Biological oxidation and the mobilization of mitochondrial calcium during the differentiation of Physarum polycephalum

We have previously reported that calcium is required for the starvation-induced differentiation of the slime mold, Physarum polycephalum. With the exception of calcium, each component of the complex starvation medium may be withheld and the organism will still differentiate into spherules. The results of the present study reveal that spherulation will proceed normally when the microplasmoidal cells are transferred from nutrient medium to a citrate buffer containing only 8 mM CaCl2. Electron microscopy and X-ray microprobe analysis reveal that there is an initial increase in the population of calcium-containing mitochondrial granules when the microplasmodia are induced to differentiate. However, as differentiation proceeds, these granules decrease in number and are virtually absent from the mitochondria of mature spherules. The accumulation and depletion of calcium-containing granules is not observed in a nondifferentiating strain of Physarum cultured under standard conditions, but is observed when this strain is first treated with a calcium-enriched nutrient medium that conditions it for spherulation. Changes in the cellular concentrations of NADH and lipid peroxides, and in the activity of superoxide dismutase, correspond temporally to the pattern of increase and depletion of the calcium-containing inclusions. The oxidative stress associated with starvation-induced spherulation may be a consequence of the active accumulation of calcium; the mobilization of this calcium may then be the event that initiates differentiation.     

Alterations in superoxide dismutase, glutathione, and peroxides in the plasmodial slime mold Physarum polycephalum during differentiation

Changes in the level of antioxidant defenses and the concentration of free radical by-products were examined in differentiating (M3cVII and LU897 X LU863), non-differentiating (LU887 X LU897), and heterokaryon microplasmodia of the slime mold Physarum polycephalum during spherulation in salts-only medium. As differentiation proceeded, superoxide dismutase activity increased by as much as 46 fold; glutathione concentration and the rate of oxygen consumption decreased; cyanide-resistant respiration, hydrogen peroxide, and organic peroxide concentrations increased. The non-differentiating culture failed to exhibit any of these changes. A heterokaryon obtained by the fusion of differentiating and non-differentiating strains was observed to differentiate at a very retarded rate and to exhibit the changes observed in the spherulating strains at a correspondingly slower rate. These observations suggest that a free radical mechanism may be involved in the differentiation of Physarum microplasmodia into spherules.     

Effects of the Free Radical Generator Paraquat On Differentiation, Superoxide Dismutase, Glutathione and Inorganic Peroxides In Microplasmodia of Physarum Polycephalum

Abstract. The herbicide paraquat was used to investigate the effects of oxidative stress on the spherulation of Physarum polycephalum microplasmodia. the responses of a white non-differentiating strain of Physarum were compared with those of a common yellow strain that readily spherulates in salts-only starvation medium. the addition of paraquat to the salts medium increased the specific activity of superoxide dismutase in both strains; it also induced an increase in the intracellular inorganic peroxide concentration in both strains. Glutathione concentration was higher in the paraquat-treated yellow strain than in the controls. Paraquat had no effect on glutathione concentration in white microplasmodia. Paraquat accelerated spherulation in yellow microplasmodia. the white microplasmodia responded to the herbicide by cleaving into structures similar to immature spherules; however, these structures were not viable. the results of this study support the hypothesis that free radicals are involved in cell state transitions.     

Effects of the free radical generator paraquat on differentiation, superoxide dismutase, glutathione and inorganic peroxides in microplasmodia of Physarum polycephalum

The herbicide paraquat was used to investigate the effects of oxidative stress on the spherulation of Physarum polycephalum microplasmodia. The responses of a white non-differentiating strain of Physarum were compared with those of a common yellow strain that readily spherulates in salts-only starvation medium. The addition of paraquat to the salts medium increased the specific activity of superoxide dismutase in both strains; it also induced an increase in the intracellular inorganic peroxide concentration in both strains. Glutathione concentration was higher in the paraquat-treated yellow strain than in the controls. Paraquat had no effect on glutathione concentration in white microplasmodia. Paraquat accelerated spherulation in yellow microplasmodia. The white microplasmodia responded to the herbicide by cleaving into structures similar to immature spherules; however, these structures were not viable. The results of this study support the hypothesis that free radicals are involved in cell state transitions.     

Involvement of Glutathione in the Differentiation of the Slime Mold Physarum polycephalum

The effects of experimentally-altered glutathione concentration on differentiation of the slime mold, Physarum polycephalum were examined. Spherulation was induced by transfer of Physarum from growth medium to a salts-only starvation medium. As differentiation proceeded, superoxide dismutase (SOD) activity in control cultures increased by as much as 21-fold. This increase in SOD activity paralleled the rate of differentiation. Glutathione (GSH) concentration decreased during differentiation by more than 80% in all cultures, regardless of the initial concentration. The rate of differentiation was inversely related to the initial GSH concentration and directly proportional to the SOD activity. These observations suggest that a free radical mechanism may be involved in the differentiation of Physarum microplasmodia into spherules.     

Superoxide dismutase activity and glutathione concentration during the calcium-induced differentiation of Physarum polycephalum microplasmodia

Microplasmodia of Physarum polycephalum differentiate into spherules when the CaCl2 concentration of their nutrient medium is increased to 54mM (high-calcium). The salts starvation medium routinely used to induce differentiation contains 8mM CaCl2. This medium will not induce spherulation in the absence of a calcium salt; no other metal is essential. High-calcium also induces the spherulation of a strain of Physarum that had not been previously observed to spherulate. The striking increase in superoxide dismutase activity (SOD) and the decrease in glutathione concentration (GSH) that are characteristic of salts-induced spherulation do not occur in salts media containing high-calcium. In the absence of calcium, no significant change in SOD is observed and very little change in GSH occurs. The immediate effect of the oxidative stress associated with spherulation may be the release of calcium stores into the cytosol. The parameters modulating this stress are, in turn, sensitive to exogenous calcium concentrations.     

Identification of mRNA in the slime mold Physarum polycephalum.

Using a differential extraction procedure which had previously been shown to yield one nucleic acid fraction enriched in cytoplasmic RNA and another enriched in nuclear RNA, we have been able to isolate two polyadenylated RNA populations from microplasmodia of Physarum polycephalum. The poly(A)-containing RNA from the cytoplasmic-enriched fraction accounts for approximately 1.2% of the cytoplasmic nucleic acid, has a number-average nucleotide size of 1339+/- 39 nucleotides, and has been shown, in a protein-synthesizing system in vitro, to be capable of directing the synthesis of peptides which have also been shown to be synthesized in vivo by microplasmodia. The poly(A)-containing RNA from the nuclear-enriched fraction has a number-average nucleotide size of 1533 +/- 104 nucleotides and represents a mixture of cytoplasmic and nuclear adenylated RNA molecules. Based upon these observations, we have identified the polyadenylated RNA isolated from the fraction enriched in cytoplasmic nuclei acid as Physarum poly(A)-containing messenger RNA.     

Differentiation response of Physarum polycephalum to macrocysts at various times in nuclear cycle

Macrocyst (spherule) formation was induced in synchronized suspension cultures of microplasmodia of Physarum polycephalum under conditions where DNA synthesis was inhibited. Plasmodia in early G2 phase of nuclear cycle were able to differentiate to spherules in the presence of an inhibitor of DNA synthesis, whereas those in late G2 phase required another round of DNA replication before they could enter into the spherulation process. These facts suggest that commitment to DNA synthesis occurred about halfway through G2 phase. The idea was also supported by the results of autoradiographic study in which spherulating plasmodia were fed with radioactive thymidine and labelled plasmodia were scored at the terminal differentiation stage.     

Uridine diphosphoglucose pyrophosphorylase activity and differentiation in the cellular slime mold Physarum polycephaluno

The specific activity of uridine 5'-triphosphate:alpha-d-glucose 1-phosphate uridyltransferase (EC 2.7.7.9) (also called uridine 5'-diphosphate [UDP]-glucose pyrophosphorylase) has been found to increase up to eightfold during spherule formation by the slime mold Physarum polycephalum. The enzyme accumulates during the first 8 to 9 h after initiation of spherule formation, declines to basal levels found in vegetative microplasmodia by 15 h, and is undetectable in completed spherules. Specific activities for UDP-glucose pyrophosphorylase in vegetative microplasmodia range from 15 to 30 nmol of UDP-glucose formed per min per mg of protein, whereas accumulated levels during spherule formation can attain a specific activity as high as 125 nmol of UDP-glucose formed per min per mg of protein. The scheduling and extent of accumulation are critically dependent on an early log-phase age of microplasmodia originally induced to form spherules. Spherule induction by 0.2 M or 0.5 M mannitol delays this schedule in a variable and unpredictable manner. Spherule-forming microplasmodia which have accumulated high levels of UDP-glucose pyrophosphorylase spontaneously excrete the enzyme when transferred to salts medium containing 0.2 M or 0.5 M mannitol. The excreted enzyme is subsequently destroyed or inactivated. Studies with preferential inhibitors of macromolecular synthesis indicate that accumulation of UDP-glucose pyrophosphorylase requires concomitant protein synthesis and prior ribonucleic acid synthesis.     

Purification and partial characterization of transglutaminase from Physarum polycephalum.

An intracellular form of calcium ion-dependent transglutaminase (R-glutaminylpeptide:amine gamma-glutaminyltransferase, EC 2.3.2.13) was purified 818-fold to apparent homogeneity from acetone powder preparations of spherules of the acellular slime mold Physarum polycephalum. The enzyme was purified by combined methods of precipitation with 15% (wt/vol) polyethylene glycol, DEAE-cellulose chromatography, and isoelectric focusing in a pH 5 to 7 gradient. The isoelectric point of the enzyme was 6.1. The molecular mass of the denatured enzyme was estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be 39.6 kDa. A molecular weight of 77,000 was found by gel filtration of the native enzyme on a Superose 12 fast protein liquid chromatography column, indicating that the native functional protein is a dimer. The purified transglutaminase catalyzed the incorporation of [14C]putrescine into protein substrates including casein, N,N'-dimethylcasein, actin purified from P. polycephalum, and actin purified from bovine muscle. Actin was the preferred substrate for the enzyme, both as a purified protein and in crude extracts prepared from P. polycephalum. With N,N'-dimethylcasein as the amine acceptor substrate, [14C]putrescine, [14C]spermidine, and [14C]spermine were all effective amine donor substrates with Km values of 49, 21.4, and 31.7 microM, respectively. All three of these polyamines demonstrated strong substrate inhibition of the enzyme activity between 100 and 200 microM. Upon starvation induced by depletion of a carbon source for growth, the specific activity of this enzyme increased sixfold during the differentiation of P. polycephalum microplasmodia to spherules. This suggests a role for transglutaminase in the construction of spherules, which have the capacity to survive starvation and dessication.     

Studies on microplasmodia of Physarum polycephalum. VII. Adhesion-dependent changes in the organization of the fibrillar actin system

Axenically-grown microplasmodia of the acellular slime mold Physarum polycephalum were used to study adhesion-dependent changes in the spatial organization of the cytoplasmic microfilament system. Results obtained by light- and electron microscopical techniques demonstrate the presence of a membrane-bound filament cortex in all microplasmodia, and the expression of additional cytoplasmic fibrils in specimens with tight contact to a substratum. The fibrils partly terminate in focal adhesion-sites and rather seem to serve a cytoskeletal than a contractile function.     

Calcium and malate are sporulation-promoting factors of Physarum polycephalum

Fruiting body formation (sporulation) is a distinctive, irreversible differentiation process in the life cycle of the slime mold Physarum polycephalum. The most important requirement for sporulation of Physarum is a period of starvation, and normally sporulation proceeds in the light. It is shown here that by omitting the liquid sporulation medium and elevating the temperature from 21 to 25 degrees C, sporulation can occur routinely in the dark. It is further shown that this autocrine signaling in the dark requires calcium ions and malate. A putative sporulation control factor was detected in conditioned media derived from plasmodia starved in the dark, which was then identified as polymalate. As an additional role for this previously detected polyanion, specific for the plasmodial state of Physarum, it is suggested that the secreted compound serves as a source for both malate and calcium ions and thus promotes sporulation without light signaling.     

Calcium binding sites in plasmodia of Physarum polycephalum as revealed by the pyroantimonate technique

Plasmodia of the acellular slime mold, Physarum polycephalum, were treated with an osmium tetroxide fixative containing potassium pyroantimonate to precipitate calcium and thereby localize calcium binding sites and sites of increased calcium concentration. Dense calcium pyroantimonate precipitates were detected within the nucleoli. The distribution of these precipitates during interphase and mitosis coincides with the distribution of the unique minichromosomes in Physarum, i.e., the numerous short pieces of extrachromosomal nucleolar chromatin containing segments of amplified DNA coding for ribosomal RNA. Calcium pyroantimonate precipitates were present as frequent dense granules in the mitochondrial matrix and as fine precipitates in the mitochondrial nucleoid. Large calcium-containing precipitates were seen within cytoplasmic vacuoles, confirming reports by others. In addition, we have identified calcium binding sites along the cytoplasmic surface of the plasma membrane. The distribution of calcium within the plasmodium is discussed in relation to the assembly of the mitotic spindle and the regulation of cell motility.     

A Fine Structure Study of Physarum polycephalum During Transformation from Sclerotium to Plasmodium: A Six-Stage Description

ABSTRACT. Physarum polycephalum is classified presently as a sarcodinid in the class Eumycetozoea. It produces a sclerotial dormant stage consisting of a crustose deposit containing nucleated spherules of cytoplasm enclosed within a honey-comb-like matrix of organic walls. When rehydrated, the sclerotium reverts to a plasmodium: 1) the spherules become increasingly vacuolated, 2) electron-dense granules become dispersed within expanding vacuoles, and 3) pseudopodial extensions develop from the periphery of the spherule cytoplasm, penetrating the fragmenting walls, and making interconnections with surrounding spherules, eventually leading to a fully reticulated plasmodium. Six stages are identified during reversion from sclerotium to plasmodium in laboratory cultures, and their successive appearance was mapped over time. The six stages are: 1) sclerotial stage with crenulated nuclei, 2) cytoplasmic activation with smooth nuclear envelopes, 3) initiation of pseudopodial protrusions, 4) pseudopodial penetration into or across walls, 5) cytoplasmic interconnections among spherules with wall disintegration, and 6) fully formed cytoplasmic network as plasmodium. Cytochrome c oxidase activity, expressed per unit protein content of the homogenate, remains fairly constant throughout the developmental sequence, whereas acid phosphatase activity, expressed per unit protein concentration, is somewhat lower in the sclerotium than in subsequent stages of development after hydration.     

Ultrastructural localization of beta-D-galactan in the nuclei of the myxomycete Physarum polycephalum.

The acellular slime mold Physarum polycephalum produces an extracellular sulfated and phosphorylated beta-D-galactan which was recently isolated from the nuclei of this organism. This polysaccharide has now been localized in the nuclei of P. polycephalum by electron microscopy using a specific "sandwich" technique: thin sections of P. polycephalum microplasmodia were incubated with the Ricinus communis lectin specific for D-galactose residues. The bound lectin was then localized with gold granules labeled with a galactose-terminated glycoprotein (desialylated ceruloplasmin). The galactan was found in the nuclei mainly associated with chromatin and, also, but to a smaller extent, in the cytoplasm and in some vacuoles. The specificity of the method was assessed by marking under the same condition the galactomannan present in the cell wall of the yeast Schizosaccharomyces pombe.     

The uptake and metabolism of uridine by the slime mould Physarum polycephalum.

1. Uridine is taken up by microplasmodia of Physarum polycephalum via a saturatable transport system with an apparent Km of 29 muM. An intracellular concentration significantly higher than that in the growth medium is attained, suggesting that the uptake is an active process. Both deoxyribonucleosides and ribonucleosides are competitive inhibitors of the uptake of uridine. 2. In contrast, the rate of entry of uridine into surface plasmodia is a linear function of the concentration of the nucleoside in the growth medium, and the uptake is not inhibited by other nucleosides. 3. As well as serving as a source of pyrimidine nucleotides for the synthesis of nucleic acids, uridine is also catabolised by P. polycephalum. Uracil accumulates in the growth medium and there is also significant conversion of C-2 of the pyrimidine ring to CO2. The proportion of uridine subject to catabolism in surface plasmodia is less than that observed for microplasmodia.     

Changes in diadenosine tetraphosphate levels in Physarum polycephalum with different oxygen concentrations.

Cellular levels of diadenosine tetraphosphate (Ap4A) were measured, by a specific high-pressure liquid chromatography method, in microplasmodia of Physarum polycephalum subjected to different degrees of hypoxia, hyperoxia, and treatment with H2O2. Ap4A levels increased three- to sevenfold under anaerobic conditions, and the microplasmodia remained viable after such treatment. Elevated levels of Ap4A returned to the basal level within 5 to 10 min upon reoxygenation of the microplasmodia. The increases in Ap4A levels were larger in stationary-phase or starved microplasmodia than in fed, log-phase microplasmodia. The maximal increase measured in log-phase microplasmodia was twofold. No significant changes in Ap4A levels occurred in microplasmodia subjected to mild hypoxia, hyperoxia, or treatment with 1 mM H2O2. These results indicate that in P. polycephalum, Ap4A may function in the metabolic response to anaerobic conditions rather than in the response to oxidative stress.     

Flow cytometry of the differentiation of Physarum polycephalum myxamoebae to cysts

Myxamoebae of Physarum polycephalum, strain Cld, were grown on agar lawns on live bacteria. Myxamoebae were harvested, fixed and stained with propidium iodide. Flow cytometry showed that, as in the case of Physarum plasmodia, there is no G1 phase during rapid exponential growth. However, an apparent G1 phase was observed at the end of exponential growth when the culture arrested with the G1 DNA content for about a day between growth and differentiation. Most myxamoebae differentiated into cysts, but some formed microplasmodia and others appeared to lose DNA. The cysts possessed the G2 phase DNA content and there was an S phase connecting the G1-arrested state with the encysted state. Encystment was blocked by hydroxyurea (HU) suggesting that DNA synthesis is essential for encystment. The natural temporary synchronization in G1 phase may provide the basis of a method for selecting mutants with a conditional block in G2 or M phases.     

Aminopeptidases of Physarum polycephalum during growth and differentiation

A soluble cell fraction from exponentially growing microplasmodia of the slime mold, Physarum polycephalum, contains 12 electrophoretically distinguishable enzymes capable of hydrolyzing the aminopeptidase substrate, l-leucyl-2-naphthylamide (LNA) at pH 6.5. These enzymes appear to represent three distinct groups of LNA isoenzymes on the basis of electrophoretic mobilities, substrate ranges, and effects of divalent cations and of EDTA on peptidase activity. When spherulation is induced by transfer of microplasmodia to a starvation medium, there is a brief increase in one form of one of the enzymes followed by complete abolition of that enzyme group. These changes in the enzymatic profile occur within 4–5 h of transfer to a starvation medium, though spherules do not appear until 15–20 h later.