Microplasmodia Of Physarum Polycephalum Research Articles

Cytotoxicity activities and chemical characteristics of exopolysaccharides and intracellular polysaccharides of Physarum polycephalum microplasmodia

BackgroundMicrobial polysaccharides have been reported to possess remarkable bioactivities. Physarum polycephalum is a species of slime mold for which the microplasmodia are capable of rapid growth and can produce a significant amount of cell wall-less biomass. There has been a limited understanding of the polysaccharides produced by microplasmodia of slime molds, including P. polycephalum. Thus, the primary objectives of this research were first to chemically characterize the exopolysaccharides (EPS) and intracellular polysaccharides (IPS) of P. polycephalum microplasmodia and then to evaluate their cytotoxicity against several cancer cell lines.ResultsThe yields of the crude EPS (4.43 ± 0.44 g/l) and partially purified (deproteinated) EPS (2.95 ± 0.85 g/l) were comparable (p > 0.05) with the respective crude IPS (3.46 ± 0.36 g/l) and partially purified IPS (2.45 ± 0.36 g/l). The average molecular weight of the EPS and IPS were 14,762 kDa and 1788 kDa. The major monomer of the EPS was galactose (80.22%), while that of the IPS was glucose (84.46%). Both crude and purified IPS samples showed significantly higher cytotoxicity toward Hela cells, especially the purified sample and none of the IPSs inhibited normal cells. Only 38.42 ± 2.84% Hela cells remained viable when treated with the partially purified IPS (1 mg/ml). However, although only 34.76 ± 6.58% MCF-7 cells were viable when exposed to the crude IPS, but the partially purified IPS displayed non-toxicity to MCF-7 cells. This suggested that the cytotoxicity toward MCF-7 would come from some component associated with the crude IPS sample (e.g. proteins, peptides or ion metals) and the purification process would have either completely removed or reduced amount of that component. Cell cycle analysis by flow cytometry suggested that the mechanism of the toxicity of the crude IPS toward MCF-7 and the partially purified IPS toward Hela cells was due to apoptosis.ConclusionsThe EPS and IPS of P. polycephalum microplasmodia had different chemical properties including carbohydrate, protein and total sulfate group contents, monosaccharide composition and molecular weights, which led to different cytotoxicity activities. The crude and partially purified IPSs would be potential materials for further study relating to cancer treatment.

Oscillatory motion of a droplet in an active poroelastic two-phase model

The onset of self-organized droplet motion is studied in a poroelastic two-phase model with free boundaries and substrate friction. In the model, an active, gel-like phase and a passive, fluid-like phase interpenetrate on small length scales. A feedback loop between a chemical regulator, mechanical deformations, and induced fluid flow gives rise to oscillatory and irregular droplet motion accompanied by spatio-temporal contraction patterns inside the droplet. By numerical simulations in one spatial dimension, we cover extended parameter regimes of active tension and substrate friction, and reproduce experimentally observed oscillation periods and amplitudes. In line with recent experiments, the model predicts alternating forward and backward fluid flow at the boundaries with reversed flow in the center. Our model is a first step towards a more detailed model of moving microplasmodia of Physarum polycephalum.

Coordination of contractility, adhesion and flow in migrating Physarum amoebae.

This work examines the relationship between spatio-temporal coordination of intracellular flow and traction stress and the speed of amoeboid locomotion of microplasmodia of Physarum polycephalum. We simultaneously perform particle image velocimetry and traction stress microscopy to measure the velocity of cytoplasmic flow and the stresses applied to the substrate by migrating Physarum microamoebae. In parallel, we develop a mathematical model of a motile cell which includes forces from the viscous cytosol, a poro-elastic, contractile cytoskeleton and adhesive interactions with the substrate. Our experiments show that flow and traction stress exhibit back-to-front-directed waves with a distinct phase difference. The model demonstrates that the direction and speed of locomotion are determined by this coordination between contraction, flow and adhesion. Using the model, we identify forms of coordination that generate model predictions consistent with experiments. We demonstrate that this coordination produces near optimal migration speed and is insensitive to heterogeneity in substrate adhesiveness. While it is generally thought that amoeboid motility is robust to changes in extracellular geometry and the nature of extracellular adhesion, our results demonstrate that coordination of adhesive forces is essential to producing robust migration.

Lack of nucleosomal structure in a DNase-I-solubilized transcriptionally active chromatin fraction of Physarum polycephalum.

Light treatment of nuclei of Physarum polycephalum microplasmodia with DNase I, at low MgCl2 concentration (less than or equal to 3% DNA acid solubility, 0.1 mM MgCl2) selectively solubilizes a defined fraction of chromatin, in the form of a macromolecular complex. This fraction (up to 15% of the total chromatin) contains a full complement of the core histones and a reduced amount of histone H1, and is enriched in the high-mobility-group type of proteins. It is preferentially associated with nascent RNA and RNA polymerase B actively engaged in transcription. Digestion of DNAase-I-solubilized chromatin by micrococcal nuclease releases a size-heterogeneous population of cleavage products, indicative of lack of a typical nucleosomal packaging. It is concluded that the procedure used allows the isolation of structurally and functionally distinct regions of Physarum chromatin.

A cDNA cloned from Physarum polycephalum encodes new type of family 3 β-glucosidase that is a fusion protein containing a calx-β motif

The microplasmodia of Physarum polycephalum express three types of β-glucosidases: secretory enzyme, a soluble cytoplasmic enzyme and a membrane-bound enzyme. We are interested in the physiological role of three enzymes. We report the sequence of cDNA for membrane β-glucosidase 1, which consists of 3825 nucleotides that includes an open reading frame encoding 1248 amino acids. The molecular weight of membrane β-glucosidase 1 was calculated to be 131,843 based on the predicted amino acid composition. Glycosyl hydrolase family 3 N-terminal and C-terminal domains were found within the N-terminal half of the membrane β-glucosidase 1 sequence and were highly homologous with the primary structures of fungal β-glucosidases. Notably, the C-terminal half of membrane β-glucosidase 1 contains two calx-β motifs, which are known to be Ca 2+ binding domains in the Drosophila Na +/Ca 2+ exchanger; an RGD sequence, which is known to be a cell attachment sequence; and a transmembrane region. In this way, Physarum membrane β-glucosidase 1 differs from all previously identified family 3 β-glucosidases. In addition to cDNA for membrane β-glucosidase 1, two other distinctly different mRNAs were also isolated. Two sequences were largely identical to cDNA for membrane β-glucosidase 1, but included a long insert sequence having a stop codon, leading to truncation of their products, which could account for other β-glucosidase forms occurred in Physarum poycephalum. Thus, the membrane β-glucosidase is a new type family 3 enzyme fused with the Calx-β domain. We propose that Calx-β domain may modulate the β-glucosidase activity in response to changes in the Ca 2+ concentration.

Beta-poly(L-malate) production by non-growing microplasmodia of Physarum polycephalum. Effects of metabolic intermediates and inhibitors.

The production of beta-poly(L-malate) (PMLA) by non-growing microplasmodia of Physarum polycephalum was investigated. Growth was minimal in culture medium devoid of nitrogen source, but PMLA production occurred at a substantial rate. The addition of metabolic intermediates, malate, fumarate, succinate, and oxaloacetate, and the omission of hematin showed considerable growth inhibition in the presence of the nitrogen source, while PMLA production per unit biomass increased significantly. The results indicated that PMLA production was dissociated from biomass production under these conditions. The stimulating effect of carbonate on PMLA production was independent on growth. Cultivation in the absence of the nitrogen source and hematin or in the presence of the metabolites may be a useful technique for efficient PMLA production at a minimum of biomass production.

β-Poly(L-malate) production by non-growing microplasmodia of Physarum polycephalum: Effects of metabolic intermediates and inhibitors

The production of β-poly(L-malate) (PMLA) by non-growing microplasmodia of Physarum polycephalum was investigated. Growth was minimal in culture medium devoid of nitrogen source, but PMLA production occurred at a substantial rate. The addition of metabolic intermediates, malate, fumarate, succinate, and oxaloacetate, and the omission of hematin showed considerable growth inhibition in the presence of the nitrogen source, while PMLA production per unit biomass increased significantly. The results indicated that PMLA production was dissociated from biomass production under these conditions. The stimulating effect of carbonate on PMLA production was independent on growth. Cultivation in the absence of the nitrogen source and hematin or in the presence of the metabolites may be a useful technique for efficient PMLA production at a minimum of biomass production.

Effects of culture conditions on β-poly( l -malate) production by Physarum polycephalum

Culture conditions for the fermentative production of β-poly(l-malate) (PMLA) by microplasmodia of Physarum polycephalum were investigated and optimized. Optimal production was achieved in the presence of CaCO3. For 1.5% (w/v) d-glucose, 1% bactotryptone and 1% CaCO3, a maximum of 1.7 g PMLA/l was secreted in 3 days. For 4.5% glucose and otherwise the same conditions, 2.7 g PMLA/l was produced in 6 days. The contribution of carbonate was inhibited by avidin. PMLA and biomass production were not strictly coupled: PMLA was also synthesized at the beginning of the stationary phase. At pH 5.5 PMLA production was twice that at pH 4.0, while biomass was not changed. Optimal temperatures were 24–28 °C.

The Actin-binding Properties of the Physarum Actin-Fragmin Complex: REGULATION BY CALCIUM, PHOSPHOLIPIDS, AND PHOSPHORYLATION

The actin-binding properties of the actin-fragmin complex from Physarum polycephalum microplasmodia were investigated with respect to regulation by Ca2+, phospholipids, and phosphorylation of the actin subunit by the endogenous actin-fragmin kinase. Fragmin possesses two high affinity actin-binding sites and probably also a third, low affinity site. Its nucleating and F-actin severing activities are inhibited by phosphatidylinositol 4,5-bisphosphate (PIP2). Actin-fragmin specifically binds PIP2 which competes with actin for the Ca(2+)-sensitive site. However, PIP2 cannot dissociate the actin-fragmin complex nor the actin2-fragmin trimer. Efficient F-actin nucleating activity by actin-fragmin is only observed with unphosphorylated actin-fragmin, in the absence of PIP2 and at high Ca2+ (> microM) concentrations. In the presence of PIP2, actin-fragmin only caps actin filaments when unphosphorylated. The results suggest that in the cell, hydrolysis of PIP2, concomitant with the increase of cytosolic Ca2+, could promote subcortical actin polymerization.

Purification and partial amino acid sequence of the actin-fragmin kinase from Physarum polycephalum.

The 80-kDa actin-fragmin kinase (AFK) was purified from Physarum polycephalum microplasmodia to apparent homogeneity through a procedure involving six chromatographic steps. Taking the activity at the first purification step as 100%, the kinase was purified more than 1500-fold, with an overall yield of 8%. The specific activity of the purified enzyme was 700 U/mg. The total amount of AFK present could be estimated as 34 ng/mg extracted protein. One of the polyclonal antibodies raised against four tryptic peptides of the purified 80-kDa AFK recognized the 80-kDa band in an immunoblot and could inhibit the AFK activity up to 100%. A minor 78-kDa protein, also displaying AFK activity, appeared to be derived from the 80-kDa AFK. A partial amino acid sequence analysis, covering up to 20% of the protein (150 amino acids), was performed and confirmed the lack of similarity with any of the sequenced kinases. These data are in agreement with the unique physical and enzymatic properties of the AFK.

Electron microscopic observations of an unusual cytoplasmic inclusion in the calcium-starved white mutant (LU887 × LU897) of Physarum polycephalum

Electron micrographs of Physarum polycephalum microplasmodia (LU887 × LU897) reveal cytoplasmic inclusions that appear “striated” at low magnifications; at higher magnifications these exhibit a structure that we have interpreted as microtubule bundles. The light and dark regions in the inclusions are due to the affinity of some microtubules for osmic acid; these appear to have dense regions while other microtubules remain electron lucent. The diameters of the microtubules are about 32–33nm; the subunits forming the tubule walls measure about 8–9nm in diameter. The diameter measurements are slightly larger than the dimensions assigned to vertebrate microtubules (28nm); however, the diameter of the subunits in the microtubule wall measures about 8–9nm which is essentially the same measurement reported for vertebrate tubulin dimers.

Isolation, characterisation and growth-related changes of an HMG-like protein from microplasmodia ofPhysarum polycephalum

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.

An unusual polyanion from Physarum polycephalum that inhibits homologous DNA polymerase alpha in vitro.

From extracts of microplasmodia of Physarum polycephalum and their culture medium, an unusual substance was isolated which inhibited homologous DNA polymerase alpha of this slime mold but not beta-like DNA polymerase and not heterologous DNA polymerases. Analysis, especially NMR spectroscopy, revealed the major component to be an anionic polyester of L-malic acid and the inhibition to be due to poly(L-malate) in binding reversibly to DNA polymerase alpha. The mode of inhibition is competitive with substrate DNA and follows an inhibition constant Ki = 10 ng/mL. Inhibition is reversed in the presence of spermine, spermidine, poly(ethylene imine), and calf thymus histone H1. According to its ester nature, the inhibitor is slightly labile at neutral and instable at acid and alkaline conditions. Its largest size corresponds to a molecular mass of 40-50 kDa, but the bulk of the material after purification has lower molecular masses. The inhibitory activity depends on the polymer size and has a minimal size requirement.

Boundary potential and membrane potential of Physarum polycephalum: effects of electrolyte ions and chloroform

The boundary potential and the membrane potential of microplasmodia of Physarum polycephalum were measured in the presence of electrolyte ions and chloroform and the results were analyzed with the aid of a new membrane model. The Donnan potential and the membrane potential, relative to the potential value at an ionic strength of 0.005, increased in manners quite similar to each other as the ionic strength of the medium increased, suggesting that the diffusion potential of microplasmodia was not noticeably affected by the variation in the ionic strength. K + ion and Na + ion seemed to exert influence dissimilarly on the diffusion potential because these two monovalent cations were found to affect differently the Donnan potential and the membrane potential of microplasmodia. As the concentration of Ca 2+ ion or Mg 2+ ion increased, the surface charge density calculated from the surface potential decreased steeply and reversed its sign after passing through the point of zero charge, due to the adsorption of the divalent cation to microplasmodia. The binding constants of Ca 2+ and Mg 2+ ions were evaluated to be 125 and 167 M −1, respectively. In contrast to the monovalent cations, the divalent cations caused no appreciable change in the diffusion potential of microplasmodia. The Donnan potential as well as the membrane potential of the model cells exhibited a sudden change of about 12 mV when 0.2 m M chloroform was added to the medium. This was interpreted as showing that the inhalation anesthetic caused a phase transition in the cell membrane.

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.

Characterization of glycopeptides from Physarum polycephalum labeled with (3H)mannose or (3H)glucosamine.

Glycopeptides from the microplasmodia of Physarum polycephalum grown in the presence of [3H]mannose or [3H]glucosamine were characterized by gel-filtration, anion-exchange and lectin-affinity chromatography before and after enzymatic or chemical treatment. Various types of glycopeptide were found in the growing plasmodia as shown by Con A affinity column chromatography, while pulse-labeled precursors were composed mainly of mannose-rich glycopeptides which were sensitive to endo H and α-mannosidase. During the processing of the glycoprotein, the mannose-rich glycopeptides changed to larger glycopeptides which were resistant to endo H and α-mannosidase. These processing reactions of the glycoprotein appeared to be blocked when the plasmodia underwent differentiation to macrocysts.

Cyanide-resistant respiration of [formula omitted] in course of starvation

The inhibition of respiration of Physarum polycephalum microplasmodia by KCN varies between 30% and 70% of initial rate of O 2 uptake, depending on the time of starvation. The kinetics of the development of cyanide-resistant respiration and its sensitivity toward salicylhydroxamic acid (SHAM) point out that CN-resistant respiration represents the activity of the alternative pathway of the electron transport. There is no evidence that during starvation the alternative pathway of respiration is active in the absence of cyanide.

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.

Structure of the extrachromosomal ribosomal RNA chromatin of Physarum polycephalum

Isolated nucleoli from exponentially growing microplasmodia of Physarum polycephalum were digested with micrococcal nuclease or DNAase I, or were photoreacted with trimethyl psoralen. In the coding region for the precursor of the ribosomal RNA, micrococcal nuclease and DNAase I digestions show predominantly a smear, and treatment with psoralen leads to a fairly continuous crosslinking of the DNA. All three assays are compatible with the absence of a typical nucleosomal array in most of the gene copies. In contrast, in the central non-transcribed spacer, except in the immediate 5′-flanking region, micrococcal nuclease and DNAase I digestions yield fragments that are multiples of a basic repeat, compatible with a nucleosomal packing of this region. The crosslinking pattern with psoralen confirms this conclusion. In addition, there are three sites over 400 base-pairs long that are inaccessible for psoralen crosslinking. Two of these sites have been mapped to the putative origins of replication. In the terminal non-transcribed spacer, except in the immediate 3′-flanking region, digestions with micrococcal nuclease and DNAase I give a smeared repeat. The crosslinking pattern after treatment with psoralen suggests that this region is packed in nucleosomes, except for about 900 base-pairs constituting the telomere regions of the linear extrachromosomal palindromic rDNA. Micrococcal nuclease digestion of the immediate 5′-flanking region shows a complete absence of any nucleosomal repeat, but digestion with DNAase I leads to a faint ten base-pair repeat. In contrast, in the 3′-flanking regions both nuclease assays indicate a chromatin structure similar to the coding region. Both flanking regions are unusual with respect to psoralen crosslinking, in that crosslinking is reduced both in chromatin and deproteinized DNA. On the basis of the known sequence-dependent psoralen crosslinking and the established sequences in these regions, crosslinking should be expected to occur. However, it does not and we therefore propose the presence of an unusual DNA conformation in these regions.

Studies on microplasmodia ofPhysarum polycephalum

The new technique of fluorescent analog cytochemistry was used to investigate the cell surface morphology (RITC-WGA staining), the organization of the microfilament system (Rh-phalloidin staining) and the spatial distribution of mitochondria (Rh-123 staining) in the various growth stages of axenically cultured living and fixed microplasmodia ofPhysarum polycephalum. The differentiation degree of the cell surface is generally size- and age-dependent: the invagination system develops by degrees from small spherical stages (50–100 μm) without invaginations to large vein-like or dumbbell-shaped specimens (300–1,000 (μm long) with extensive invagination systems. The microfilaments are always organized in a cortical system along the entire cell surface and sometimes in a fibrillar system as well, extending throughout the cytoplasmic matrix. Results on living microplasmodia demonstrate that the cortical microfilament system is mainly involved in motive force generation for changes of cell surface morphology and protoplasmic streaming activity, whereas the fibrillar system rather serves a stabilizing and adhering function. Moreover, the functional differences of the two microfilament systems are indicated by the position of a large population of stationary mitochondria in close vicinity to the cell surface, thus pointing to a reasonable arrangement of the energy-supplying and energy-transforming system.