Polysphondylium Research Articles

The presence and location of sporopollenin in fruiting bodies of the cellular slime moulds.

The presence of an acetolysis-resistant polymer (sporopollenin) in the cellular slime moulds is demonstrated. This polymer is located on the stalk sheath of fruiting bodies as a bundle of fine fibrils (4-5 nm diameter). The location and structure of sporopollenin in spores are shown to vary considerably, depending upon the species. In Polysphondylium violaceum spores, sporopollenin is composed of fine spicules (4-5 nm in diameter, 25-50 nm long) that cover both the outermost layer of spore wall and the inner surface of the cell membrane. The sporopollenin of Dictyostelium discoideum spores is located preferentially close to the inner surface of the cell membrane, forming a mass of electron-opaque fine granules (4-5 nm in diameter). D. mucoroides spores, however, appear not to possess a tight network of sporopollenin, since they were less resistant to acetolysis than those of the other species. The biological significance of the results is discussed with special reference to fruiting body formation.

Extracellular cyclic AMP during development of the cellular slime mold Polysphondylium violaceum: comparison of accumulation in the wild type and an aggregation-defective mutant.

Cyclic AMP was synthesized by Polysphondylium violaceum after starvation and during the preaggregation stage of development. Most of the newly synthesized cyclic AMP accumulated in the extracellular medium, with very little change in the intracellular cyclic AMP concentration. The addition of 10(-3) to 10(-6) M exogenous cyclic AMP to starved amoebae caused a 20 to 50% decrease in the number of aggregation centers formed compared with untreated controls. An aggregation-defective mutant of P. violaceum (strain aggA586) excreted or accumulated very little cyclic AMP. Strain aggA586 aggregated normally in the presence of a dialyzable, excreted product (D factor) produced by wild-type amoebae. When the mutant was incubated with D factor, cyclic AMP accumulated in the medium, and the amount accumulated depended on the amount of D factor added to the mutant amoebae.

The timing of phenotypic suppression of an aggregation defect by an aggregation-stimulating factor from Polyspboadylium violaceum

A class of aggregation deficient mutants of Polysphondylium violaceum ( aggA) had previously been shown to aggregate in the presence of an excreted, dialyzable product (D factor) prepared from wild type amoebae. We have characterized further the development of the aggA mutants in liquid culture. In the absence of an external source of D factor, aggA mutants never become aggregation competent. D factor must be added to the mutants in order for them to be able to aggregate when removed from liquid culture and plated on a surface. The ability of D factor to stimulate the development of aggregation competence can be illustrated with both the aggA mutant and wild type amoebae. D factor is only required by the aggA mutants at a late stage in development of aggregation competence and does not have to be present continuously during incubation. Wild type amoebae provided with additional D factor become aggregation competent earlier than amoebae incubated without additional D factor. These data suggest that the amoebae develop most of the biochemical functions necessary in order to aggregate and that D factor is necessary to trigger aggregation. One of these biochemical functions, development of aggregation-specific adhesion sites, has been shown to occur in the aggA mutant in the absence of D factor.

The Role of Light and an Aggregation Stimulating Factor During Aggregation of Polysphondylium violaceum

SUMMARY: Light has been shown to affect aggregation and founder cell development in the cellular slime mould Polysphondylium violaceum. An aggregation-stimulating factor, D factor, also affects aggregation of P. violaceum wild-type and aggregation-defective mutants in the complementation group aggA. Both light and D factor cause premature aggregation of amoebae and increase the number of aggregate centres formed. In addition, D factor is able to stimulate aggregation of wild-type amoebae at densities where aggregation would not normally occur. Several experiments show that amoebae are more sensitive to D factor in the presence of light, with optimum aggregation occurring in the presence of both light and D factor. Development of founder cells has been observed in the aggA mutants in the absence of D factor. However, these founder cells are inactive and do not produce aggregates until D factor is added. This class of inactive founder cells can be detected in wild-type amoebae. Production of D factor is also dependent on light with a 20- to 60-fold decrease in production over that occurring in darkness. A complex relationship between D factor, light and founder cell differentiation is established by this study.

Chemical identity of the acrasin of the cellular slime mold Polysphondylium violaceum.

The aggregation chemoattractant (or acrasin) of Polysphondylium violaceum, a species of cellular slime mold that does not respond chemotactically to cAMP, has been identified. It was extracted and purified from aggregating amoebae, then analyzed for amino acid composition and by IR and mass spectrometry. The active molecule is N-propionyl-gamma-L-glutamyl-L-ornithine-delta-lactam ethyl ester (Mr, 327), which we have named glorin. The compound has been synthesized and shows normal chemotactic activity with the amoebae of P. violaceum.

Calcium, cellular adhesion and aggregation competence in the cellular slime mold Polysphondylium violaceum

Cellular adhesion in Polysphondylium violaceum is mediated by Ca 2+ ions. The extent of cell adhesion exhibited by developing P. violaceum is greater in the presence of 0.5 mM Ca 2+ than in the absence of Ca 2+. Vegetative amebae exhibit some adhesive properties, although the cellular interactions expressed by vegetative amebae are not as extensive as those exhibited by developing amebae. If the amebae are incubated in the presence of chelators (EGTA or EDTA) cellular adhesion is prevented and the amebae remain as single cells. Vegetative cell adhesion is blocked by 1 mM EGTA, whereas blocking adhesion in developing cells requires 5- to 10-fold greater concentrations of EGTA. The acquisition of developmental adhesive properties occurs even if the amebae are incubated in the presence of EGTA, suggesting that Ca 2+ is required for interaction between adhesion sites but not for their formation. P. violaceum amebae become aggregationcompetent (aggregate immediately when placed on a solid surface) at the same time that the developmental adhesion sites are expressed, even when incubated in the presence of EGTA. Thus it seems unlikely that cellular adhesion is required to develop aggregation competence.

Acid proteinases in various species of cellular slime mold

The proteolytic activities of the cellular slime molds Dictyostelium mucoroides, Dictyostelium purpureum, Polysphondylium pallidum, and Polysphondylium violaceum have been examined. Myxamoebae possessed activity against Hide Powder Azure at pH 2–5 which was enhanced by dithiothreitol: this enhancement was small in Dictyostelium species but three- to four-fold in the Polysphondylium species. Following electrophoresis on polyacrylamide gels containing denatured haemoglobin five or more proteinases could be detected in each species. Activity against Hide Powder Azure was inhibited severely by HgCl 2 and to a lesser extent by other thiol proteinase inhibitors such as N-α- p-tosyl- l-lysine chloromethyl ketone-HCl, antipain, and leupeptin. Inhibitors of aspartyl and serine proteinases had no effect. All proteinases visualized on gels were inhibited by HgCl 2, and some, but not the major one of each species, were sensitive to the other thiol proteinase inhibitors. Extracts of fruiting bodies retained acid proteolytic activity. New proteinases were detected in D. mucoroides; there was a relative increase in one proteinase in P. violaceum but three proteinases were lost during fruiting body formation in P. pallidum. During microcyst formation in P. pallidum there was a decrease in proteolytic activity but most of the myxamoebal proteinases could be detected. Overall the results demonstrate that the cellular slime molds possess similar types of proteinase although there were significant differences between the actual proteinases observed in individual species.

CELLULAR SLIME MOLDS OF SWITZERLAND. II. DISTRIBUTION IN FOREST SOILS

A detailed analysis of occurrence and distribution of cellular slime molds in soils of different forest types of Switzerland is given. Specific prevalence patterns representative for each forest type were determined. The degree of complexity of these prevalence patterns parallels tree diversity of the respective forest communities. A comparison of the prevalence patterns shows that by proceeding from species‐rich to species‐poor sites the frequent and common species disappear, whereas the infrequent and rare species are conserved. Thus, these latter species, which probably represent specialists, appear to be of considerable ecological significance.Dictyostelium mucoroides is the overwhelming dominant in all forest types. Polysphondylium violaceum and D. minutum are abundant in the deciduous forests. These three species account for 87% of the clones isolated. Two species are characteristic of conifer forests, D. aureo‐stipes var. Helvetium and D. polycarpum. Other species encounterd and listed in decreasing order are D. fasciculatum. P. filamentosum, D. sphaerocephalum, D. giganteum, Acytostelium leptosomum, D. aureum var. luteolum and D. polycephalum. Diversity of species is greatest in the mesic beech forests of the Midland, Jura or Forealps regions but reduced populations containing unique cellular slime molds occur in the highest spruce, larch, and pine forests of the Alps.

Species-specific cell surface changes during the development in the cellular slime moulds.

Concanavalin A-mediated cell surface changes during development have been studied in four species of the cellular slime moulds, Dictyostelium discoideum, D. mucoroides, D. purpureum and Polysphondylium violaceum. Strains belonging to the same species showed a similar pattern of changes during the course of development. The pattern was similar for D. discoideum and D. mucoroides. These two species showed a gradual decrease in Con A-mediated cell agglutination as development proceeded. In D. purpureum and P. violaceum, however, the cell agglutinability increased at the beginning of development and then decreased thereafter. The results therefore indicate that cell-surface changes during development are species-specific.

Evidence for aggregation center induction by the ionophore A23187 in the cellular slime mold Polysphondylium violaceum

A thin glass fiber coated with the ionophore A23187 placed among preaggregation amoebae of Polysphondylium violaceum will induce numerous aggregation centers along the fiber. Both calcium and magnesium ions appear to be involved in this induction. A23187 is aiso seen to disrupt the normal cell streaming process by producing secondary centers in the aggregation streams.

A one-dimensional pattern in the cellular slime mould Polysphondylium pallidum

The cellular slime mould Polysphondylium pallidum has a fruiting body with whorls of branches spaced at regular intervals along the stalk (Fig. 1). The spacing between successive whorls is quite regular and under genetic control1. Because stalk cell number per interval correlates strongly with interval length, we have studied the influence of ploidy on the distance between whorls to determine if cell counting could account for the spacing. It is known that diploid spores and amoebae of Dictyostelium discoideum and Polysphondylium violaceum are roughly twice the size of their haploid parents2,3, and we find that stalk cells in P. pallidum follow the same pattern. Our results show that the number of stalk cells between successive whorls in haploids is approximately twice the number in diploids, while the spacing remains the same. This clearly indicates that spacing is not achieved by a cell counting mechanism. Instead, it appears to depend on distance.

Evidence for differential cellular adhesion as the mechanism of sorting-out of various cellular slime mold species

Various authors have shown previously that if the amoebae of two species of slime molds are mixed they have the ability to sort themselves out. In the work reported here, the sorting-out of cells of several slime mold species was examined in clumps of cells in suspension. Cells of four species, Dictyostelium discoideum (Dd), D. mucoroides (Dm), D. purpureum (Dp), and Polysphondylium violaceum (Pv), were mixed in pairs in suspension and clumps of cells formed. Dd and Pv cells sorted out completely and formed separate clumps, each of single species. Both Dd and Dm, when mixed separately with Dp, formed clumps containing both species. Sorting-out took place in these clumps such that the cells of Dd and Dm partially enveloped the Dp cells. Finally, in the Dd-Dm mixtures, the Dm cells always sorted out such that they surrounded the Dd cells. When mixed in a 1:2 ratio (Dd:Dm) the Dm cells formed a complete shell around a sphere of Dd cells. Sorting-out of cells in clumps in suspension can occur by either of two possible mechanisms: response of cells to a chemotactic gradient or differences in cell surface strengths of adhesion (Steinberg, 1964). Mixing of two species in a clump of cells and observing the process of sorting-out permits one to distinguish between these two mechanisms (Steinberg, 1964). By such an analysis it was found that the sorting-out observed in mixtures of Dd and Dm is consistent with the mechanism of differential cellular adhesion. The major reasons for this are (1) when the adhesive properties of the cells are known to change the Dd cells began to move inside the clumps, (2) the Dd cells coalesced into islands rather than streaming inward independently, and (3) the Dd cells and cell masses did not lie at the center of the clumps but rather lay randomly within the clumps. The partial envelopment observed in the Dd-Dp and Dm-Dp mixtures and the separate clumps formed by the Dd-Pv mixtures are also consistent with differential cellular adhesion. They represent cases in which the interspecific strengths of adhesion are low (Dd-Dp and Dm-Dp) and near zero (Dv-Pv).

Cyclic nucleotides and cyclic nucleotide phosphodiesterase during development of Polysphondylium violaceum

Polysphondylium violaceum is shown to produce and excrete cyclic nucleotides and to produce a cell-associated cyclic nucleotide phosphodiesterase(s). The amount of adenosine 3′,5′-cyclic monophosphate (cAMP) excreted by the amebae reaches a maximum during development when aggregation centers are just forming and then falls off rapidly. Measurements of total cAMP show that the amount synthesized increases more than 15-fold throughout development with the majority of the increase coming during the culmination stages. Guanosine 3′,5′-cyclic monophosphate (cGMP) is either not excreted or is excreted at levels below our limits of detection. An increase in the total cGMP synthesized occurs at mid-aggregation when two or three sharp peaks of synthesis are observed. However, development of P. violaceum is not affected by the addition of high concentrations of either cAMP or cGMP (or their dibutyryl derivatives) to the medium despite the fact that the cells produce these nucleotides. Cell-associated cyclic nucleotide phosphodiesterase activity, which hydrolyses both cAMP and cGMP, is greatest at the onset of starvation with a second increase in activity during aggregation.

Immunohistochemical Localization of Cyclic GMP in Aggregating Polysphondylium violaceum

Using indirect immunofluorescence technique, it has been possible to localize cyclic GMP in Polysphondylium violaceum cells. The bound cyclic nucleotide is localized throughout the cell during early stages, however, this staining increases and there is marked localization of cyclic GMP in the nuclear areas of the cells when aggregation is in full swing. Over 90% of the cells exhibited intense nuclear staining by 6 h and this decreased to less than 10% by 10 h.

Intracellular cyclic GMP-binding proteins in cellular slime molds

The complexity of cyclic GMP-binding activity in the 48,000 X g supernatant of three species of the cellular slime molds (Dictyostelium discoideum, Dictyostelium rosarium, and Polysphondylium violaceum) was studied by gel filtration chromatography on AcA 34 Ultrogel. All these species have in common a cyclic GMP-binding protein of molecular weight of about 2.5 X 10(5) which specifically binds this nucleotide. In addition, Scatchard plots of assays carried out with the 48,000 X g supernatant of these species exhibit cyclic GMP-binding activity with an apparent dissociation constant of about 1 nM. None of the cyclic GMP-binding proteins separated by chromatography on AcA 34 Ultrogel was associated to protein kinase activity stimulation. In view of the cyclic GMP function during chemotactic transduction in the cellular slime molds, the possible molecular function for this 2.5 X 10(5)-dalton cyclic GMP-binding protein is discussed.

Inactivation of the chemoattractant folic acid by cellular slime molds and identification of the reaction product.

Enzymes inactivating the chemoattractants folic acid and pterin were detected in extracellular, intracellular, and particulate fractions obtained from Dictyostelium discoideum strains NC4 and AX-2 and Polysphondylium violaceum. The products of the inactivation reaction were analyzed by thin-layer chromatography and UV spectroscopy. Results obtained indicate that folic acid and pterin were deaminated to 2-deamino folic acid and lumazine, respectively.

Distribution of Cellular Slime Molds in Wisconsin Prairie Soils

Cellular slime molds were isolated from 75% of soil samples from 50 prairie remnants in southern Wisconsin. Total populations (cells per g of soil) were greatest in wet sites, intermediate in wet-mesic and mesic sites, and least in dry-mesic sites. Populations were slightly higher in dry than in dry-mesic sites, possibly because soils in dry sites had higher water-retaining capacities than soils in dry-mesic sites. Dominant species included Polysphondylium violaceum in wet prairies, Dictyostelium giganteum and P. violaceum in mesic prairies, and P. pallidum and D. sphaerocephalum in dry prairies. Dictyostelium sp. (YS) and P. pallidum both had greater populations at the two extremes of the moisture gradient than in mesic sites. Other species isolated included D. discoideum, D. lacteum, D. mucoroides, D. polycephalum, D. purpureum and D. rosarium. Average species diversity ranged from 3.2 species per site in dry-mesic prairies to 4.6 per site in wet-mesic prairies.

Mutual cohesion and cell sorting-out among four species of cellular slime moulds.

Interspecific cell cohesion among 4 species of cellular slime moulds, Dictyostelium discoideum, D. mucoroides, D. purpureum and Polysphondylium violaceum has been studied. Binary mixtures of aggregation-stage cells of the 4 species were shaken in suspension, one species of each pair being labelled with [3H]thymidine. Cell aggregates were sampled at intervals over 24 h and their composition examined by autoradiography. The following results were obtained: (i) Cells of each species were capable of cohesion with those of the other 3 species. (ii) In general cells of both species in any mixture were present in aggregates after 1 h, but were not localized according to species. (iii) Within 8-h aggregates cells of different species were regionally localized, i.e. sorting-out appeared to have taken place. (iv) 24-h aggregates were more varied: in mixtures of Dictyostelium species, the different species were localized within the aggregates; in mixtures of Dictyostelium species with Polsphondylium, there was a tendency for cells of the different species to become segregated into completely separate aggregates. The significance of these results in relation to both previous descriptive work and recent biochemical studies on the mechanism of slime mould cell cohesion is discussed.

Cyclic GMP regulation and responses of [formula omitted] to chemoattractants

In cells of the cellular slime mold Polysphondylium violaceum an attractant, which is released during the aggregation stage, causes a transient rise of the cyclic GMP concentration. Cells of this organism develop in shaken suspensions after they have finished growth. Cell development is not accompanied by an increase in the EDTA stability of cell adhesion. Both the developmental regulation and the specificity of chemotactic responses is reflected in the light scattering patterns recorded in cell suspensions: Folic acid causes a strong response in early preaggregation cells and the Polysphondylium attractant does the same in aggregation competent cells, whereas cyclic AMP is inactive in both stages.

The regulation of cellular slime mold development: A factor causing development of Polysphondylium violaceum aggregation-defective mutants

aggA mutants of Polysphondylium violaceum develop normally in synergistic mixtures with other aggregation-defective mutants. Cell to cell contact is not necessary for development. A small dialyzable factor(s) produced by wild-type and other aggregation-defective mutants triggers development of aggA mutants. This factor (D factor) is developmentally regulated, appearing early in development and then disappearing. Mutants require D factor until aggregation has just begun and then they can continue even in the absence of added factor. D factor is produced by many, but not all species of cellular slime molds and is developmentally regulated in Dictyostelium discoideum as well as P. violaceum.