

Reversib,lity o f the growth inhibition Having defined the antimitotic effect of nonodazole it was interesting to examine the reversibility of the growth inhibition. For this purpose, D. discoideum amoebae were incubated in HL-5 medium containing 5 gg/ml nocodazole at a density of 5 × l 0 s cells/ml. Samples were taken at intervals and cells washed once in 0.4% NaC1 solution and either resuspended in fresh HL-5 (where growth was followed for 48 h) or plated clonally on agar containing A. aerogenes to determine the a m o u n t of plaque-forming units. Figure 2 shows that 11 hours of incubation in a nocodazole-containing medium totally prevents cell growth for at least 48 h; at the same time, only 1.4% of the cell population was still able to multiply in n o n axenic conditions. This number falls to 0.8% after 30 h.

Macromolecular synthesis o f inhibited cells To study the effect of nocodazole on DNA synthesis in D. discoideum amoebae, under pulse labelling conditions, cells were incubated with 6 ~g/ml of the drug for 21 h, washed once and cultivated in medium without nocodazole for an additional 27 h. Figure 3 shows that [methyl-~H]thymidine incorporation is greatly reduced 7--9 h after the treatment and DNA synthesis continues to be lower than normal after the inhibitor is washed out. Other evidence that DNA synthesis is inhibited in nocodazole-treated cells comes from continuous PFU xlOG/mt .6 Cm)

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labelling experiments (Fig. 4a,b,c). The results show that incorporation of [methyl-3H]thymidine into TCA-precipitable material stops a few hours after the addition of nocodazole. Incorporation does not restart for at least 27 h after the removal of the inhibitor. A somewhat different pattern can be seen in experiments of continuous labelling with [3H] uridine and [ 14C]leucine, which show that even though cell multiplication stops, RNA and protein synthesis continues for at least 25 h after the removal of the inhibitor. One reason for this may be that during this time cell size increases {Cappuccinelli et al. unpublished).

Differentiation of mitotically arrested cells When cells, mitotically arrested with an inhibitory concentration of nocodazole, are allowed to develop either on Millipore filters or on agar slides, the period of differentiation depends on the nocodazole concentration (Fig. 5). Stalk cells and spores are formed although an alteration in spore morphology is evident (Fig. 5b,c). The longer period of differentiation (e.g. cells treated with 6 /~g/ml nocodazole develop in 48 h instead of 24) is mainly due to an increase in the length of the slug phase. With higher concentrations of nocodazole (8 ug/ml), differentiation is prevented and cells show an extensive alteration in spore coat secretion (Cappuccinelli, unpublished results). Finally, to show that differentiation in stalk and spore cells can take place

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TIME (hrs) Fig. 5. The m o r p h o g e n e t i c sequence of n o c o d a z o l e inhibited D . d i s c o i d e u m Ax-2 a m o e b a e . Axenic cells were i n c u b a t e d for 24 h in the presence o f increasing c o n c e n t r a t i o n o f nocodazole, washed and allowed to develop on agar slides. Insert p h o t o g r a p h s s h o w spores (a) and part o f a stalk (b) f o r m e d after t r e a t m e n t w i t h 6 ~g/ml nocodazole. A l t e r a t i o n in spore size and shape is evident. Insert bars represent 10 ~m.

w i t h o u t an increase in cell number (probably due to cell division), the a m o u n t of D. discoideum amoebae was determined during development on Millipore filters and compared to controls. Figure 6 shows that nocodazoletreated cells do not increase in number during the developmental period; on the other hand, there is a decrease of approx. 30% of the cell population. This pattern differs totally from controls which have a growth curve remarkably similar to that obtained in comparable conditions by Zada-Hames and Ashworth (1978}. DISCUSSION

Our experiments on the development of mitotically-inhibited D. discoideum amoebae show that mitosis is not an indispensable part of the cell differentiation cycle, and may help to clarify the role of cell division during the differentiation of slime moulds. In fact, the results so far obtained on this subject are conflicting. On one hand, earlier authors concluded that cell division was absent during the developmental phase of D. discoideum (Olive, 1902; Raper, 1941). On the other hand, studies with bacterically-grown strains, showed t h a t although cell division may occur during development, it

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is n o t i m p o r t a n t for d i f f e r e n t i a t i o n ( B o n n e r and Frascella, 1 9 5 2 ; Wilson and Ross, 1957; Sussman and Sussman, 1960). M o r e r e c e n t l y , h o w e v e r , ZadaHames and A s h w o r t h (1978), using the axenic strain Ax-2, d e m o n s t r a t e d t h a t a d o u b l i n g o f cell n u m b e r due t o t r u e m i t o t i c division o c c u r s during developm e n t in a way indicate it is an integral part o f the d i f f e r e n t i a t i o n p r o g r a m m e . T o o b t a i n large a m o u n t s o f m i t o t i c a l l y - i n h i b i t e d a m o e b a e we did n o t use inhibitors o f DNA synthesis, such as elongate p y r i m i d i n e derivatives t h a t m a y also inhibit RNA synthesis and t h e r e f o r e gene t r a n s c r i p t i o n (Cozzarelli, 1 9 7 7 ) , as previous a u t h o r s did ( L o o m i s , 1971; Zada-Hames and A s h w o r t h , 1 9 7 8 ) , b u t we used n o c o d a z o l e which is able to p r e v e n t mitosis b y interfering with m i c r o t u b u l e s (De B r a b a n d e r et al., 1976). In particular, it binds t o t u b u l i n where it shares the same binding site with colchicine ( H o e b e k e et al., 1976). A l t h o u g h in m a m m a l i a n cells its action is rapidly reversible (De B r a b a n d e r et al., 1976), the m i t o t i c inhibition it p r o d u c e s in D. d i s c o i d e u m c a n n o t in the m a j o r i t y o f cells be reversed. This m a y d e p e n d on an irreversible denatu-

251 r a t i o n o f D. discoideum t u b u l i n which differs f r o m m a m m a l i a n tubulin, even t h o u g h it can be c o n s i d e r e d a closely-related p r o t e i n (Cappuccinelli and Hames, 1978; Cappuccinelli, et al., 1 9 7 8 ) , or, m o r e likely, it could be a consequence o f the extensive c h r o m o s o m a l breakage taking place in a cell after a long t r e a t m e n t with n o c o d a z o l e . T o g e t h e r with m i t o t i c inhibition, cells show an inhibition of D N A synthesis, while RNA and p r o t e i n synthesis continues. Since the e f f e c t of n o c o dazole on cell division is p r o b a b l y due t o the d i s r u p t i o n o f the m i t o t i c spindle, c h r o m a t i d a l separation does n o t o c c u r , the cells have a d o u b l e D N A c o n t e n t and can t h e r e f o r e be c o n s i d e r e d as being in a f u n c t i o n a l G2 stage. In this situation t h e y can a c c o m p l i s h t h e i r d e v e l o p m e n t a l p r o g r a m m e p r o d u c i n g d i f f e r e n t i a t e d cells s y n c h r o n o u s l y . T h e r e is, h o w e v e r , a d e l a y in fruiting b o d y c o n s t r u c t i o n p r o b a b l y due t o an i m p a i r m e n t o f spore coat secretion. In fact, in m a n y cell t y p e s , secretion is o f t e n d e p e n d e n t on the integrity o f the m i c r o t u b u l a r s y s t e m (for r e f e r e n c e s see S t e p h e n s and Edds, 1 9 7 6 ) . In conclusion, o u r results show t h a t mitosis, n a t u r a l l y occurring during d e v e l o p m e n t , is n o t an absolute r e q u i r e m e n t for cell d i f f e r e n t i a t i o n in D. discoideum Ax-2. T h e r e f o r e a c o n n e c t i o n b e t w e e n d e v e l o p m e n t a l events and certain phases o f the cell cycle, seen in p r o k a r y o t e s , such as Caulobacter crescentus (Osier and N e w t o n , 1977), and in e u k a r y o t i c systems such as the m a m m a r y gland {Vanderhaar and T o p p e r , 1 9 7 4 ) , m y o b l a s t s (Okazaki and H o l t z e r , 1 9 6 6 ) and tracheal cells of plants {Shininger, 1975} does n o t seem t o be necessary for D. discoideum d i f f e r e n t i a t i o n . ACKNOWLEDGEMENTS We thank Dr. Gall Clinton for reading the manuscript. This work was supported by a grant (No. 70.02027.04) from the Italian Research Council (CNR). REFERENCES Bonner, J.T.: The Cellular Slime Moulds (Princeton University Press, Princeton) 2nd. ed. (1969). Bonnet, J.T. and E.B. Franscella: J. Exp. Zooi. 121,561--571 (1952). Cappuccinelli, P. and J.M. Ashworth,: Exp. Cell Res. 103, 387--393 (1976). Cappuccinelli, P. and B.D. Hames,: Biochem. J. 169,499--504 (1978). Cappuccinelli, P., G. Martinotti and B.D. Haines,: FEBS Lett. 91, 153--157 (1978). Cappuccinelli, P., M. Fighetti and S. Rubino: FEMS Microbiol. Letters; in press (1978). Cozzarelli, N.R.: Annu. Rev. Biochem. 46, 641--648 (1977). De Brabander, M.J., R.M.L. Van de Veire, F.E.M. Aerts, M. Borges and P.A.J. Janssen: Cancer Res. 36,905--911 (1976). Hoebeke, J., M. Van Nijen and M. De Brabander: Biochem. Biophys. Res. Commun. 69, 319--322 (1976). Katz, E.R. and L.W. Bourguignon: Develop. Biol. 36, 82--87 (1974). Loomis, W.F.: Dictyostelium discoideum: a Developmental System (Academic Press, New York and London), ('1975). Loomis, W.F.: Exp. Cell Res. 64,484--486 (1971). Okazaki, K. and H. Holtzer: Proc. Natl. Acad. Sci. U.S.A. 56, 1484--1490 (1966). Olive, E.W.: Proc. Boston Soc. Nat. Hist. 30, 451--513, (1902).

252 Osley, M.A. and A. Newton: Proc. Natl. Acad. Sci. U.S.A. 74, 124--128 (1977). Raper, K.B.: Growth 5,41--76 (1941). Shininger, T.L.: Dev. Biol. 4 5 , 1 3 7 - - 1 5 0 (1975). Sussman, M.: In: Methods in Cell Biology vol. 2, ed. D.M. Prescott (Academic Press, New York), pp. 397--410 (1966). Sussman, R.R. and M. Sussman : J. Gem Microbioi. 23,287--293 (1960). Stephens, R.E. and K.T. Edds: Physiol. Rev. 56,709--777 (1976). Watts, D.J. and J.M. Ashworth: Biochem. J. 119,171--174 (1970). Wilson, C.M. and I.K. Ross: Am. J. Bot. 4 4 , 3 4 5 - - 3 5 0 (1957). Vonderhaar, B.K. and Y.J. Topper: J. Cell Biol. 63,707--712 (1974). Zada-Hames, I. and J.M. Ashworth: Develop. Biol. 63,307--320 (1978).