No growth cycle in Physarum?

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Experimental

Cell Research 135 (1981) 399-406

NO GROWTH

CYCLE

G. WEGENER’ ‘Institut

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Zoologie

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Unilvrsifiit,

D-6500 D-8700

IN PHYSARUM?

and H. W. SAUER2 Mainz,

Wiirzburg,

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Universittir.

Germany

SUMMARY The activities of a number of enzymes have been determined in growing plasmodia of Physururn polycephulum at 1 h intervals during the naturally synchronous nuclear division cycle. The enzymes selected represent the main pathways of energy metabolism, they do not require posttranslational steps for activation, nor are they directly involved in DNA replication, mitosis or differentiation. We found constant activity levels for all enzymes tested and discuss the lack of endogenous oscillations in the synthesis of the corresponding proteins with respect to the growthcycle concept.

The cell-cycle concept, originally proposed by Howard & Pelt [I] has formally combined the essential events that must take place in a cell after it has divided and before it can divide again: to duplicate its chromatin, to double in mass and to build up the mitotic apparatus (MA). Under norma1 conditions cell growth, DNA replication and mitosis (i.e. nuclear and cell division) are coupled, yielding the four consecutive stages mitosis, Gl, S, and G2 phase which make up the cell cycle. Cell-cycle studies on a wide variety of systems ranging from bacteria, over unicellular eukaryotic organisms, including yeast and Tetrahymena, to established tissue culture cells, have been conceptually summarized by Mitchison [2] and more recently by Prescott [3]. For biochemical work many individuals or cells have to be synchronized, and two different strategies have been developed for this purpose. (1) Selection synchrony, where cells are collected in a certain stage of their cycle by procedures which were

designed to minimize interference with ongoing growth processes; (2) induction synchrony, where a whole population of cells accumulates at a distinct point in the cell cycle, either before division (as in the heat shock procedure) or at division (after colchicine treatment), or before or at S phase (by treatment with excess thymidine or hydroxyurea respectively). Any of these procedures leads to cells that are synchronized with respect to some cell cycle events, yet none of these cell populations is undergoing undisturbed and balanced growth any more. Mitchison has expanded an earlier conclusion reached by Swann [4] that growth can continue even if a cell does not synthesize its DNA. He has formalized this obvious uncoupling of cell cycle phenomena in terms of two cycles that constitute the classical cell cycle concept [2, 51. In his view there is the DNA-division cycle (DDC) that takes care of DNA replication and mitosis, and a separate growth cycle, supposedly another cyclic series of events

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that run parallel to the DDC, yet are cycle interval, as defined by the DDC [5]. coupled to it. They served as landmarks and were defined In the meantime there is firm evidence as growth cycle markers when the abrupt of the interdependence of several events change in activity took place, even if the in the DDC, particularly in the yeast sys- DDC had been experimentally halted. Howtems where genetic analyses have revealed ever, it is implicit in the hypothesis of the a number of cdc genes (ceil divisions cycle), growth cycle, as defined by Mitchison [2, which must function in the correct order in 51, that its markers show up in artificially order to drive a cell of the budding yeast synchronized cells which are no longer in [6] or the fission yeast [7] through their balanced growth and lose synchrony within respective DDC. In both systems two check the following DDC. Therefore, the handling points or transition points have been de- of cell populations rather than a growth fined, one in Gl phase (the start) and one in cycle could have caused the enzyme G2 phase, although in the fission yeast the changes observed. The plasmodia of Phyfirst may be hidden, i.e. it becomes cryptic sarum, with their naturally synchronous under certain conditions [7]. In many other mitotic nuclear divisions, might be an adesystems these two transition points have quate experimental system to probe the also been described (see [3], for review). growth cycle concept in an undisturbed in Physarum, plasThere are also numerous examples where a organism. Moreover, modia differentiation is strictly correlated Gl phase may be absent [3], e.g. cleavage division in early embryos, rapid divisions in with starvation but not with growth [ 10, 111. some plant meristems, and in well-fed uniWe have chosen to analyse several enzymes of the energy metabolism which are cellular organisms, like amoebae, and of course the true slime mold Physarum (see clearly not involved in the DNA-division [8], for a recent review). Even in these cycle nor in differentiation and which are cases, cell proliferation can still be well de- present in non-limiting concentrations durscribed by the cell cycle terminology as ing a naturally synchronous mitotic cycle. Furthermore, all the selected enzymes obbeing Gl (-). Some years ago it was questioned wheth- viously do not need activation steps after er cells cycle at all and some evidence for synthesis, and they are stable even in crude a random event at a point similar to the extracts for at least 6 h at 0°C. Therefore, transition point in Gl phase in the con- it can be assumed that a positive and strong ventional concept has been postulated [9]. correlation exists between activity and enEven in this hypothesis, a discontinuity zyme protein concentration in these rapidly during the lifetime of a cell (a transition growing cultures, and any abrupt changes point) must occur after which it has be- would provide evidence for the expression of the respective structural gene and supcome committed to divide. The growth cycle concept was based on port the growth cycle concept. the observation that many parameters, seemingly unrelated to the DDC-such as MATERIALS AND METHODS most enzymes studied in synchronized Physurum (strain M&,,) was grown as a suspension cells, two-thirds of about 130 enzymes microplasmodia in semidefined medium and macroanalysed by 1973-showed an activity in- ofplasmodia were prepared as surface cultures accordcrease at predictable time points along a cell ing to standard procedures [12. 131. At 26°C the gen-

No growth eration time was 9 h + 25 min and nuclei throughout the plasmodium passed through metaphase within 10 min. Most experiments were done between the second and the third post-fusion mitosis and four individual plasmodia were analysed (discarding the inocula) at each point at 3-h intervals. In a second series, four synchronous plasmodia were used and from each of them an aliquot was taken at 1-h intervals. The material was weighed, washed in cold Na-Kphosphate buffer (20 mM, pH 7.3) and homogenized by sonication in 10 vol of the same buffer. By centrifugation at 30000 g for 30 min the homogenate was separated into a supematant and a pellet fraction, the latter was extracted again and after centrifugation the two supematants were pooled. The following enzymes representing the main pathways of energy metabolism were tested in both fractions emolovine established ODtical tests [14, 151. (1) Hexokinase (HK. EC 2.7.1.1) vieldine the catalytic capacity of glucose phosphorylation; (2yphosphoglucomutase (PGM, EC 2.7.5.1) which balances the levels of glucose phosphates and therefore contributes to the metabolic pathways of glycogen synthesis and degradation alike; (3) glucose-6-phosphate dehydrogenase (G-6-PDH, EC 1.1. I .49) of the pentose phosphate pathway, yielding pentoses and reduced NADP for synthetic processes; (4) pyruvate kinase (PK, EC 2.7.1.40) of glycolysis; (5) lactate dehydrogenase (LDH, EC 1.1.1.27) of the anaerobic glycolysis; (6) glyceraldehydephosphate dehydrogenase (GAPDH, EC 1.2.1.12) acting in both glycolysis and gluconeogenesis; (7) citrate synthase (CS, EC 4.1.3.7) of the Krebs cycle; (8) 3-hydroxyacyl-CoA dehydrogenase (HOADH, EC 1.1.1.35) of the P-oxidation of fatty acids; (9) glutamate dehydrogenase (GluDH, EC I .4.1.3) of the amino acid metabolism. The enzyme activities remaining in the pellet fraction were negligible for all enzymes tested. The enzyme activities were stable for more than 6 h in homogenates and supernatants kept at 0°C under the chosen conditions. In control experiments it was shown that the intracellular compartmentation of these enzvmes is the same as in other eukaryotic systems by the differential extraction procedure described bv Pette r161. Therefore, we assume that all enzymes analysed are fully represented in the suuematant used for the enzyme assays. The enzyme activities, measured at 2X, are expressed in U (umolelmin) and based on protein, determined by Lowry’s method [17], and wet weight. It had previously been shown that total protein increased continuously and doubles completely during one cell cycle under optimal growth conditions

1181.

RESULTS

AND

DISCUSSION

As can be seen in fig. 1, none of the nine enzyme activities (GluDH is not shown) changes significantly during the mitotic cycle of Physarum. Therefore, not one of these enzymes can be a step enzyme,

cycle in Physarum

401

since, at the chosen points of the mitotic cycle, the low enzyme level before and the high level after the step would have been detected. Six of the enzymes are located in the cytoplasm. Our results on glucose-6phosphate dehydrogenase confirm the earlier observation of a steady level of that enzyme in Physurum [ 191. This is also seen in selection synchronized mouse neoplastic mast cells [20], as well as in colcemidtreated and mechanically collected rat hepatoma cells [21], whereas synchronization by colcemid and brief trypsinization revealed three peaks in Chinese hamster ovary cells [22]. In the latter system LDH has also been detected as three peaks, but again, in selection synchronized mouse cells this enzyme was shown to be continuous [20]. These authors stress their observation that discontinuities of the cell cycle may disappear after changing from induction to selection methods for synchronization. In Physarum a continuous level of LDH activity is observed. The possibility that discontinuous synthesis of different isoenzymes becomes averaged as a continuous pattern could be excluded, since the Physarum enzyme was not separable into isoenzymes on polyacrylamide gels. Therefore, the steady activity level is assumed to reflect continuous synthesis during growth. However, a slight increase in specific activity is observed as the plasmodium gets older. Our attempts to induce the enzyme under either hypoxic conditions or by supplying its substrate to the medium has been unsuccessful, as reported in a related study [23]. The increase in specific activity of LDH is obviously related to starvation or ageing of Physnrum. This is even more obvious for glutamate dehydrogenase (GluDH), for which a massive increase in activity during starvation has been proven as de novo synthesis by E-rr, Cell

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Ulmg

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protein

inoculum has not been included in plasmodial samples used in this study. In a mouse cell line (P815) this enzyme has been described as a step after selection synchrony [20]. Two other enzymes, hexokinase and glyceraldehydephosphate dehydrogenase. which are linear in Physarum have been described as step enzymes in yeast after induction synchrony by X-rays [26]. The two mitochondrial enzymes of fig. 1h, citrate synthase of the citrate cycle and 3hydroxyacyl-CoA dehydrogenase, involved in the P-oxidation of fatty acids, are also linear enzymes in the mitotic cycle of

a I

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Fig. I. Some enzyme activities during the mitotic cycle of Physarum. Enzyme activities (U/mg protein, 25°C) were determined at 3 h intervals between the 2nd and 3rd post-fusion mitosis (M II-M III). Mean values, together with the S.D. of four independent assays performed with extracts from individual plasmodia are given. In (a) cytosolic enzymes involved in the metabolism of glucose are gathered; in (b) two mitochondrial enzymes and the G-6-PDH of the pentose phosphate pathway are shown,

density labelling [24]. An earlier claim of a ‘step’ of this enzyme in the midcycle of Physarum [25], which we could not confirm, is now interpreted as an enzyme increase in the ageing and slime-producing center (the inoculum) of a macroplasmodium (results not shown, and Htittermann, personal communication). Therefore, the

Physarum.

Since sharp enzyme peaks, though unlikely for the chosen abundant household enzymes, could have escaped detection in the 3 h intervals of fig. 1 a, b, the linear pattern has been confirmed at hourly intervals in a second series of experiments (see Materials and Methods). As an example the results are given only for one enzyme, glyceraldehydephosphate dehydrogenase (fig. 2). As the enzyme activities reflect catalytic capacities, the metabolic organization of a tissue or an organism can be derived therefrom. In this respect Physarum shows only a small degree of metabolic specialization. All the metabolic pathways tested are well represented and the difference between the enzymes with highest and lowest activities is by a factor of ten or less. In other systems analysed so far, like protozoa 1271, yeast [28], animal tissues [15, 28, 291, the differences between enzyme activities are far more prominent, reaching factors of lo*-103. The high activity of phosphoglucomutase underlines the importance of glycogen for this organism. Compared with other systems the glucose 6-phosphate dehydrogenase activity is relatively high,

No gao\c-th cycle in Physavum U/g

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Fig. 2. Activity of glyceraldehyde dehydrogenase during the mitotic cycle of Physarum(in U/g wet weight at 25°C). Aliquots were taken between the second and third post-fusion mitosis (M II-M III) at I h intervals

from each of four synchronous cultures (marked by different symbols); the mean values of the four cultures are connected by a line. All the other enzymes gave virtually the same results.

which accords with the need of synthetic power in growing cultures. A more comprehensive account of the metabolic organization and regulation of energy metabolism of Physarum will be given elsewhere. In summary, the Physarum enzymes tested in this paper and several others (summarized by Huttermann [23]) as well as RNA-polymerases [30] do not change during the undisturbed mitotic cycle, and wherever changes occur in the shape of a step, they seem to be correlated with starvation, bringing the plasmodium off balance and lengthening the cell cycle as well. Other investigations of Physarum which compare the pattern of proteins derived from nuclei [31] and the cytoplasm [32] during the mitotic cycle again showed correlation with growth conditions, but not with the cell cycle. A similar observation has recently also been reported in the fission yeast system (Schizosaccllnromgces pombe) by Mitchison, where a decisive control experiment has clearly shown that many enzymes display steps even in a seemingly undisturbed

population after gentle centrifugation, irrespective of their cell cycle phase [33]. In addition, very many pulse-labelled proteins in bacteria [34] and yeast [35], as revealed by two-dimensional gel electrophoresis (5-700 spots), seem to be synthesized continuously throughout the cell cycle. It now seems that in Physarum, as also perhaps in other balanced proliferating cell populations, there are no cyclic changes in enzyme activity or protein synthesis which might serve as landmarks for the growth cycle. The observed discontinuities could rather be explained as resulting from interference with the cell cycle and different activity patterns may indicate a different sensitivity of the respective organism or cell population to its treatment. Furthermore, it can be argued, from the most coherent model of mitotic cycle control in Physarum, that neither is there any need for it conceptually. In this model, also called the nuclear sites titration model [36], it is assumed that growth is monitored by continuous accumulation-in itself a steady

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state of both continuous synthesis and deg- lational modification (possibly a dephosradation-f a mitotic stimulator substance phorylation) [43, 441, while the enzyme which titrates a fixed number of nuclear peak, though a good time marker for mitosites, thus triggering mitosis. The disconsis, is not essential for S phase, as 24 TKtinuous event causing the nuclei in the mutants of the structural genes grow well plasmodium to cycle is the postulated dou- with less than 10% of the normal enzyme bling of nuclear receptor sites, yet no comlevel [45’]. The histone kinase activity ponent associated with growth. change is also due to an activation of preHowever, cyclic changes in enzyme ac- existing enzyme [46]. These observations tivity have been noted, particularly in gly- on bona fide DDC enzymes in Physarum, colysis [37], which are not related to together with the theory of peak enzymes changes in enzyme synthesis, but obviously being the consequence of either transcripreflect allosteric regulation of metabolic tion of the gene [47] or oscillatory dereprespathways. They were best analysed in ex- sion [48], may invite a different interpretatracts and have a brief period of about a tjon: swift enzyme activity changes are realminute or two. There has been a report of ly enzyme activation and inactivation that cycling glycolysis in Physarum [38]; yet are not regulated on the genome level exit turned out to be an artifact caused by clusively. This interpretation could also exthe superposition of a true endogenous plain the two step peaks of guanylate oscillator in Physarum: the shuttle streamcyclase observed in S phase and G2 phase ing with a l-3 min period. This phenomin the Physarum mitotic cycle [49]. enon has been closely analysed [39]. To On the other hand, a steady activity level date, the oscillator is not known, but it as for those examples of stable enzymes has not been correlated with growth or the chosen in this study, does not need to re8-10 h mitotic cycle in Physarum. Furtherflect constant transcription of the respecmore, the key enzyme for glycolytic osciltive genes either. If so, one might expect lations, the phosphofructokinase (PFK) of an increase in enzyme activity after the Physarum, cannot be modulated in its ac- structural gene has become doubled. Howtivity as in other systems (unpublished re- ever, a gene dosage effect, indicative of sults). Such is also the case for the cellular continuous-linear synthesis in the termislime mold Dictyostelium [40]. nology of Mitchison [2, 5, 501, has not been seen in our analysis, nor in any other enThis leaves us with some peak enzymes in Physarum which have been assigned zyme of Physarum. It seems, therefore, positions in the DNA division cycle (DDC). that most of the enzymes of various meTwo examples for peak enzymes are thymitabolic pathways, and even those cyclic dine kinase [19] which correlates well with changes which occur once per cycle are S phase, and histone kinase (phosphorylatcontrolled beyond transcription of the reing H,-histone) which increases IO-fold in spective genes. However, there are several activity in the mid-cell cycle [41, 421 and examples of an increase in enzyme activmay be involved in preparation for chro- ity, mostly by more than by a factor of two mosome condensation. It is now clear that (as would be expected as a consequence the thymidine kinase gene is transcribed at of gene dosage) which might be due to about 1.5 h before mitosis, and that the de nova synthesis (as judged from inhibitor activity peak in S phase follows post-transeffects). Of these enzymes, ODC (ornithine Exp

Cell

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No gro+z?th cycle in Physarum decarboxylase), an enzyme involved in polyamine synthesis [5 I], NAD pyrophosphorylase [52], and poly ADP ribose polymerase (possibly involved in post-translational modifications of nuclear proteins [53]) display increases in activity in S phase, whereas an RNase shows a step just after S phase [54]. In addition, a high level of DNA polymerase [55] and RNA polymerase B [56], at least in nuclear preparations parallels S phase in the Physarum mitotic cycle. It can be speculated that these changes in enzyme activity are examples of replication-transcription-coupling, a phenomenon previously described on the transcription level in the mitotic cycle of Physarum [56581. Taken together with the well established temporal order of DNA replication [59] it may turn out that some structural genes are expressed in a definite temporal sequence during the nuclear division cycle of Physurum. This argument might also apply to the observed ordered expression of several enzymes in the budding yeast studied by Halvorson and co-workers [60, 611. At any rate, such a hypothesis would stress the interdependence of DNA replication, an event typical of the DNA-division cycle (DDC), with gene expression, leaving no evidence of a separate cycle of oscillating gene regulation as originally proposed in the cell cycle concept. We are indebted to Michael Koepsell for skilful help with some of the experiments. This work was supported by Deutsche Forschungsgemeinschaft, Bonn.

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