JOURNAL OF CELLULAR PHYSIOLOGY 145120-128 (1990)

Regulation of Tubulin Synthesis During the Cell Cycle in the Synchronous Plasmodia of Physarum polycephalum B. DUCOMMUN, J. CANCE, AND M. WRIGHT* laboratoire de Pharrnacologie et de Toxicologie Fondamentales, Centre National de la Recherche Scientifique, 3 I400 Toulouse, France Regulation of a- and p-tubulin isotype synthesis during the cell cycle has been studied in the myxomycete Physarum polycephalum, by subjecting synchronous plasmodia to temperature shifts and pharmacological perturbations. Temperature shifts interfered with the regulation of tubulin synthesis. Inhibition of DNA synthesis prevents tubulin degradation after completion of the cell cycle (Ducornrnun and Wright, Eur. J. Cell Biol., 50:48-55, 1989) but did not perturb the initiation of tubulin synthesis. The constant increase of tubulin synthesis in the presence of tubulin-sequestering drugs and the decrease of tubulin synthesis during a treatment with aphidicolin in late G2 phase suggest the existence of an autoregulatory mechanism of tubulin synthesis. Moreover, the microtubule poison methyl benzimidazole carbamate dissociated synthesis of the a,-tubulin isotype from the generally strictly coordinated synthesis of all tubulin isotypes during the transient interruption of mitosis. These observations show that a rnicrotubular poison can perturb regulation of the synthesis of specific isotubulins.

Investigation of the cell cycle of eukaryotic cells is hindered by the difficulties of obtaining easily synchronized cells without the artefactual effects due to the synchronizing rocedure (Mitchison, 1971). In this respect, the abso Ute and natural synchrony of the giant plasmodia of the myxomycete Physarurn (Howard, 1932; Guttes and Guttes, 1964) is particularly useful for analysing the regulation of proteins known to be synthesized during a limited part of the cell cycle (i.e., thymidine kinase, histones, and tubulins (Sachsenmaier and Ives, 1965; Laffler et al., 1981; Chang et al., 1983; Loidl and Grobner, 1987). It has been well documented that in Physarurn synchronous plasmodia, external perturbations generally delay the onset of mitosis (Sachsenmaier and Rusch, 1964; Sachsenmaier and Becker, 1965; Cummins et al., 1965; Brewer and Rusch, 1968; Hildebrandt and Sauer, 1973; Grobner and Loidl, 1982). Among them, a few agents have a limited action on macromolecular biosyntheses, such as total RNA and protein syntheses (Eon-Gerhardt et al., 1981a,b; Wright and Tollon, 1982,1988,1989).These agents have been used extensively in the case of thymidine kinase (EonGerhardt et al., 1981a,b; Wright and Tollon, 1979, 1988,1989) and histones (Loidl and Grobner, 1987), in order to define at the phenomenological level the regulatory pathways playing a role in the onset and in the arrest of the synthesis of these proteins. For example, inhibitors of DNA replication prevent the progression of nuclei from prophase to metaphase without disturbing the timing of thymidine kinase synthesis (Eon-Gerhardt et al., 1981a; Wright and Tollon, 1988, 1989).By contrast, temperature shifts and microtubu-

f

0 1990 WILEY-LISS,INC.

lar poisons prevent both the onset of prophase and the increase of thymidine kinase synthesis (Wright and Tollon, 1979; Eon-Gerhardt et al., 1981b). Despite numerous studies performed at the transcriptional and translational levels (Laffler et al., 1981; Schedl et al., 1984; Green and Dove, 19881, the synthesis of tubulins has received limited attention as a cell cycle marker in Physarurn (Carrino and Laffler, 1985). The synthesis of the different tubulin isotypes (a1,az, pl, and p2)increased coordinately from mid-G2 phase to mitosis and decreased abruptly after mitosis (Laffler et al., 1981; Burland et al., 1983; Chang et al., 1983; Laffler, 1987). This transient increase of tubulin synthesis is under a set of regulations acting partly at the transcriptional level, but mainly at the translational level in Physarum plasmodia (Green and Dove, 1988). Furthermore, the tubulin messenger RN A (mRNA) (Green and Dove, 1988) and the tubulin molecule itself (Ducommun and Wright, 1988,1989) have been shown to be stable before mitosis but to have a short half-life during the postmitotic period (Green and Dove, 1988; Carrino and Laffler, 1985; Ducommun and Wright, 1988, 1989). However, it has not been shown, in Physarum, whether tubulin regulates its own synthesis, although this has been documented extensively in mammalian cells (Cleveland et al., 1981; Caron et al., 1985, 1986; Gay et al., 1987; Patcher et al., 1987; Yen et al., 1988; Gay et al., 1989).

Received April 16, 1990; accepted July 2, 1990. *To whom reprint requestskorrespondence should be addressed.

REGULATION OF TUBULIN SYNTHESIS DURING CELL CYCLE IN PHYSARUM POLYCEPHALUM

We previously investigated the effects of external perturbations on the cyclic variations of tubulin halflife during the cell cycle (Ducommun and Wright, 1989). In view of the other possible mechanisms regulating the quantity of tubulin, we focused our interest on the relationships between the syntheses of the different tubulin isotypes since they result from the combination of different regulatory mechanisms operating during transcription and translation (Green and Dove, 1988). As tubulin is stable in late G2 phase, i.e., during its period of synthesis, pulse incorporation of radioactive methionine could give some information about the variation of its rate of synthesis. The syntheses of isotubulins were useful as cell cycle markers because microtubule poisons such as methyl benzimidazole carbamate and griseofulvin indirectly perturb the synthesis of thymidine kinase (Eon-Gerhardt et al., 1981b), another protein that is also synthesized transiently during the late G2 period. The results described here, suggest that (1) the regulation of tubulin synthesis during the cell cycle involves the same regulatory pathway triggering thymidine kinase synthesis and the entry of nuclei into prophase stage; (2) the regulation of tubulin synthesis is independent of another regulatory pathway involving S phase and the entry of nuclei into metaphase; (3) the regulation of tubulin synthesis is more complex than previously assumed. In particular, we have shown for the first time, that, in the presence of the microtubule poison methyl benzimidazole carbarnate, the syntheses of the different tubulin isotypes apparently occur in an uncoordinated way. MATERIALS AND METHODS Plasmodia1 strain Microplasmodia were derived from CL amoebae (Wheals, 1970) and were grown at 22°C in 500 ml vials containing 150 ml liquid semidefined medium (Daniel and Rusch, 1961; Daniel and Baldwin, 1964) subjected to a rotatory shaking of 100 rpm. Mitotically synchronous plasmodia were prepared from shaken cultures essentially according to Guttes and Guttes (1964). Microplasmodia were concentrated by decantation, and 1 ml of the pellet was added in the center of a membrane which was placed at 22", 27", or 32°C on a metallic grid without medium in order to favor microplasmodial fusion (F). After 2 h of starvation, the membrane was placed on agar semidefined medium. Cellulose nitrate membranes were used in all cases (Sartorius 11306, 0.45-kM pore size), except for treatment with methyl benzimidazole carbamate where cellulose acetate membranes (Sartorius 11106, 0.45 p,M) were used (Eon-Gerhardt et al., 1981b). Mitosis was followed by phase-contrast observations of smears (Guttes and Guttes, 1964) mounted in 95% v/v ethanol/ glycerol. Determination of the relative synthesis of tubulin isoforms For each determination, 7 ~1 of water containing 25 p,Ci of 35S-labelled methionine (CEA, SMM-31, 800 Ci/mmole) was spread on a 7-mm circular plasmodia1 disk cut with a cork borer from the synchronous plasmodium. After an incubation for 1 h at growth

121

temperature in a humidified atmosphere, the plasmodial circle was kept frozen at -70°C. Preparation of extract and two-dimensional gel electrophoresis was performed as described by Laffler et al. (1981). An aliquot of the extract, corresponding to 400,000 cpm (20-100 pg of protein), was submitted to two-dimensional gel electrophoresis. Then gels were treated for 30 min with Amplify (Amersham), dried, and autoradiographed (film Kodak X-omat S) for 7-10 days, in order to locate the area of interest. The spots corresponding to the actin and the tubulin isoforms a,, aZ,and pz (the P1-isotype was not recovered due to its low abundancy) were cut out, incubated for 24 h at 60°C in 1 ml of 110% Hz02. Then, after addition of 10 ml Aquasure (NEN) and a 24-h incubation at 4"C, radioactivity was measured by the external standard ratio method. Approximately 10,000-15,000 cpm was recovered in the actin spot, while 300-400 cpm was measured in the al-and P2:tubulin spots. The amount of radioactivity determined using this method was proportional to the amount of radioactivity present in the gel. For example, increasing amounts of radioactive crude extracts, obtained after 35S-methionine incorporation into a plasmodium, were mixed with polyacrylamide before polymerization. This gel was subsequently treated as described by Laffler et al. (19811, and the amount of radioactivity was determined in 12.5-mm2 disks; 90, 160, 230, 325, 450, 480, 710, 730, and 850 cpm were measured in the presence of 2,5,10,15,20,25,30,35, and 40 p,l of radioactive extract in the gel, while 50 cpm were measured in the gel without any radioactive extract. Moreover, the amounts of radioactivity recovered in the actin, al-,a2-,and p,-tubulin spots were proportional to the quantities of protein resolved by two-dimensional gel electrophoresis. After incorporation of 35S-methionine in a synchronous plasmodium during late G2 phase, different amounts of radioactive crude extract (10,40, 60, and 80 ~ 1were ) analyzed by two-dimensional gel electrophoresis. Radioactivity determined in the spots corresponding to actin and isotubulin chains al,aZ,and p2 increased linearly with the amount of extract submitted to gel electrophoresis (actin: 380, 850, 1,230, and 1,350 cpm; a,-isotubulin: 150, 290, 470, and 470 cpm; ci2-isotubulin: 140, 210, 290, and 330 cpm; p,-tubulin: 150, 230, 330, and 400 cpm). Corrections for minor variations in sample loading and recovery were made by normalizing the radioactivity recovered in the al,a2,and P2 spots to the radioactivity recovered in the actin spot, which showed no systematic variations under the conditions used (Laffler et al., 1981). The relative synthesis of tubulin was calculated according to the following formula: Amount of radioactivity in the a,-,a2-,or.p,-tubulin spots corresponding to 4,000 cpm in the actin spot : 100

Moreover, determination of the variations of the rate of tubulin synthesis in a control plasmodium was performed in each set of experiments in order to check whether the absolute and relative incorporation of radioactive methionine in the actin and isotubulin spots el, cia, and pz were consistent with previous experiments. When griseofulvin and methyl benzimidazole carbamate were used, control experiments were carried out in the presence of 0.25 and 0.5% (vlv)

122

DUCOMMUN ET AL. TABLE 1. Pool of a,-and p-tubulin isotypes during metaphase according to growth temperature's2

Temperature Isotype ul-isotype pisotypes

22oc

27OC

29°C

32°C

0.30 (0.02) 0.25 (0.04)

0.25 (0.01) 0.24 (0.03)

0.25 (0.04)

0.26 (0.03) 0.29 (0.03)

Not done

'Thequantitiesoftubulinisotypes areindicatedas the percentageoftotalproteinin the extract (Ww/w). The al-isotype was quantitated with the monoclonal antibody

TPH4, while the 5-isotypeshave been quantitated with the monoclonal antibody KMX1, after monodimensional gel electrophoresisasdescribed under Materialsand Methods. *Numbers in parentheses are the standard deviation for at least three independent measurements.

Fig. 1. Variation of the relative rate of synthesis of tubulin at different temperatures. A: Autoradiography of a two-dimensional gel electrophoregram of a plasmodial extract labelled in vivo a t 32°C for 1h before metaphase with "S-methionine. The spots of interest that were subsequently cut out are shown with circles. A actin. al,a2,PI, and p,: al-, a,-, PI-, and p,-isotubulins. The pH value decreases from the left to the right, while the molecular weights decrease from the top to the bottom of the autoradiogram. B: Variation of the average relative rate of synthesis of all tubulin isotypes (normalized to the actin spot) a t 22°C (m), 27°C (el and 32°C (A),according to the period of the cell cycle. The third synchronous mitosis (M,) represents the third mitosis occurring after the formation of the synchronous plasmodia by fusion of asynchronous microplasmodia.

dimethyl sulfoxide, the solvent used to dissolve these drugs. Determination of the pool of tubulin The amount of tubulin was measured in crude extracts using a modification of the method of Fukuda and Iwata (1986), as described by Ducommun and Wright (1989). Schematically, unlabelled crude extracts were analyzed using one dimensional gel electrophoresis. Then polypeptides were electrotransferred to a nitrocellulose membrane and tubulin was reacted with tubulin specific antibodies which were quantitated with 1251-labelledsheep antibodies against mouse immunoglobulins. Absolute determinations of tubulin amounts were obtained using known quantities of purified amoeba1 tubulin in the same electrophoretic gel. Two monospecific monoclonal antibodies have been used. The antibody TPH4 recognized the a,-tubulin isoform only (Planques et al., 1989). The antibody KMXl recognized all p-tubulin isotypes, but to different extents, the P,-tubulin isotype being more strongly detected than the p,-tubulin isotype (Birkett et al., 1985).

RESULTS Effects of temperature shifts Determination of tubulin synthesis was first performed at different physiological temperatures during the cell cycle of plasmodia grown at 22", 27", and 32°C (Fig. 1).The rate of tubulin synthesis was estimated, after two-dimensional gel electrophoresis, as the incorporation of 35S-labelledmethionine in the al-, a,-,and p,-isotubulin spots relative to the incorporation measured in the actin spot, The maximum relative incorporation of radioactive methionine increased exponentially, in a coordinated fashion, with temperature in al, az,and p2 spots (Fig. 1).However, the pool of a- and p-tubulin isoforms did not vary significantly from 22" to 32°C (Table 11, and no tubulin degradation occurred during late interphase both at 22" and 32°C (Ducommun and Wright, 1989). The exact significance of this discrepancy is still not clear. Most experiments were performed at 22" and 32°C to monitor the influence of temperature. Although results were consistent in all of these experiments, the relative incorporation of 35S-labelledmethionine after a temperature shift was interpreted with caution and was used only as a qualitative assessment of tubulin synthesis. The onset of prophase in plasmodia was influenced greatly by an increase in temperature from 22°C to 32°C (Wri ht and Tollon, 1978): a temperature shift to 32"C, 2.5 before the corresponding metaphase in a control segment of the same plasmodium maintained at 22"C, delayed metaphase by 5.75 h. In the 22°C control plasmodium segment, tubulin synthesis has already been triggered by this time (Fig. 2). However, during the 3 h after the beginning of the treatment at 32"C,the apparent rate of S-methionine incorporation in the various tubulin isoforms diminished considerably. Incorporation of radioactive methionine in the tubulin isoforms then recovered slightly and was maintained at a reduced rate (relative to the rate of incorporation immediately prior to the treatment) until tubulin synthesis ceased at the onset of the delayed metaphase (Fig. 2). Effect of microtubule poisons Out of a variety of drugs active on tubulin, griseofulvin and methyl benzimidazole carbamate are the only members of the group which affect Physarum plasmodial microtubules in vivo (Wright et al., 1976). At the concentrations used to delay mitosis, griseoful-

a

REGULATION OF TUBULIN SYNTHESIS DURING CELL CYCLE IN PHYSARUM POLYCEPHALUM

$c 1

m/@\

Time Ihl

fi

A

b M j 122"CI

C°23'22 F HI

H2

H3( 32OC 1

M3122"CI 22Y

Time(h l

t

&

"C 1

32OC

t Fig. 2. Variation of the relative rate of synthesis of tubulin isotypes after a temperature shift from 22" to 32°C. Control mitosis [M,(22"C)I and mitosis occurring in the plasmodial part submitted to the temperature shift [M,(32"C)I are indicated with open and solid arrowheads, respectively. ., relative rate of synthesis of a,-isotype; A , relative rate of synthesis of a,-isotype; 0, relative rate of synthesis of &-isotype. At the beginning of the experiment (vertical arrow), 2.5 h before the third synchronous mitosis occurring at 22"C, i.e., when the synthesis of tubulin has already been triggered, a synchronous plasmodium was split in two parts. One part remained on agar medium at 22°C and provided control data for mitotic stages. The other part was the experimental plasmodium submitted to the temperature shift to 32°C. As expected from previous studies (Wright and Tollon, 19781, mitosis occurring in the plasmodial part submitted to the temperature shift to 32°C [M, (32"C)I was delayed and occurred 5.75 h after the control metaphase [M, (22"C)I. The scheme of the experiment is shown on the flowchart: F, end of the starvation period corresponding to the formation of the plasmodium via fusion of the microplamodia; M, successive synchronous mitoses (metaphases); the subscript numbers ( 1 3 )are used to indicate the successive mitoses relative to the fusion of the microplasmodia (MI to M,). The branching in the flow chart corresponds to the transfer of a plasmodial part to the new conditions as indicated on the right of the new branch (32"C, in this case). The crosshatched bar on the flow chart indicates the period during which tubulin synthesis was measured. The same symbols are used in Figures 3 to 6.

vin had no effect on RNA and protein syntheses, while methyl benzimidazole carbamate reduced RNA and protein syntheses by 15% (Eon-Gerhardt et al., 1981b). When a plasmodium was placed on agar medium with 0.2 mM griseofulvin in G2 phase, mitosis was delayed (Eon-Gerhardt et al., 1981b),but tubulin synthesis was not prevented and the kinetics of the relative incorporation of 35S-labelled methionine in al-,az-, and pztubulin spots were not modified (Fig. 3). However the relative incorporation of radioactive methionine in tubulin did not stop concomitantly with the mitosis occurring in the control plasmodial part (M3C) but increased until the occurrence of the delayed mitosis (M3T) in the treated plasmodial part. At this point, there was a coordinated decrease in the rates of methionine incorporation in all three tubulin isotypes. Similarly, when a plasmodium was placed on agar medium containing 0.1 mM methyl benzimidazole carbamate, mitosis was delayed (Eon-Gerhardt et al., 1981b) without any interference with the onset of tubulin synthesis (Fig. 4A). Incorporation of 35S-labelled methionine in tubulin ceased, not simultaneously with the onset of the control mitosis (M3C),but concomitantly with the delayed mitosis (M3&This also occurred in the plasmodium treated with griseofulvin (Fig. 3). The relative rate of incorporation of 35Smethionine in the three tubulin isotypes increased

123

M3c

FY

h T

Control "1

M3TGriseofulvin (TI

t

Fig. 3. Variation of the relative rate of synthesis of tubulin isotypes during treatment with griseofulvin. Control mitosis (M,,J occurring in the control plasmodial part (0.254 (v/v) dimethyl sulfoxide) is indicated with a n open arrowhead, while the delayed and extended mitosis (M3T) occurring in the presence of 200 pM griseofulvin is indicated with a solid horizontal bar. ., A, : relative rate of synthesis a2-,and p,-tubulin isotypes, respectively. At the beginning of of al-, the experiment (vertical arrow), 6 h before the control mitosis (MSc), a plasmodium grown at 27°C was split in two parts. One part was put on the agar medium with 0.25% (v/v)dimethyl sulfoxide and provided control data for mitotic stages. The other part was the experimental plasmodium exposed to 200 pM griseofulvin and 0.25% (v/v)dimethyl sulfoxide. As expected from previous studies (Eon-Cerhardt et al., 1981b), mitosis (MST) occurring in the presence of griseofulvin was delayed and occurred 2.5 h after the control metaphase (MS,-).

more than usual, reaching a value three times that of the control plasmodium. Moreover, in the presence of methyl benzimidazole carbamate, mitosis was not only delayed but the course of mitosis was abnormal. Nuclei were transiently blocked, 2.5-3.5 h, in an unusual mitotic stage consisting of condensed chromatin and multiple spindle poles (Planques et al., 1989). At the beginning of this period, the incorporation of radioactive methionine in the a2- and P2-tubulin isoforms decreased sharply to the background level (Fig. 4B), while its incorporation in the al-isotubulin decreased somewhat but remained near the maximum value observed in a control plasmodium until the completion of the treated and delayed mitosis (M3T).Despite the apparent increase of the incorporation of radioactive methionine in all tubulin isotypes in the presence of microtubule poisons, the relative pool of tubulin measured in metaphase showed no significant variation. The pool of tubulin was evaluated with the monoclonal antibody TPH4, specific for the al-tubulin isoform. The amount of al-isotype was slightly lower in the presence of griseofulvin and methyl benzimidazole carbamate (0.28 and 0.27 2 0.01% (w/w),respectively) than in the control plasmodium grown in the presence of 0.25-0.596 (v/v) dimethyl sulfoxide (0.35 2 0.03% w/w). Effect of hydroxyurea and fluorodeoxyuridine These two substances decreased the rate of DNA synthesis when treatment of the plasmodium began before S phase (Hildebrandt and Sauer, 19731, i.e., during mitosis or the G2 phase of the preceding cell cycle (there is no G1 phase in Physarum plasmodia (Braun et al., 1965)). In both cases, the first mitosis

DUCOMMUN ET AL.

124

\

'.

\

\

I I

A Fig. 4. Variation of the relative rate of synthesis of tubulin isotypes during treatment with methyl benzimidazole carbamate. Control mitosis (M3J occurring in the control plasmodial part (0.5% (vlv) dimethyl sulfoxide) is indicated with an open arrowhead, while the delayed and extended mitosis (M3T)occurring in the presence of 100 FM methyl benzimidazole carbamate (MBC) is indicated by a solid horizontal bar. 1,A, 0: relative rate of synthesis of q-, a2-and pztubulin isotypes, respectively. A At the beginning of the experiment (vertical arrow), 5.5 h before the control mitosis (M3& a plasmodium grown at 27°C was split in two parts. One part was put on agar medium with 0.25% (vh) dimethyl sulfoxide and provided control data for mitotic stages. The other part was the experimental plasmodium exposed to 100 pM methyl benzimidazole carbamate and 0.25%(vlv) dimethyl sulfoxide. As expected from previous studies (Eon-Gerhardt et al., 1981b), mitosis occurring in the presence of methyl benzimidazole carbamate (M3T) was delayed and occurred 2 h after control mitosis (M3& Moreover, mitosis perturbed with methyl benzimidazole carbamate was highly abnormal (Planques et al., 1989) and extended over a 2.5-h period. B The experiment is similar to the previous one, except that the variation of the rate of tubulin synthesis was recorded in the plasmodial part treated with methyl benzimida-

zole carbamate every 0.5 h after the third control metaphase (MSc). The plasmodium was exposed to methyl benzimidazole carbamate 3 h before the third control metaphase (M3,.). The treated mitosis (M3T) was delayed of 2.5 h and extended over a 3.3-h period. Each photograph corresponds to the autoradiogram of a two-dimensional gel electrophoregram of a plasmodial extract after in vivo labelling with 35S-methionine in the presence of methyl benzimidazole carbamate. A: actin. al,a2,pl, pz: al-,az-,PI-, and p,-isotubulins, respectively.

(MZT)after the beginning of the treatment occurred normally, i.e., concomitantly with the control mitosis (MZc).However, the mitosis following the perturbed S and G2 phases was delayed. Significantly, nuclei entered into a very early prophase stage and then remained in this stage for several hours (Wright and Tollon, 1988, 1989). During treatment with 10 mM hydroxyurea, metaphase was delayed but eventually occurred (Wright and Tollon, 1988) while, in the presence of 40 p,M fluorodeoxyuridine, nuclei remained blocked in early prophase for a t least 50 h (Wright and T o h n , 1989). All these effects were attributable to the action of hydroxyurea and fluorodeoxyuridine on DNA synthesis, since they were not observed after addition of the four deoxynucleosides or thymidine, respectively (Wright and Tollon, 1988, 1989). The incorporation of radioactive methionine during treatment with hydroxyurea showed a distinct and constant synthesis of the q-, aZ-,and Pz-tubulin isoforms during the entire metaphase delay (8h) but a t a rate below the maximum rate observed in a control plasmodium (Fig. 5 ) . More-

over, the synthesis of the different tubulin isoforms was stopped in a coordinated manner when nuclei completed mitosis (Fig. 5 ) . The relative amount of the tubulin a,-isotype pool during metaphase in the plasmodium treated with hydroxyurea (0.20 f 0.01% w/w) was not different from the value measured during metaphase in an untreated control plasmodium (0.25 f 0.03% w/w). Similar observations were made when DNA synthesis was slowed down with fluorodeoxyuridine. In this case, no decrease of tubulin synthesis occurred during a t least 10 h; nuclei never proceeded through metaphase during this period (data not shown). Effect of aphidicolin Aphidicolin is known to diminish DNA synthesis in Physarum and to inhibit DNA polymerase activities partially in crude extracts of Physarum plasmodia (Wright et al., 1981). When aphidicolin was applied 30 min before the second control mitosis (Mzc), as had been done with hydroxyurea and fluorodeoxyuridine,

Timelh)

REGULATION OF TUBULIN SYNTHESIS DURING CELL CYCLE IN PHYSAR U M POLYCEPHALUM

125

I

Tirnelh)

h M3c

n '3T

M1

M2C

M3c

'LC

,(,

MLC

HU IT1

t

Fig. 5. Variation of the relative rate of synthesis of tubulin isotypes during treatment with hydroxyurea. Control metaphases (MZc and M4c) occurring in the control plasmodial parts are indicated with open arrowheads. The delayed and extended mitosis (M,d occurring in the presence of 10 mM hydroxyurea (HU) is indicated by a solid horizontal bar. ,. A , 0: relative rate of synthesis of a l - , a2-,and p,-tubulin isotypes, respectively. At the beginning of the experiment, 3 h before the second synchronous metaphase (M2c), a plasmodium grown at 27°C was split in two parts. One part was put on agar medium in order to provide control data for mitotic stages, while the other part was exposed to 10 mM hydroxyurea. As expected from previous studies (Wright and Tollon, 1988), the first mitosis occurring in the presence of hydroxyurea (MZT)was not disturbed, while the second mitosis occurring in the presence of hydroxyurea (M3T)was delayed to 8 hand was asynchronous.

Time fhl

b M3c

l'

k 'l

2'

'3'

=

Control(C) Aphidlcolin (TI

t

Fig. 6. Variation of the relative rate of synthesis of tubulin isotypes during treatment with aphidicolin. Control metaphase (M3c) occurring in the control plasmodial part is indicated with an arrowhead. The delayed mitosis occurring in the presence of 200 pM aphidicolin is indicated by a solid horizontal bar. B, A ,. :relative rate of synthesis of a,? a2-, and P,-tubulin isotypes. At the beginning ofthe experiment (vertical arrow), 4 h before the third synchronous metaphase (M,,-), a plasmodium grown at 27°C was split in two parts. One part was put on agar medium with 0.5% Wv) dimethyl sulfoxide and provided control data for mitotic stages. The other part was exposed to 200 KM aphidicolin in 0.5%(vh) dimethyl sulfoxide. As expected from previous studies (Eon-Gerhardt et al., 1981a), mitosis occurring in the presence of aphidicolin (M,) was delayed and occurred 6 h after the control mitosis (M3c).

the nuclei entered an early prophase stage, and metaphase onset was delayed of several hours (EonGerhardt et al., 1981a). We observed an increase incorporation of 35S-methionine in tubulin in the treated plasmodium during the period defined by the two successive control mitoses M,c and M4C (data not shown). However, it has been demonstrated (Eon-

Fig. 7. Relative variation of the pool of tubulin during treatment with aphidicolin. At the beginning of the experiment (vertical arrow), 3.5 h before the third synchronous metaphase (M3J, a plasmodium grown at 27°C was exposed to 200 pM aphidicolin in 0.5% (viv) dimethyl sulfoxide. In the treated plasmodium, metaphase (M,) occurred 6 h after the control mitosis. The amount of a,-isotubulin was determined after monodimensional gel electrophoresis (see under Materials and Methods) and recorded as the amount of tubulin relative to the initial quantity of tubulin present at the beginning of the experiment.

Gerhardt et al., 1981a)that aphidicolin was also able to delay mitosis when the treatment was applied to a plasmodium in early or mid-G2 phase, i.e., at least 3 h before the following metaphase. In that case, the nuclei of the plasmodium treated with aphidicolin in G2 phase also entered an early prophase stage, and metaphase onset (M34 was delayed by several hours (Eon-Gerhardt et al., 1981a).Under these conditions (Fig. 61,the relative incorporation of 35S-labelledmethionine in a!-, a2,and P2-tubulin spots increased, as expected, in similarity with the control plasmodium. The incorporation of 35S-methionine in tubulin spots then diminished until the beginning of the control mitosis (M?c). However, a significant, although lower, incorporation of 35S-methionine was still observed in tubulin spots and stopped simultaneously with the onset of the delayed metaphase (M3T) in the treated plasmodial segment. The relative amount of the a,-tubulin isoform probed in the plasmodium treated with aphidicolin increased until the control plasmodial segment entered mitosis. The amount of a,-tubulin then remained constant until it increased at the initiation of the delayed metaphase (Fig. 7). DISCUSSION Tubulin synthesis occurs transiently in late G2 phase during the synchronous cell cycle of Physurum plasmodia as demonstrated by pulse labelling with radioactive methionine (Laffler et al., 1981). Tubulin synthesis begins to increase 4 h before metaphase and then stops abruptly after mitosis (Laffler et al., 1981). Although Physarum is unable to synthesize its own methionine (Daniel et al., 1963), variations of the endogenous methionine pool are likely to occur in some of the conditions used. Thus, incorporation of radioactive methionine into tubulin, as carried out by Laffler et al. (19811, was quantitatively determined relative to the incorporation into actin of radiolabelled methionine.

126

DUCOMMUN ET AL.

Actin synthesis has been reported to be constant throughout the cell cycle (Laffler et al., 1981). This study, in which tubulin synthesis has been investigated to define elements of the cell cycle inADhidicolin REGULATORY PATHWAY 11 volved in tubulin regulation, has provided two important conclusions. First, tubulin synthesis is regulated by those cell cycle pathways necessary for the entry of the cell into prophase that are sensitive to temperature shifts from 22"to 32°C. Temperature shifts applied in late G2 phase, once tubulin synthesis had begun, transiently reduced tubulin synthesis, which then resumed and continued until the onset of the delayed mitosis. A similar effect has been observed with thyridine midine kinase (Wright and Tollon, 1979). Our observaand tion is in agreement with descriptions of the effects of heat shock on tubulin synthesis (Carrino and Laffler, 1985), although the action of a temporary temperature increase to a nonpermissive temperature is more complicated in analysis than the effects of a temperature shift between two physiological temperatures, both permitting a normal cell cycle (Wright and Tollon, 1978). Second, tubulin synthesis, similar to that of thymidine kinase, is not regulated by the cell cycle pathway necessary for metaphase entry and which is perturbed by drugs preventing DNA synthesis (EonMethyl Gerhardt et al., 1981a; Wright and Tollon, 1988,1989). benzimidazde 'carbarnate and When S phase was prolonged by a treatment with griseofulvin hydroxyurea and fluorodeoxyuridine, tubulin synthesis was not prevented and occurred at a constant rate. Tubulin synthesis ceased only after the completion of the delayed metaphase. This also occurred in treatments with hydroxyurea. These conclusions demon\ .& I strate a similarity in the regulation between the increase of thymidine kinase activity and syntheses of REGULATORY PATHWAY I tubulin al,a2,and p2 isoforms. In all cases, synthesis was regulated by the cell cycle athway sensitive to temperature shifts and indepen ent of the pathway involving DNA synthesis (Fig. 8). Despite the strong similarities between the regulation of thymidine kinase and tubulin syntheses, other observations demonstrate that the regulation of tubu- Fig. 8. Schematic diagram of the regulatory pathways of some cell lin isotypes in Physarum could involve an autoregula- cycle events in Physarum plasmodia. The different stages of mitosis tory mechanism such as has been demonstrated in (VEP, P, M, A, and T) correspond to very early prophase, prophase, mammalian cells (Cleveland et al., 1981; Pittenger and metaphase, anaphase and telophase, respectively. According to preCleveland, 1985). Aphidicolin is a poison of DNA vious studies (Wright and Tollon, 1979),it has been assumed that the regulatory pathway (I) acts through a concentration dependent synthesis that is able to delay mitosis when the treat- first mechanism. Thus, this regulatory pathway can be stopped at any time ment is applied either before S phase or at least 3 h by submitting the plasmodium to a temperature shift between the two before metaphase. Although aphidicolin delays physiological temperatures 2242°C or to a transient heat shock from metaphase, it does not prevent either the entry of 27"C, the optimal temperature, to 37°C. By contrast, the second regulatory pathway (11) cannot be perturbed once DNA synthesis has nuclei into a very early prophase stage or the initiation been completed. Fluorodeoxyuridine and hydroxyurea act on S-phase of thymidine kinase synthesis (Eon-Gerhardt et al., only, while aphidicolin is supposed to act also on a possible discrete 1981a). This is also the case with other inhibitors of and latter event of DNA synthesis occurring during the G2 phase, 3 h DNA synthetic events. From these observations and before metaphase (Eon-Gerhardt et al., 1981a; Wright and Tollon, 1988, 1989).The pre-S-phase stage corresponds to a period of the cell the similarities between the regulation of tubulin and cycle, during which the replicating machinery, although ready, has thymidine kinase syntheses, one would expect that, in not yet been triggered (Brewer and Rusch, 1968; Wright and Tollon, the presence of aphidicolin, the triggering of tubulin 1978;Wolf et al., 1979).In this diagram, the two regulatory pathways (I and 11) are shown as operating independently for their larger part, synthesis would occur normally and would stop com- but this is not meant to imply that in an unperturbed cell cycle there pletely only after the completion of the delayed mitosis. is no coordinationbetween the two pathways. In contrast to a previous This is effectively what has been observed despite the model (Wright and Tollon, 1989),the involvement of tubulin has been differences of the relative rate of tubulin synthesis in assumed to imply an increase of the tubulin pool, while temperature shifts would possibly act indirectly or directly (dashed line) on the presence of aphidicolin and hydroxyurea. However, thymidine kinase synthesis. It is suggested that microtubule poisons in the presence of aphidicolin, the rate of synthesis (methyl benzimidazole carbamate and griseofulvin) act on tubulin increased more than usual during the first 3 h of the synthesis (autoregulation) and on thymidine kinase synthesis by treatment and then decreased to stabilize at a lower sequestering or decreasing the amount of active tubulin.

\

\t' I

B

REGULATION OF TUBULIN SYNTHESIS DURING CELL CYCLE IN PHYSARUM POLYCEPHALUM

rate throughout the mitotic delay. In agreement with these observations, the pool of the a,-tubulin isoform was constant during the entire mitotic delay. This observation could be understood if tubulin was able to regulate its own synthesis as occurs in mammalian cells (Cleveland et al., 1981; Pittenger and Cleveland, 1985). Two microtubule poisons, griseofulvin (Gull and Trinci, 1974) and methyl benzimidazole carbamate (Wright et al., 1976), are known to prevent the normal assembly of the plasmodial microtubules. In both cases, these microtubule poisons delay mitosis (Eon-Gerhardt et al., 1981b)and permit formation of abnormal tubulin assemblies such as macrotubules (Wright et al., 1976; Hebert et al., 1980) and tubulin paracrystals (Gull and Trinci, 1974). In contrast with the induction of thymidine kinase synthesis (which was delayed to the same extent as mitosis when plasmodia were treated with these two drugs; Eon-Gerhardt et al., 1981b), the triggering of tubulin synthesis was not perturbed and occurred about 4 h before the metaphase observed in the control plasmodial segment. In contrast to the action of aphidicolin, in the presence of microtubule poisons, the relative rate of tubulin synthesis increased until the appearance of nuclei exhibiting condensed chromatin. This observation could be explained if tubulin, being sequestered, stored in an inactive form, or possibly accumulated in the nuclear pool, was unable to allow the regulation of its own synthesis as in the case of treatment with aphidicolin. Although the triggering of thymidine kinase and tubulin syntheses were both perturbed by temperature shifts, only the triggering of thymidine kinase synthesis was perturbed with microtubule poisons. As discussed previously (Eon-Gerhardt et al., 1981b), it is unlikely that two chemically unrelated substances such as methyl benzimidazole carbamate and griseofulvin could both act on tubulin and on another independent target involved in the triggering of thymidine kinase synthesis. Thus, we hypothesize that, in the presence of either of these microtubule poisons, tubulin was sequestered or occurred in an inactive form that was unable to permit directly or indirectly the triggering of thymidine kinase synthesis (Fig. 8).Not only does methyl benzimidazole carbamate delay mitosis (Eon-Gerhart et al., 1981b1, but mitotic stages are abnormal (Planques et al., 1989). After prophase and chromatin condensation, the mitotic apparatus is blocked transiently during a 1-to 2-h period as a prominent aster surrounded with condensed chromosomes and several additional minor microtubule organizing centers. When plasmodia entered this abnormal mitotic stage, the rate of tubulin synthesis decreased abruptly. The rate of synthesis of a,- and p,-tubulin isotypes decreased t o the basal level, but the synthesis of the a,-tubulin isot pe decreased to a lesser extent. This uncoordinated be avior of the a,-tubulin isotype could be explained in the following way: the signal permitting the complete arrest of tubulin synthesis is clearly postmitotic as shown by the complete arrest of the al-tubulin isotype after completion of mitosis in the presence of methyl benzimidazole carbamate. This signal could correspond to the normal signal triggering the coordinated arrest of all isotubulin syntheses at the end of mitosis in a normal cell cycle.

K

127

Thus, the early arrest of az- and P,-tubulin isotype syntheses could result from the feedback regulation of tubulin on its own synthesis. However, in the presence of methyl benzimidazole carbamate, the regulation of the al-isotype could be less efficient, and its synthesis (Cleveland et al., 1981; Yen et al., 1988; Gay et al., 1989) or degradation (Burke et al., 1989) no longer coordinated with the other tubulin isotypes. To account for the results of our investigations, it has been hypothesized that two distinct signals lead to a partial or complete arrest of tubulin synthesis. The first signal would be usually cryptic and could act only when the rate of tubulin synthesis is higher than usual as is the case during treatment with aphidicolin or methyl benzimidazole carbamate. The second signal would act at the onset of the S phase occurring after mitosis has been completed during each cell cycle. Laffler (1987) has reported that the arrest of tubulin synthesis after mitosis could involve the presence of a diffusible negative factor which could turn off tubulin gene transcription during S phase. It is not yet clear how this hypothesis could account for the transient decrease of tubulin synthesis after heat shocks (Carrino and Laffler, 1985) and temperature shifts. Although the rate of tubulin gene transcription decreases after mitosis, transcription of the tubulin genes is still active (Green and Dove, 1988) suggesting that the large decrease of the level of tubulin mRNA after mitosis would result from its destabilization rather than to the absence of synthesis. These observations are consistent with the regulation of tubulin synthesis, which has been analysed at the molecular level in mammalian cells (Cleveland et al., 1981; Yen et al., 1988; Gay et al., 1989). Thus, it is not clear whether the two signals which apparently operate during the arrest of tubulin synthesis in Physarum plasmodia act by a distinct mechanism at the molecular level or are merely the result of the action of different factors on a common basic regulation.

ACKNOWLEDGMENTS Mrs. Y. Tollon and Dr. C. Birkett are gratefully acknowledged for their gifts of monoclonal antibodies. This work was supported by L'Association pour la Recherche sur le Cancer and La Ligue Nationale Frangaise contre le Cancer.

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