Electron microscopic and microprobe analysis of calciuminduced differentiation of the white mutant (LU887 x LU897) strain of Physarum polycephalum

Differentiation of the white mutant (LU887 x LU897) strain of Physarum polycephalum leading to spherule formation can be induced by CaCl(2) if the concentration in the nutrient medium is increased by 5mM prior to the transfer to a non-nutrient salts medium. All stages previously reported for the typical (M(3)cVII) strain of Physarum polycephalum from microplasmodia to spherules are seen but the mutant lacks the synchrony that the replacement technique induces in the typical strain. X-ray microanalyses locate calcium and phosphorus in granules in mitochondria and in the cytoplasm of specimens fixed without osmium. Mitochondria accumulate calcium-containing granules during early differentiation and appear to be essentially without granules in mature spherules. Mobilization of mitochondrial calcium is implicated in the initiation of differentiation. A longitudinally striated cytoplasmic inclusion is abundant in microplasmodia grown in media that have not been supplemented with additional calcium and is seen more rarely during calcium-induced spherulation. Whether or not this inclusion represents cytoplasmic contractile elements is unknown. The calcium-treated mutant strain, previously considered non-differentiating, may prove to be a good alternate model for the study of factors influencing differentiation. It was employed earlier as a control in studies of strains that readily spherulate in response to routine procedures.     

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.     

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.     

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.     

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.     

Induction of spherule formation in Physarum polycephalum by polyols

A method has been developed for inducing spherule formation (spherulation) in the myxomycete Physarum polycephalum by transferring the culture to synthetic medium containing 0.5 m mannitol or other polyols. This morphogenetic process occurred within 12 to 35 hr after the inducer was added. The mature spherules existed as distinct morphogenetic units, in contrast to the clusters of spherules formed during starvation. Ninety per cent of the spherules germinated by 24 hr in synthetic medium. The changes in the synthesis of ribonucleic acid (RNA), deoxyribonucleic acid (DNA), and protein during plasmodial growth, spherulation, and germination of spherules are described. When spherule formation was completed, RNA, protein, and DNA decreased, compared with the values at the beginning of the conversion. The incorporation of (3)H-uridine into trichloroacetic acid-insoluble material was different in each of these periods, and this incorporation was sensitive to actinomycin D. The amount of glycogen increased during growth, whereas it decreased during spherulation. (14)C-glucose could be taken up by the cells in the presence of the inducer, and mannitol could not replace glucose as a source of energy. The mode of action of mannitol and its mechanism of induction are discussed.     

Variance of ploidy in mitochondrial nucleus during spherulation in Physarum polycephalum

In Physarum polycephalum, microplasmodia differentiated into spherules when cultures were aged for 8–10 days. Respiration rates of the microplasmodia decreased rapidly with ageing to a 90% decrease in oxygen consumption over 9 days. We studied this phenomena by isolating and characterizing mitochondria from microplasmodia and spherules at different stages of spherulation. Oxygen uptake by the isolated mitochondria decreased with spherulation. Morphological and biochemical analyses showed that mitochondrial differentiation to inactive state was characterized by a decrease not only in dimension but also of content (DNA, RNA and protein). Diminutive mitochondria contained small particle-shaped mitochondrial nuclei. The DNA content, measured by microscopic fluorometry, was about 1.15 and 0.58 × 10⁻¹⁰ g, which corresponded to about 16 and 8 genome copies, respectively (e.g., 32 genome copies per mitochondrion at mitochondrial G1). Restriction endonuclease analysis showed that the physical structure and methylation pattern of the mtDNA had not changed although the DNA content per mitochondrion had decreased remarkably with spherulation. This showed that changes in the ploidy level of the mitochondrial nucleus during spherulation were due to reduction in the number of whole mitochondrial genomes.     

Levels of histone H4 mRNA during spherulation and germination of Physarum polycephalum

The level of histone H4 mRNA was measured during spherulation and germination of Physarum polycephalum cultures. Histone H4 mRNA is present in prespherules as well as in mature and germinating spherules. During this differentiation process the cells have a 4C or G₂-phase DNA content and therefore no DNA synthesis occurs. The presence of histone mRNA in the dormant cells shows that Physarum prepares long in advance for resumption of vegetative growth.     

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.     

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.     

Accumulation of phosphate-containing granules in the nucleoid area of Pseudomonas aeruginosa

Many electron-dense granules were found in the nucleoid area of Pseudomonas aeruginosa strain K by electron microscopy with the technique of the freeze-substitution method. These granules contained phosphorus and calcium as determined by X-ray microanalysis. The size and the numbers of the granules decreased when the bacteria was cultured in the medium from which phosphate-containing compounds were depleted. From these observations we concluded that the granule was a phosphate-containing granule and possibly a polyphosphate granule. The excellent preservation of the fine structures by the freeze-substitution technique enables us to show very small polyphosphate granules in the nucleoid area of the bacterial cells which cannot be revealed by the conventional chemical fixation method. As we could not see the granules in other bacteria cultured in nutrient medium such as Serratia, Escherichia, Bacillus and Vibrios, the accumulation of the phosphate granules in Ps. aeruginosa might be a unique character of this bacteria and might be related to the growing capability of this bacteria in extremely low nutrient supply.     

Scanning electron microscopy of spherules of Physarum polycephalum

The ultrastructure of starvation-induced sclerotic and mannitol-induced spherules was studied by a scanning electron microscope (SEM). Each spherule in the berry-like sclerotium is not rounded and has lobe-like forms, whereas the separate spherules are significantly bigger and rounded. Bead-like granules were observed inside and on the spherules. Broken spherules observed in the SEM show ultrastructure of cell wall, cytoplasm, vacuoles and granules, nuclei and nucleoli.     

Molecular cloning of mRNAs expressed specifically during spherulation of Physarum polycephalum

A cDNA library was constructed using the poly(A)+ RNA extracted from spherulating Physarum polycephalum microplasmodia. This library (740 clones) was screened by differential hybridization with 32P-labeled poly(A)+ RNA from growing plasmodia and developing spherules. The results showed that at least 30% of the clones corresponded to mRNAs expressed specifically in spherulating plasmodia. The 35 spherulation-specific cDNA clones giving the strongest hybridization signals were analysed. From this group, four different sequences complementary to very abundant mRNAs were identified. They each accounted for 1.5% of 4.5% of all the clones in the library and probably represented the most abundant spherulation-specific mRNAs. In addition, four less abundant mRNAs were identified from stage-specific clones giving weaker hybridization signals. These sequences represented individually between 0.3% and 0.7% of the clones in the library. Northern blots showed that these eight different sequences were absent from plasmodia and were most abundant 24-36 h after the induction of spherulation. Similar results were also obtained when spherulation was induced by the addition of a sublethal concentration of ferrous iron ions to the growth medium. Hybridization of the spherule-specific clones to Southern blots of genomic DNA suggested the presence of one copy for each gene.     

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.     

The rapid intranuclear accumulation of preexisting proteins in response to high plasmodial density in Physarum polycephalum

When actively growing microplasmodia of the lower eukaryote Physarum polycephalum are gently pelleted and allowed to stand at high plasmodial densities for 45 min, three specific nuclear acidic proteins undergo dramatic quantitative changes. Two major proteins of molecular weight 46 000 and 94 000 increase 110 and 320%, respectively. The increase in these two proteins is not markedly attenuated during periods when 88% total protein synthesis is blocked by cycloheximide, and the specific radioactivities of these proteins from prelabeled and continuously labeled control and pelleted plasmodia are essentially identical. A third protein of molecular weight 34 000 decreases by 51 % during the 45 min period and when cycloheximide is present, a 36% decrease in this protein still occurs. The rapid changes which occur in these three proteins in response to high plasmodial density also develop, together with many other changes, during plasmodial differentiation, but only after about 6 h of starvation. It is concluded that the rapid increase in the 46 000 and 94 000 mol. wt proteins results from protein transfer phenomena rather than de novo synthesis and that these proteins perhaps function in the early reorganization of cell metabolism rather than in structural differentiation. In further comparative studies it has been observed that mature spherules of P. polycephalum contain a major acidic protein not present in growing or differentiating plasmodia and also that the complement of residual acidic proteins differs in starvation-induced vs cold-induced spherules.     

Superoxide dismutase induces differentiation in microplasmodia of the slime mold Physarum polycephalum

Evidence is presented that supports a role for the enzyme superoxide dismutase (SOD) in the differentiation of the slime mold, Physarum polycephalum. SOD activity increases 46-fold during differentiation. A strain of Physarum that does not differentiate exhibits no change in SOD activity. Addition of SOD, via liposomes, to the nondifferentiating strain induces differentiation; this effect is enhanced by an inhibitor of glutathione synthesis. Other antioxidants selected for study failed to induce differentiation. Conversely, oxidative treatments including introduction of D-amino acid oxidase, via liposomes, induced differentiation. Cellular oxidation is the probable cause of the SOD effect.