Studies on isoenzyme pattern during differentiation (spherulation) of Physarum polycephalum

Plasmodial homogenates of the true slime mold Physarum polycephalum grown on a liquid medium contain carbohydrates which form a complex with protein under conditions of acrylamide electrophoresis and thus make isoenzyme studies from those extracts impossible. A method, using mild homogenization and centrifugation on top of a 30% sucrose solution was developed. This treatment leaves most of the soluble cytoplasmic enzymes in the upper layer above the sucrose, which then can be used for successful isoenzyme or protein studies with polyacrylamide electrophoresis.The activity changes and isoenzyme pattern of 16 different enzymic activities were studied during differentiation (spherulation) of Physarum polycephalum, induced either by starvation or by mannitol. Only one enzyme, esterase, exhibited a conspicuous change in isoenzyme pattern during development.     

Differential protein synthesis during differentiation (spherulation) of Physarum polycephalum: Lack of synthesis of glucose-6-phosphate dehydrogenase

Summary In the neutral lipid fraction of Hydrogenomonas eutrapha strain H 16, two esters of phthalic acid were found and separated by column chromatography.By totally labelling chemolithotrophically growing bacteria with ¹⁴CO₂ it was demonstrated that these phthalic acid esters are not genuine bacterial products but of exogenous origin.It is supposed that these esters which are used as plasticizers for various plastics originate from such materials used in the preparation and storage of demineralized water.     

Flow cytometric determination of nuclear DNA content during differentiation (spherulation and germination) of the myxomycete Physarum polycephalum

Summary Using flow cytometry, spherulating nuclei of Physarum isolated at the beginning of spherule wall formation were found to exhibit a DNA content corresponding to the G₂ phase of the cell cycle, although 8% lower. Before the first mitosis after spherule germination, a very slight incorporation of ³H thymidine into DNA was observed that was too weak to correspond to S phase, strongly suggesting that nuclei are stopped in G₂ phase inside the spherules. The lower value of nuclear DNA content found using flow cytometry of germinating spherules may not be related to DNA quantity, but may be due to a difference in chromatin organization during growth or spherulation, resulting in interference with the staining.     

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.     

Amino acid pool and protein turnover during differentiation (spherulation) ofPhysarum polycephalum

The composition of the amino acid pool during spherulation was determined. It changes in size and in composition, the concentration of each amino acid behaving individually. The first response to the onset of spherulation either by starvation or osmotic shock (0.5 M mannitol) always is a decrease of the pool's size, which during further starvation expands for a short period and then decreases again. During development induces by mannitol in the presence of external amino acids, the pool size increases continuously after the initial depletion.As shown by radioactive labeling, amino acids were actively released from the plasmodium into a medium containing amino acids, but retained by the microplasmodia in an amino acid-free medium. The kinetics of the uptake of radioactive amino acids from the medium is biphasic, indicating the existence of multiple pools. Even after a labeling period of 8 h the amino acid pool is not yet in equilibrium with the medium. The possibility of a compartimentation of the pool was confirmed by density labeling of two different enzymes.Whereas the turnover of total protein is only very low during growth, it is rather high in spherulating microplasmodia. At least 70% of the originally existing protein is degraded during this development, while, simultaneously, at least 50% of the protein present after 24 h starvation is newly synthesized during that period.     

Melanin biosynthesis during differentiation of Physarum polycephalum.

Melanin synthesis in the myxomycete Physarum polycephalum occurs during sporulation but not during spherule formation. Melanin-like pigment was extracted from spores. An almost identical substance of polyphenols was extracted from spherules and characterized by its ultraviolet and infrared absorbance spectra. Polyphenol oxidase activity in spherules was very low and showed only one weak isoenzyme band in isoelectric focusing polyacrylamide gels. A much higher activity, and an increasing number of isoenzymes, were detected in sporulating cultures after illumination during the differentiation process. The addition of melanin precursors resulted in the synthesis of brownish-yellow spherules, probably containing dopachrome, whereas the addition of polyphenol oxidase inhibitors resulted in yellow sporangia. The results indicate that melanin synthesis is probably only a stage in maturation but not an essential part of the morphogenetic process itself.     

Induction of synchronous differentiation (encystment) in Physarum polycephalum myxamoebae

Encystment of Physarum polycephalum myxamoebae, grown under nearly identical physiological conditions as plasmodia is induced by transfer to a salts medium containing 0.5 M mannitol or mannose. After 24 h induction approximately 50% of amoebae had differentiated to cells which were identified to be young cysts by light and electron microscopy. Several other polyols, sugars, biogenic amines, and a starvation period from 24 h to one week caused no reproducible cyst formation. In contrast to the formation of dormant forms in the plasmodial stage of the life cycle, the induction of cysts and their germination to amoebae are not inhibited neither by actinomycin C nor by cycloheximide. In addition, the isoenzyme spectra of aminopeptidases and acid proteases remain nearly identical in growing and differentiating amoebae.Abbreviations SD semi-defined BSS basal salts solution The investigation is a part of the Ph. D. thesis of A. Haars, Göttingen, 1976     

Cell-cycle-specific activity of cyclic nucleotide phosphodiesterase in Physarum polycephalum

The cell-cycle-related activities of the cAMP- and cGMP-dependent phosphodiesterases of Physarum polycephalum were assayed. The activities of plasmodial homogenate and of selected subcellular fractions were measured. The results suggested the presence of both cAMP- and cGMP-dependent phosphodiesterase in the isolated nuclei of P. polycephalum. In addition, they reveal that the cAMP- and cGMP-dependent phosphodiesterase activities of the subcellular fractions fluctuate throughout the cell cycle. The whole-cell homogenates exhibit no cell-cycle-related changes in the presence of 5 X 10(-4) M cGMP. Kinetic data suggest the presence of multiple phosphodiesterase activities in the homogenate and its particulate fractions for the cGMP-dependent enzyme. Multiple cAMP activities are also suggested for the particulate fractions. The Km values indicate that the substrate affinities of the phosphodiesterases from P. polycephalum are similar to those found previously in mammalian systems.