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Myxomycetes are eukaryotic microorganisms containing characteristics akin to both fungi and amoebae. They can complete their whole life cycles while being cultured on agar media, and under-laboratory conditions, which favors taxonomic, phylogenetic, and cytological researches. Here, we describe the life cycles of two such species: Didymium squamulosum collected from the field and Physarum rigidum cultured from moist chamber both belonging to the Order Physarales. Three per cent oat-agar media (OAM) was used to culture the plasmodia until they aggregated and were almost starved. Natural light was then applied to the plasmodia to induce fructification. Their life cycles share the same common stages, namely: spore, myxamoebae, swarm cell, plasmodia, and sporulation. In this study, we describe the morphogenesis from spore to spore of two species by differential interference contrast (DIC) and stereoscopic microscopies, as well as discuss the differences between the development of both species and interspecies. We found that the spore germination method of both species was the same. However, there were differences noted in time taken and fruiting body formation. Unlike P. rigidum, the species D. squamulosum did not require natural light stimulation. Moreover, the maturation process of both species had similar color transitions but exhibited distinct morphology in each developmental stage except during the swarm cell stage., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The plasmodial slime molds is the largest group in the phylum Amoebozoa. Its life cycle includes the plasmodial trophic stage and the spore-bearing fruiting bodies. However, only a few species have their complete life cycle known in details so far. This study is the first reporting the morphogenesis of Didymium laxifilum and Physarum album. Spores, from field-collected sporangia, were incubated into hanging drop cultures for viewing germination and axenic oat agar plates for viewing plasmodial development and sporulation. The spores of D. laxifilum and P. album germinated by method of V-shape split and minute pore, respectively. The amoeboflagellates, released from spores, were observed in water film. The phaneroplasmodia of two species developed into a number of sporangia by subhypothallic type on oat agar culture. The main interspecific difference of morphogenesis was also discussed., (© 2016 The Author(s) Journal of Eukaryotic Microbiology © 2016 International Society of Protistologists.)

A new widespread myxomycete species, Physarum pseudonotabile, inhabiting the arid regions of the Eurasia, South and North America is described and illustrated. Tentatively assigned to Ph. notabile T. Macbr., a phylogeny based on the small ribosomal subunit (SSU) and elongation factor 1 alpha (EF1a) genes placed the new species in a clade far from Ph. notabile. Ph. pseudonotabile was found to be frequent in surveys based on the moist chamber culture technique with samples of litter, bark and herbivore dung collected in dry steppe and deserts of the Caspian lowland (Russia), Kazakhstan, Mongolia, China, Spain, Argentina and USA. The main morphological difference between Ph. pseudonotabile and Ph. notabile lies in spore ornamentation. Spores of the former species display irregularly distributed verrucae, whereas the latter species possesses spores with dense and regularly arranged spinulae. In addition, the ecological preferences of the two species differ. Ph. pseudonotabile inhabits the bark of living plants and ground litter in arid regions, whereas Ph. notabile is found on coarse woody debris in boreal and temperate forests. Although the new species appears to be closest to Ph. notabile morphologically, the phylogenetic analysis reveals Ph. pusillum and Ph. nivale as the closest relatives. In addition, the molecular investigations revealed a considerable amount of hidden diversity within species of Physarum with gray lime flakes. Currently we have only sufficient material to assess the morphological variation of Ph. pseudonotabile but expect that more taxa within this clade may emerge within studies combining morphological and molecular analyses.

A new nivicolous species of Physarum was discovered during the study of myxomycetes in the Patagonian Andes of South America. It is described herein under the name Physarum andinum. The species is characterized by stalked sporophores or more rarely sessile sporocarps or short plasmodiocarps. The sporocarps are strikingly large, reaching 2.6 mm tall and 3 mm diam when open, and have a peridium with three layers, the internal layer being clearly visible and opening separately. Physarum andinum was found at five localities in Argentina as well as in herbarium material collected about 100 y ago in Chile. The new species is reminiscent of the non-nivicolous species Physarum brunneolum, but the latter forms smaller sporophores, has darker spores and the three layers of the peridium are adhered and open together. The characters of the new species were examined under stereomicroscope, light microscope and scanning electron microscope and micrographs of relevant details are included.

The elemental composition of spores, peridium walls, and lime nodes of Physarum compressum sporocarps, cultivated on rabbit dung as a natural growing environment for the slime mold and on artificial agar medium, was compared to evaluate differences that may be dependent on substrates. Whole fruiting bodies and samples of both experimental media were extracted with nitric acid or Parr digest bomb, respectively, and analyzed by means of total X-ray reflection fluorescence (TXRF). Electron probe microanalysis (EPMA) of spores, peridium walls, and lime nodes structure was carried out with the scanning electron microscope equipped with energy-dispersive spectrometer. Because of minute sizes and roughness of investigated structures, Monte Carlo simulations were utilized to establish analytical conditions of EPMA. Biological and geological standards were used in the quantification of element concentrations. According to TXRF, the fruiting bodies from agar medium revealed lower concentrations of K, Ca, Cr, Mn, and Fe in relation to fruiting bodies from the dung, reflecting elemental relationships in the experimental media. According to EPMA, the highest Ca concentration was found in the lime nodes followed by the peridium and the spores. Culturing of the slime molds on the rabbit dung indicated higher concentration of Ca in the lime nodes and peridium walls when compared with those obtained from the sporocarps grown on agar media. The opposite relation was found for the spores. The concentration of Na, Mg, P, S, and Cl was generally lower in all structures of the sporocarps harvested from the dung than from the agar medium. K was in higher concentration in analyzed structures from dung than from agar. Different element uptake (except for Ca and K) was revealed by the two methods: TXRF and EPMA.

The plasmodium of Physarum polycephalum has attracted much attention due its intelligent adaptive behavior. In this study, we constructed a model of the organism and attempted to simulate its locomotion and morphogenetic behavior. By modifying our previous model, we were able to get closer to the actual behavior. We also compared the behavior of the model with that of the real organism, demonstrating remarkable similarity between the two., (2010 Elsevier Ireland Ltd. All rights reserved.)

The ontogeny of the myxomycete Physarum globuliferum was observed on corn meal agar and hanging drop cultures without adding sterile oat flakes, bacteria or other microorganisms. Its complete life cycle including spore germination, myxamoebae, swarm cells, plasmodial development, and maturity of fructifications was demonstrated. Details of spore-to-spore development are described and illustrated.

The Physarum machine is a biological computing device, which employs plasmodium of Physarum polycephalum as an unconventional computing substrate. A reaction-diffusion computer is a chemical computing device that computes by propagating diffusive or excitation wave fronts. Reaction-diffusion computers, despite being computationally universal machines, are unable to construct certain classes of proximity graphs without the assistance of an external computing device. I demonstrate that the problem can be solved if the reaction-diffusion system is enclosed in a membrane with few 'growth points', sites guiding the pattern propagation. Experimental approximation of spanning trees by P. polycephalum slime mold demonstrates the feasibility of the approach. Findings provided advance theory of reaction-diffusion computation by enriching it with ideas of slime mold computation.

Myxomycetes are protists whose life cycle depends on aerially dispersed spores that germinate into motile myxamoebae, which then pair and fuse to form a larger, motile plasmodium. The plasmodium seeks out a suitable fruiting site (usually atop vegetative material or detritus) and transforms into fruiting bodies that release the spores. In this paper we report the first known instance of a myxomycete, in this case Physarum pusillum, sporulating on the body of a living animal, the cryptic lizard Corytophanes cristatus, which was collected in eastern Honduras in 2003.

The plasmodium of Physarum polycephalum grows without cytokinesis and shows an active cytoplasmic streaming under wet and nutritious conditions. It can undergo reversible differentiation into several types of dormancy to survive in adverse environments. Temperature change or osmotic stress leads to cytoplasmic division of the plasmodium into cells containing one or more nuclei: these form a macrocyst, the spherule. Desiccation also induces cell division of the plasmodium followed by formation of a sclerotium, a dormant body resistant to dry stress. More than half of the actin in a sclerotium is phosphorylated at a single site, threonine 203, resulting in loss of its ability to polymerize into actin filaments. In the present study, actin phosphorylation was found in the sclerotium but not in either the plasmodium or in the spherule. This result suggests that phosphorylation of sclerotium actin may be related to the mechanism associated with desiccation resistance rather than morphological changes through cell compartmentalization in the macrocyst formation. Moreover. dephosphorylation of the phosphorylated form of sclerotium actin began within 5 min after addition of water. Dephosphorylation was not affected by sucrose and sorbitol sugars, but was inhibited by ammonium bicarbonate, ammonium phosphate, sodium phosphate, NaCl, and KCl in a dose-dependent manner. On the other hand, in germination of the sclerotium there was measurable sensitivity to both carbohydrates and salts. Actin dephosphorylation seems to be one of the important processes in the early phase of sclerotium germination.

Even for humans it is not easy to solve a maze. But the plasmodium of true slime mold, an amoeba-like unicellular organism, has shown an amazing ability to do so. This implies that an algorithm and a high computing capacity are included in the unicellular organism. In this report, we discuss information processing in the microorganism to focus on the issue as to whether the maze-solving behavior is akin to primitive intelligence.

Genetic evidence has shown the presence of a common spindle pole organiser in Physarum amoebae and plasmodia. But the typical centrosome and mitosis observed in amoebae are replaced in plasmodia by an intranuclear mitosis devoid of any structurally defined organelle. The fate of gamma-tubulin and of another component (TPH17) of the centrosome of Physarum amoebae was investigated in the nuclei of synchronous plasmodia. These two amoebal centrosomal elements were present in the nuclear compartment during the entire cell cycle and exhibited similar relocalisation from metaphase to telophase. Three preparation methods showed that gamma-tubulin containing material was dispersed in the nucleoplasm during interphase. It constituted an intranuclear thread-like structure. In contrast, the TPH17 epitope exhibited a localisation close to the nucleolus. In late G2-phase, the gamma-tubulin containing elements condensed in a single organelle which further divided. Intranuclear microtubules appeared before the condensation of the gamma-tubulin material and treatment with microtubule poisons suggested that microtubules were required in this process. The TPH17 epitope relocalised in the intranuclear spindle later than the gamma-tubulin containing material suggesting a maturation process of the mitotic poles. The decondensation of the gamma-tubulin material and of the material containing the TPH17 epitope occurred immediately after telophase. Hence in the absence of a structurally defined centrosome homologue, the microtubule nucleating material undergoes a cycle of condensation and decondensation during the cell cycle.

The microbial eukaryote Physarum polycephalum displays several distinct cell types in its life cycle, including amoebae, flagellates and plasmodia. Despite its relative simplicity, Physarum has a tubulin gene family of complexity comparable to that of Drosophila. We have identified beta-tubulin cDNAs from Physarum that are derived from the betA beta-tubulin locus and encode beta 1A tubulin. We have also identified a partial cDNA for the unlinked betB beta-tubulin gene, which encodes beta 1B tubulin. The polypeptide sequences encoded by betA and betB show 99% identity, but the nucleotide sequences show only 85% identity, consistent with an ancient duplication of these genes. The betB gene is expressed in amoebae, flagellates and plasmodia, whereas betA is expressed only in amoebae and flagellates. During the amoeba-flagellate transition the level of betA transcript increases over 100-fold, while the level of betB transcript changes very little. Thus Physarum has a mechanism for regulating the level of discrete beta-tubulin transcripts differentially during flagellate development. A need for this differential regulation could account for the maintenance of the virtually isocoding betA and betB beta-tubulin genes.

An alanine, lysine and glutamic acid-rich nuclear protein (P2) of Mr approximately 19,500 co-extracts with the histones from nuclei of Physarum polycephalum when using the CaCl2 method for histone extraction [1] and was found to have the composition previously ascribed to a putative histone H1(0) isolated from microplasmodia using 5% PCA (Yasuda, H., Mueller, R.D., Logan, K.A. and Bradbury, E.M. (1986) J. Biol. Chem. 261, 2349-2354). P2 has very similar electrophoretic properties to chicken erythrocyte histone H5, calf thymus histone H1(0) and the Physarum HMG-like protein AS-2, but does not appear to be immunologically or structurally similar to H5 or H1(0). An increase in the abundance of P2 was observed during exponential growth in microplasmodia, reaching an approximately 1:1 ratio with histone H1 by 48 h of culture. Standard amino acid analysis and NMR show that P2 is more HMG-like than H1-like and CD measurements demonstrated that P2 contains only 5% secondary structure in its maximally structured state and is, therefore, essentially unstructured under in vivo conditions. Also possible clustering of acidic residues is detected using CD and may be of functional significance. Analysis of post-translational modification of P2 shows that it is phosphorylated at up to three sites as isolated from immature spherules. The relationship of P2 to the HMG family of proteins and AS-2 is discussed.

Physarum amoebae possess an extranuclear spindle-organizing centre (abbreviated SPOC), located in a typical centrosome with a pair of associated centrioles while plasmodia possess an intranuclear SPOC without centrioles. In order to ascertain whether, during conjugation, the plasmodial SPOC is derived from the amoebal one or is not related to it, we have constructed amoebal strains possessing two and three SPOCs and we have used as a genetic marker the frequency of polycentric metaphases in order to evaluate the number of SPOCs in the plasmodia. The results of both symmetrical crosses, i.e. between amoebae possessing the same number of SPOCs, and asymmetrical crosses, i.e. between amoebae possessing a different number of SPOCs, show that: (1) the number of SPOCs in plasmodia is dependent upon the number of SPOCs in either one of the two parental amoeba; (2) in no cross does the number of plasmodial SPOCs equal the sum of the parental amoebal SPOCs, but it corresponds to that of only one parent without any polarity of transmission in asymmetrical crosses. These results are consistent with the following model: (1) plasmodial SPOCs are derived from the amoebal ones; and (2) one set of parental SPOCs is lost, destroyed or inactivated in the zygote.

A type I topoisomerase has been purified from nuclei of a slime mold Physarum polycephalum and its activity was tested during spherulation. The final preparation contained a single polypeptide of about 100 kDa. Basic properties of Physarum topoisomerase I (substrate specificity, ionic requirement, sensitivity to inhibitors) were similar to those of topoisomerases from higher eukaryotes. Specific features of Physarum enzyme were that it was rapidly inactivated at 45 degrees C and did not react with antibodies against human topoisomerase I. The activity of topoisomerase I in developed dormant spherules decreased approx. 2-fold, as compared with a 4-fold decrease of RNA and a 10-fold decrease of DNA synthesis. Basic properties of the enzyme remained unchanged during spherulation.

The intracellular acid-soluble purine and pyrimidine derivatives of myxamoebae-swarm cells of Physarum flavicomum were investigated during growth, microcyst formation, and during adenine-inhibition of encystment, using high performance liquid chromatography (HPLC). We also studied the incorporation of exogenous radioactive adenine into the acid soluble purine derivatives and S-adenosyl-sulphur compounds separated by HPLC. The most abundant ribonucleoside monophosphate was AMP in the growing and 15 h encysting cells (NC), while it was UMP in the 15 h adenine-inhibited cells (AIC). ADP was the nucleoside diphosphate present in the greatest quantity in the growing and NC cells but it was CDP in the AIC. The nucleoside triphosphate in highest concentration was ATP, UTP, and GTP in growing, NC, and AIC, respectively. Guanosine was the most abundant nucleoside in all cells. The nucleobase occurring in greatest concentration was cytosine, cytosine and guanine, and adenine in the growing, NC, and AIC, respectively. The AMP content in the 15 h AIC was 2.1-fold higher than that of adenosine. The 15 h NC had the lowest adenylate energy charge, a value of 0.54 +/- 0.02, while the values for growing cells and the AIC were 0.62 +/- 0.02 and 0.76 +/- 0.01, respectively. [14C]-Adenine labelling studies (15 h) revealed the occurrence of purine nucleotide interconversion, as the label was detected not only in adenosine, AMP, ADP, ATP, but also in guanine, guanosine, GMP, GDP, GTP, as well as, in inosine monophosphate and xanthosine monophosphate. The percentage incorporation of the radiolabelled adenine into AMP was higher than into adenosine. An increased intracellular level of guanine nucleotides is associated with the inhibition of encystment. The extracellular adenine, rather than internal adenine sources, appears to be the primary precursor of nucleotide for S-adenosylmethionine synthesis during adenine-inhibition of encystment.

The development of uninucleate amoebae into multinucleate plasmodia in myxomycetes is called the amoebal-plasmodial transition (APT). During the APT in Physarum polycephalum the ability to form flagellar axonemes is lost; the astral, open mitosis is replaced by the anastral, closed mitosis; and cytoskeletal microtubules disappear. These changes are accompanied by alterations in the repertoire of expressed tubulins. Using immunofluorescence microscopy we have studied the timing of loss and accumulation of developmentally regulated tubulin isotypes in relation to other cellular events during the APT. We specifically asked whether changes in the composition of microtubules are correlated with changes in their organization. The plasmodium-specific beta 2-tubulin can first be detected in microtubules of uninucleate cells after they become committed to plasmodium formation. However, rare cells are observed that exhibit beta 2-tubulin at earlier or only at later stages of development. Amoeba-specific acetylated alpha 3-tubulin disappears gradually during development. Individual cells differ in the timing of loss of this isotype: alpha 3-tubulin is present in the majority of uninucleate cells, in a fraction of binucleate and quadrinucleate cells, and is absent from larger multinucleate cells. Cytoplasmic microtubules in uninucleate cells are organized by a single microtubule-organizing center (MTOC) juxtaposed to the nucleus. Binucleate cells and quadrinucleate cells exhibit variable numbers of MTOCs. Cytoplasmic microtubules persist during the APT until the stage of plasmodia containing at least 100 nuclei. The lack of a strict correlation between the changes in tubulin composition and changes in organization of microtubular structures indicates that accumulation of beta 2-tubulin and disappearance of alpha 3-tubulin isotypes are not sufficient to bring about reorganization of microtubules during development. Individual cells in a developing population differ not only in the succession of accumulation and loss of developmentally regulated tubulins, but also in the sequences of other cellular changes occurring during the APT.

H1 and P2 (an H1 degree/HMG-like protein) accumulate during exponential growth of Physarum microplasmodia (unpublished results), indicating that these proteins may play a role in differentiation (spherulation). To test this hypothesis, pulse labelling using [14C]lysine was used to determine whether any differential histone synthesis occurs during salts-induced spherulation. A peak in the uptake of [14C]lysine into microplasmodia was detected between 12 and 24 h following salts-induction. During the same interval, incorporation of label into the CaCl2-extracted histones occurred, with H1 being synthesised at approx. 3 times the level of the core histones and P2. Densitometry of SDS-PAGE gels showed that high levels of H1 were maintained up to 40 h in salts medium, beyond the observed peak in synthesis. The synthesis and accumulation of high levels of H1 during early spherulation indicates a role for this histone in the initiation and maintenance of a transcriptionally inactive differentiated state.

In Physarum flavicomum Berk., haploid myxamoebae convert to dormant microcysts under conditions of nutrient imbalance. Exogenous adenine increases the intracellular content of S-adenosylmethionine (SAM) and inhibits this process. However, treatments that reduce the intracellular SAM levels relieve the inhibition of encystment induced by adenine. SAM plays a major metabolic role in cellular transmethylation reactions. In this study, we compared the DNA methylation patterns of growing cells, encysting cells, adenine-inhibited cells, and cysts using three different approaches: incubation of the cells with [14C]methylmethionine and detection of the labeled methyl group in purified DNA samples; analyses of DNA base composition by high performance liquid chromatography; and restriction endonuclease analyses of DNA. We found that DNA from the adenine-treated cells was labelled 1.3 times more with [14C]methylmethionine than was the DNA of untreated encysting cells. The DNA G + C content of this species was about 41%. The DNA of growing cells had the highest 5-methylcytosine (5MC) content, while DNA from the cysts had the lowest (about 27% that of growing cells). Adenine-inhibited cells had about 1.2 times more DNA-5MC than did encysting cells. Using the restriction enzymes SmaI, PvuI, and XhoI (which are inhibited by C residue methylation), we found that cyst DNA had more cutting sites than did amoebal DNA. By using the restriction enzyme DpnI which cuts DNA at GmATC sites, we found that cyst DNA, but not growing cell DNA, contained N6-methyladenine.

In Physarum flavicomum Berk., growing amoebae convert to dormant cysts under conditions of nutrient imbalance. Exogenous adenine inhibits the process and the cells produce an elevated intracellular concentration of S-adenosylmethionine. Evidence indicates that the increased level of S-adenosylmethionine is responsible for the disruption of the normal developmental process. One of the biological functions of S-adenosylmethionine is in polyamine synthesis and it is known that a well-controlled intracellular concentration of polyamines is essential for normal cell growth and differentiation. In this study, high-performance liquid chromatography was used to determine the intracellular polyamine patterns in growing cells, adenine-treated and normal encysting cells, and dormant cysts. Putrescine and spermidine were the most abundant polyamines found in the cells; growing cells had the highest level, adenine-treated cells had a 1.5 to 2.0 times higher level than normal encysting cells, while cysts had the lowest (only 3 and 12% of that of growing cells). Cadaverine and N1-acetylspermidine were found in all the cells and their levels decreased during encystment. Acetylputrescine was found in growing cells only and acetylspermine was found in all cells except cysts. Acetylcadaverine, N8-acetylspermidine, 1,3-diaminopropane, and spermine were not detected in any of the cells.

Previous studies from this laboratory suggested that in Physarum flavicomum an S-adenosylmethionine (SAM) related metabolic flux is involved in the developmental control of microcyst formation. In the present study, this phenomenon was further analyzed by comparing the metabolic effects of compounds found to favor, stimulate, or inhibit the normal encystment process. The compounds utilized were adenine, cycloleucine, L-ethionine, DL-selenomethionine, cadaverine, cyclohexylammonium sulfate, papaverine, mycophenolic acid, decoyinine, putrescine, spermidine, and spermine. Following incubation with the compounds or combinations of the compounds, the intracellular levels of SAM and SAM metabolites were determined using high performance liquid chromatography. The actual level of the intracellular SAM pool varied considerably (notably after 15 h of incubation), depending on the compound(s) in the incubation solution, but was always highest during inhibition of encystment. SAM was undetectable or barely detectable during stimulation of encystment or under conditions favoring encystment. In cells exposed to L-ethionine, we detected intracellular S-adenosylethionine rather than SAM. The intracellular methionine pool in cells encysting normally for 15 h was found to be 1.3 mumol/pg DNA. Both SAM and S-adenosylhomocysteine (SAH) were present in growing amoebae at concentrations greater than that of cells undergoing normal encystment. The developmental transition from growing amoebae to dormant cysts in P. flavicomum is metabolically characterized by the adjustment of the intracellular concentration of SAM and SAH to a minimal critical level of 0.31 and 0.70 fmol/pg DNA, respectively.

Time-lapse cinematography and immunofluorescence microscopy were used to study cellular events during amoebal fusions and sexual plasmodium development in Physarum polycephalum. Amoebal fusions occurred frequently in mixtures of strains heteroallelic or homoallelic for the mating-type locus matA, but plasmodia developed only in the matA-heteroallelic cultures. These observations confirmed that matA controls development of fusion cells rather than cell fusion. Analysis of cell pedigrees showed that, in both types of culture, amoebae fused at any stage of the cell cycle except mitosis. In matA-heteroallelic fusion cells, nuclear fusion occurred in interphase about 2 h after cell fusion; interphase nuclear fusion did not occur in matA-homoallelic fusion cells. The diploid zygote, formed by nuclear fusion in matA-heteroallelic fusion cells, entered an extended period of cell growth which ended in the formation of a binucleate plasmodium by mitosis without cytokinesis. In contrast, no extension to the cell cycle was observed in matA-homoallelic fusion cells and mitosis was always accompanied by cytokinesis. In matA-homoallelic cultures, many of the binucleate fusion cells split apart without mitosis, regenerating pairs of uninucleate amoebae; in the remaining fusion cells, the nuclei entered mitosis synchronously and spindle fusion sometimes occurred, giving rise to a variety of products. Immunofluorescence microscopy showed that matA-heteroallelic fusion cells possessed two amoebal microtubule organizing centres, and that most zygotes possessed only one; amoebal microtubule organization was lost gradually over several cell cycles. In matA-homoallelic cultures, all the cells retained amoebal microtubule organization.

The kinetics of nuclease digestion of Physarum polycephalum nuclei by staphylococcal nuclease and DNase I has been studied at different stages of the cell cycle. Significant differences in the digestion behaviour of nuclei from metaphase and interphase have been detected with DNase I but not with staphylococcal nuclease. Furthermore the structure of newly replicated DNA in S phase differs from the bulk in that it is more easily degraded to acid-soluble products by either staphylococcal nuclease or by DNAase I. At least four types of chromatin structure can be distinguished by our digestion kinetics experiments.

The effects of the microtubule inhibitors colchicine, parbendazole and nocodazole on the growth of myxamoebae of Physarum polycephalum were closely paralleled by the effects of these drugs on the assembly in vitro of purified amoebal microtubule protein. Colchicine at 100 microM did not inhibit amoebal growth and did not inhibit formation, or depolymerization, of amoebal microtubules. The benzimidazole carbamate derivatives nocodazole and parbendazole were very effective in both inhibiting growth and inhibiting the assembly in vitro of amoebal microtubule protein. Parbendazole was the most effective.

The physical and nutritional requirements of the antibiotic-producing slime mold Physarum gyrosum were examined to develop a liquid medium for this myxomycete. Liquid culture is desired to expedite a useful scale of production of antibiotic materials for ease of isolation and structure study. Culture conditions were selected to favor antibiotic production rather than maximum growth. The medium devised consisted of 0.010 M potassium phosphate buffer (pH 6.0), 2% bakers' yeast, and 0.2% glucose and was supplemented with either 10(-7) M hemoglobin (preferred) or 2.0 ml of live Escherichia coli per 100 ml of culture medium grown to a steady-state population in nutrient broth. The slime mold, which contained some E. coli carried along with the inoculum, was allowed to grow as a surface plasmodium at 20 degrees C in the dark with weekly subculturing for stocks or for 10 days for antibiotic production. P. gyrosum produced the same antibiotic materials when grown in liquid medium as it did when grown on agar plates. A seeded plate disk assay against Bacillus cereus was employed to follow antibiotic activity.

1. The rate of protein synthesis changes very little during the first 2-3 h (S phase) of the nuclear division cycle in plasmodia of Physarum polycephalum and then increases continuously during G2 phase, so that by the end of the cycle the rate has doubled relative to that in S phase. Protein synthesis appears to continue during mitosis. 2. Fractionation of extracts of plasmodia, labeled with [3H]lysine for 1 h, by two-dimensional electrophoresis indicated that most if not all proteins are synthesised throughout the nuclear division cycle. However, two metabolically stable polypeptides, the synthesis of which occurs predominantly in Gs phase, were detected. 3. Using a double-labelling procedure, the differential rates of synthesis of 30 relatively abundant polypeptides were measured in relation to the nuclear division cycle. As a group, their differential rates of synthesis increase during the cycle so that their actual rates of synthesis increase 4-6-fold. This implies that their synthesis is regulated over and above any simple change due to a doubling in the number of genes during S phase.

An expression of UDP-glucose:poriferasterol glucosyltransferase activity associated with differentiation of a true slime mold, Physarum polycephalum, from haploid myxoamoebae to diploid plasmodia was demonstrated. In the haploid cells, this enzyme activity was not detected, but after conjugation of the myxoamoebae, the enzyme activity was expressed and increased definitely. In the plasmodial stage, high enzyme activity was maintained constantly. The enzyme was partially purified (35-fold purification, and 28% yield), and molecular weight of 72,000, pH optimum of 7.0, and some characteristics were demonstrated.

Translational regulation of protein synthesis accompanies sclerotization in Physarum polycephalum. Plasmodial and sclerotial poly(A)+ RNA were translated in a message-dependent wheat germ lysate in the presence of the cap analogue 7-methylguanosine-triphosphate to determine whether 5' structural alterations in mRNA accompany translational repression. The translation of plasmodial and sclerotial poly(A)+ RNA was reduced to identical levels suggesting that both RNA populations are capped. The 5'-termini of plasmodial and sclerotial poly(A)+ RNA were identified as m7G5'ppp5'Cm. Alterations in the 5'-cap of mRNA during sclerotization do not appear to be responsible for translational dormancy.

The structure of chromatin in the growing myxomycetes P. polycephalum can be interpreted on the basis of three types of substructure: (i) soluble in a low ionic strength buffer and 2 mM EDTA (I), (ii) soluble in 0.6 M NaCl (II) and (iii) non-soluble and nuclease-insensitive (III). The ratio of these structures in the nuclear pellet obtained after autolysis was found to be 71 : 0, 4 : 12 for the young and 7.7 : 62 : 20.6 for the old cultures, respectively. The ageing of the culture involves transition of substructure I to substructure II. The substructures differ in the nuclease sensitivity. The specific features of enzyme fragmentation of chromatin from P. polycephalum microplasmodium nuclei are discussed.

Tubulin synthesis in the naturally synchronous plasmodium of Physarum polycephalum is a markedly periodic event restricted to the late G2 period of the cell cycle. Mitosis in the plasmodium is intranuclear, and there are no cytoplasmic microtubules at any stage of the cell cycle. We have combined a biochemical investigation of the synthesis of the plasmodial tubulin isotypes and their participation in the mitotic spindle with a microscopic study (immunofluorescence) of the development of spindle microtubules throughout the cell cycle. We have shown that all four tubulin isotypes identified in the plasmodium (alpha 1, alpha 2, beta 1 and beta 2) are present in the mitotic spindle. The stoichiometry of isotype usage in the mitotic spindle generally reflects the overall abundance of isotypes in the plasmodium as a whole: beta 2 greater than alpha 1 greater than alpha 2 greater than beta 1. We have also shown that tubulins synthesized in the G2 period of one cell cycle can be incorporated into the spindles of the immediately ensuing mitosis and have sufficient biological longevity to allow participation in the mitotic divisions of future cell cycles. Thus, the phenomenon of periodic tubulin synthesis does not reflect a restricted use of tubulin to the cell cycle in which it was synthesized. The major polymerization of tubulin in the nucleus occurred less than 30 min before metaphase. A novel tubulin-containing structure was, however, present in the nucleus approximately 60 min before metaphase. Polymerized tubulin is rapidly removed from the nucleus following nucleokinesis.

The haploid phase (myxamoebae-swarm cells) of the myxomycete Physarum flavicomum grew readily in chemical defined liquid media. The minimal medium contained salts, glucose, biotin, thiamine, hematin, glycine, L-arginine and L-methionine. Cell yields of 1.4 times 10(7) cells/ml were obtained in this medium in aerobic shake culture. These cells consumed about 35 muliters of oxygen/mg protein-hr in the minimal medium. The morphology of cells maintained in this medium appeared to be "normal". L-valine replaced either glycine or L-methionine in the minimal medium but the growth rates and cell yields were reduced. Growth rates increased in media containing four, seven, or fourteen amino acids.

The expression of an alpha-tubulin gene (altB1 (N alpha Tu) [(1987) J. Mol. Biol. 193, 427-438]) of Physarum polycephalum (strain CLdAXE) was found to be governed by a developmental switch since mRNA transcripts were detected, by S1 nuclease analysis, in the plasmodial but not the amoebal phase of the life-cycle. The conclusion that the altB1 (N alpha Tu) allele codes for a plasmodial specific alpha-tubulin isotype is supported by recent amino acid sequence data.

We reported previously that myosins from amoebal and plasmodial stages in the life cycle of Physarum polycephalum differ in the primary structure of heavy chains and phosphorylatable 18,000 Mr light chains, while Ca-binding 14,000 Mr light chains are common to both myosins (Kohama & Takano-Ohmuro, Proc Jpn acad 60B (1984) 431; Kohama et al., J biol chem 260 (1986) 8022). We have carried out immunofluorescence microscopical studies upon differentiating cultures of amoebic colonies, which show apogamic amoebo-plasmodial differentiation as follows: Typical amoebae differentiate into mono-nucleate intermediate cells with swollen nuclei and then into two or multi-nucleate young plasmodia (Anderson et al., Protoplasma 89 (1976) 29. Antibodies against plasmodial myosin heavy chain (PMHC) and 18,000 Mr plasmodial myosin light chain (PMLC18) stained intermediate cells and young plasmodia, but not typical amoebae. On the other hand, antibody against amoebal myosin heavy chain (AMHC) stained typical amoebae and intermediate cells--but not young plasmodia. Thus staining was detected using antibodies against both PMHC and AMHC in intermediate cells. Intermediate cells were also stained by antibody against another plasmodium-specific cytoskeletal protein, viz., high molecular weight actin-binding protein (HMWP). We propose that synthesis of myosin subunits switches immediately from amoebal to plasmodial type in mono-nucleate cells with swollen nuclei. This myosin switching is associated with the initiation of HMWP synthesis.

Isolated muscle and Physarum actins were labeled with various fluorochromes and microinjected into living stages of Physarum polycephalum (caffeine-droplets, endoplasmic drops, thin-spread macroplasmodia). Subsequent analysis of the intracellular redistribution by fluorescence microscopy, video-enhancement and digital image processing revealed RITC (rhodamineisothiocyanate) actin to be the most reliable molecular probe for the marking of microfilaments. In relaxed caffeine-droplets, the RITC-actin first diffuses randomly and then is locally incorporated into a thin cortical layer at the internal face of the plasma membrane. During Ca2+-induced contraction the fluorescent layer starts to detach from the plasma membrane, thus causing separation of central granuloplasm from peripheral hyaloplasm. Thin sections of both, relaxed and contracted specimens demonstrated that the RITC-actin layer in living droplets exactly coincides with a sheath of more or less oriented microfilaments. In contrast, RITC-bovine serum albumin (BSA) injected as control is excluded from those regions which show intense fluorescence with RITC-actin and the presence of an actin network with EM. Successful incorporation of the molecular probe into stages of Physarum polycephalum other than caffeine droplets was not yet achieved. The results obtained by fluorescent analog cytochemistry (FAC) are discussed with regard to the spatial organization of the actin system in acellular slime molds.

In growing plasmodia of the myxomycete Physarum polycephalum (G2-phase), three distinct classes of nuclei with a relative DNA content of 1x, 2x, and 4x are observed in the presumed haploid strain CL. The 2x and 4x species comprise up to 35% and 5% of the nuclei. Quantitative cytofluorometric studies of nuclei isolated in either G2- or S-phase or after FUDR treatment (G1 arrest) show that the three nuclear populations undergo a synchronous mitotic cycle and that the relative DNA content of the nuclear fractions in G-2 phase reflects the 2c, 4c, and 8c state. The heterogeneity of the nuclear population does, however, seem to be restricted to the growth phase. During a starvation period of 4 days that always preceeds sporulation (and also meiosis), the 4c nuclear population is reduced to 7%, 8c nuclei are no longer detected. These results suggest that a mechanism exists in Physarum for the selective detection and elimination of polyploid nuclei.

1. The proteins of amoebae and plasmodia of strain CL of Physarum polycephalum have been compared by two-dimensional gel electrophoresis. Both forms of the organism were labelled by growth on formalin-killed bacteria labelled with [35S]sulphate, [3H]lysine or [14C]lysine. Plasmodia were also labelled from radioactive lysine in the medium. 2. Of 306 relatively abundant proteins examined, 26% were phase-specific, that is they were found only in amoebae or in plasmodia. About a quarter of these apparent differences in gene expression may be due to minor changes in charge and/or size. 3. Amongst the 74% of the proteins present in both amoebae and plasmodia, there are substantial differences in differential rates of synthesis and these have been measured for a representative set of proteins by a double-label procedure.

Amoebae of the acellular slime mold Physarum polycephalum convert to plasmodia both asexually and sexually. Genetic analysis of a mutant that exhibits enhanced asexual plasmodium formation is reported. The mutant carries a single lesion (gad-11) located 12.3 map units from mt, a gene that controls mating specificity in sexual plasmodium formation. The mutation, which was isolated in an mt3 strain, is also expressed in mth and mt4 strains.

In a standard growing medium, the specific activities of acid-phosphatase, beta-galactosidase and beta-glucosidase of Physarum polycephalum increase during the growth of the culture. The decrease of the pH of the culture medium during the growth has no effect on the variations of these hydrolase activities. In a glucose-starved medium, the specific activity of beta-galactosidase increases up to 350% of its initial value in 24 h, whereas the specific activities of acid phosphatase and beta-glucosidase stay near their minimal level.

Surface and internal changes associated with transition from myxamoebal to microcyst stages in Physarum polycephalum have been observed with transmission and scanning electron microscopy. Nuclear morphology, granules studded with ribosomal-like particles, synthesis of polysaccharides which are deposited exterior to the cell, and origin of material for the cell wall appear to be similar in the amoeba and in the plasmodium. In addition, the viability of the strain used in this research after being allowed to remain in a quiescent or encysted stage was observed to be approximately six months.

When synchronous plasmodia of the myxomycete Physarum polycephalum were submitted to temperature shifts from 22 degrees C to 32 degrees C, the highest physiological temperature, protein synthesis was increased during at least 10 h. Moreover during 2 h, four proteins (69, 74, 82 and 105 kDa) showed a transient increase of their synthesis, independently of the period of the temperature shift during the cell cycle. The stability of these proteins and the susceptibility of their synthesis to actinomycin suggested that they corresponded to four different proteins. Temperature shifts from 22 degrees C or 29 degrees C to 37 degrees C, a non-physiological temperature, demonstrated that the 69-kDa, 74-kDa, 82-kDa and 105-kDa proteins were identical to the four heat-shock proteins which could be detected in Physarum. Although the physiological significance of these heat-shock proteins remained unclear, comparison between the extent of their synthesis and the length of the mitotic delays induced by various temperature shifts ruled out a direct relationship between mitotic delays and synthesis of the 74-kDa, 82-kDa and 105-kDa proteins.

Using a selfing strain of Physarum polycephalum that forms haploid plasmodia, we have isolated temperature-sensitive growth mutants in two ways. The negative selectant, netropsin, was used to enrich for temperature-sensitive mutants among a population of mutagenized amoebae, and, separately, a nonselective screening method was used to isolate plasmodial temperature-sensitive mutants among clonal plasmodia derived from mutagenized amoebae. Complementation in heterokaryons was used to sort the mutants into nine functional groups. When transferred to the restrictive temperature, two mutants immediately lysed, whereas the remainder slowed or stopped growing. Of the two lytic mutants, one affected both amoebae and plasmodia, and the other affected plasmodia alone. The growth-defective mutants were examined for protein and deoxyribonucleic acid synthesis and for aberrations in mitotic behavior. One mutant may be defective in both protein and deoxyribonucleic acid synthesis, and another only in deoxyribonucleic acid synthesis. The latter shows a striking reduction in the frequency of postmitotic reconstruction nuclei at the restrictive temperature. We believe that this mutant, MA67, is affected in a step in the nuclear replication cycle occurring late in G2. Execution of this step is necessary for both mitosis and chromosome replication.

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.