Evidence for an induced plasma membrane mating receptor in the myxomycete Didymium iridis.

Haploid myxamoeboid mating types, CR5-11A11 and HON 1-2A1, of the acellular myxomycete Didymium iridis, were isolated and grown in liquid culture. Previously it has been postulated that a molecule, possibly a hormone, is responsible for the induction of mating competency through receptor site synthesis. The intention of this study was to isolate the molecule and to determine its mode of action. Experiments were designed in order to learn whether it acts only upon the cells which produce it (self-induction) or does it have an effect only on opposite mating types (cross-induction). It was found that plasmodia can only form on solid media, although zygotes are readily formed in liquid media. Moreover, competency of cells to fuse, or to form receptors required that they be haploid and in a specific phase of the cell cycle, namely G1 of interphase. The use of high pressure liquid chromatography techniques was also employed in order to investigate the inducer molecule. It was demonstrated that the inducer molecule was similarly produced by both mating types, and its production was essentially paralleled by cell numbers. It was also discovered that mating competency is self-induced and these results are discussed.

Studies of cell-surface glorin receptors, glorin degradation, and glorin-induced cellular responses during development of Polysphondylium violaceum.

The chemoattractant mediating cell aggregation in the slime mold Polysphondylium violaceum is N-propionyl-gamma-L-glutamyl-L-ornithine-delta-lactam ethylester (glorin). Here we examine the binding properties of tritiated glorin to intact P. violaceum cells. Scatchard analysis of binding data yielded slightly curvilinear plots with Kd values in the range of 20 and 100 nM. The number of glorin receptors increased from 35,000 in the vegetative stage to 45,000 per cell during aggregation. Later, during culmination receptor numbers decreased to undetectable levels (less than 1000). The receptor binding kinetics show binding equilibrium within 30 s at 0 degrees C, and ligand dissociation occurs from two kinetically distinct receptors whose half-times were 2 s for 72% of the bound glorin and 28 s for the remainder. The enzymatic degradation of glorin did not affect binding data during incubations of up to 1 min at 0 degrees C. Two glorinase activities were observed. An ornithine delta-lactam cleaving activity with a Km of ca. 10(-4) M and a propionic acid removing activity (Km 10(-5) M), both of which were detected mainly on the cell surface. Cleavage of the lactam occurred at a higher rate than removal of propionic acid. Lactam-cleaved glorin showed no chemotactic activity nor did it bind to cell-surface glorin receptors. Cell-surface-bound glorinase activity and glorin-induced cGMP synthesis were developmentally regulated, peaking at aggregation. In the most sensitive stage half-maximal responses (cGMP synthesis, chemotaxis, light-scattering) were elicited in the 10-100 nM range. Neither cAMP synthesis nor glorin-induced glorin synthesis was observed. Guanine nucleotides specifically modulated glorin receptor binding on isolated membranes, and, conversely, glorin modulated GTP gamma S binding to membrane preparations. Our results support the notion that glorin mediates chemotactic cell aggregation in P. violaceum acting via cell-surface receptors, G-proteins, and cGMP accumulation.

Enzyme-gold affinity labelling of cellulose.

The enzyme-linked colloidal gold affinity labelling technique was tested as a method to localize cellulose on thin sections of plant cell walls and slime mold spores. Commercially available cellulase from cultures of Trichoderma reesei, the main components being cellobiohydrolase I and II (CBH I, CBH II) and endoglucanase (EG), was linked to colloidal gold by using standard techniques and applied as a dilute, buffered suspension to thin sections. After brief exposure, e.g., 15-30 minutes, cellulose exposed on the surface of sections was labelled with the enzyme-gold complex. Poststaining did not appear to have a deleterious effect on the labelled sections. The specificity of labelling was demonstrated by its complete inhibition when carboxymethylcellulose was incorporated in the labelling mixture, by lack of labelling of 1,4-beta-mannans or 1,3-beta-xylans in noncellulosic walls of marine algae, by lack of labelling of 1,4-beta-glucans in chitin, by much lower labelling density when done at 4 degrees C, and by lack of labelling when sections were predigested with cellulase. Labelling with the crude commercial cellulase was compared to labelling with purified CBH I-, CBH II-, and EG-linked colloidal gold, and the labelling pattern was similar. This method was found useful on conventionally fixed material and required no special preparation other than the use of inert (Ni or Au) grids and 0.5% gelatin to reduce nonspecific binding of the gold complex. Labelling was similar in the several embedding resins tested: LR White, Lowicryl K4M, Epon 812, and Spurr's.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

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.

Identification and purification of an endogenous receptor for the lectin pallidin from Polysphondylium pallidum.

We report the identification and purification of an endogenous carbohydrate-containing receptor of pallidin, the cell surface lectin implicated in mediating cell-cell adhesion in the cellular slime mold Polysphondylium pallidum. The receptor is identified in an aqueous extract of crude P. pallidum membranes as a potent inhibitor of the hemagglutination activity of pallidin. The inhibitor is purified to apparent homogeneity by affinity precipitation with pallidin followed by fractionation of the solubilized precipitate on Sepharose 4B. The hemagglutination inhibitor (HAI) is metabolically radiolabeled, indicating that it is a biosynthetic product of the amoebae and not an ingested food substance. The HAI is released into the extracellular medium by living, differentiated amoebae. This release is markedly facilitated by the addition of D-galactose, a specific saccharide that binds to pallidin. Hence, the HAI appears to have an in situ association with pallidin at the cell surface. Exogenously added HAI promotes the agglutination of differentiated amoebae in a gyrated suspension at very low concentrations. The results are consistent with a model of cell-cell adhesion in which the HAI is a multivalent, extracellular aggregation factor that is recognized by pallidin molecules on adjacent cells. The HAI would then be analogues to the aggregation factors identified in marine sponges.

Cellular differentiation and pattern formation in the absence of morphogenesis in the cellular slime mould Polysphondylium pallidum: evidence for a biochemical tip (organizer) in submerged aggregates.

When amoebae of Polysphondylium pallidum WS320 are placed in nonnutrient buffer in roller tube culture they form spherical or ellipsoidal aggregates. At first the aggregates demonstrate a "loose" morphology but by 12 h, with the formation a a cellulose-containing, peripheral sheath, they become "tight" aggregates. At this time stalk differentiation begins. Using various methods for the resolution of prespore (ultrastructure, spore antigen immunofluorescence, periodic acid - Schiff staining) and prestalk (ultrastructure, alkaline phosphatase histochemistry, neutral red staining, Calcofluor fluorescence) cell localization, the pattern of cell differentiation in submerged aggregates was shown to be essentially identical to that of normal pseudoplasmodia. Furthermore, using a cAMP bioassay it was revealed that the submerged aggregates, while devoid of a morphological tip, do possess a biochemical tip which is correlated with sites of neutral red staining and stalk cell differentiation. As a result of these studies, an earlier argument that the tip of the pseudoplasmodium is not essential for the establishment of pattern or in the "organization" of cellular differentiation during slime mould development is contradicted.

Purification and characterization of multiple species (isolectins) of a slime mold lectin implicated in intercellular adhesion.

An improved purification procedure for the carbohydrate-binding proteins (lectins) of cohesive Polysphondylium pallidum cells has been devised. The procedure uses extraction of cells with lactose-containing buffer followed by ammonium sulfate precipitation and affinity chromatography of the redissolved precipitate on a column of acid-treated Sepharose 6B. All hemagglutination activity is adsorbed to the column and recoveries are about 70% of the activity of the starting cell lysate. Sodium dodecyl sulfate-gel electrophoresis of the protein obtained with this procedure resolved three subunits with molecular weights of 26,500 (A), 26,000 (B), and 25,000 (C). Three species are resolved by isoelectric focusing with apparent pI values of 6.4 (I), 7.3 (II), and 7.5 (III) which contain Subunits A, B, and C in the following ratios: I, B:C at 2:1; II, A:B at 2:1, and III, A:B at 1:2. All three isoforms agglutinate rabbit and human type O erythrocytes and are thus isolectins. Isoforms II and III are separated from Isoform I by galactose-gradient elution of the Sepharose 6B column. Isoforms II and III aggregate extensively (nonamers and multiples thereof), but reduction with 2-mercaptoethanol reverses this process yielding a single species of Mr = 73,000 (trimer). Isoform I exists as trimers and hexamers and reduction has no effect on this distribution. Amino acid compositions and tryptic peptide maps of S-[14C]carboxymethyl-isolectins indicate that Subunits A and B are very similar and may represent the same peptide chain, while Subunit C is a peptide quite distinct from A and B.

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.

Localization of actin in Dictyostelium amebas by immunofluorescence.

Antibody prepared against avian smooth muscle actin has been used to localize actin in the slime mold, Dictyostelium discoideum. The distribution of actin in migrating cells is different from that in feeding cells. Migrating amebas display fluorescence primarily in advancing regions whereas feeding amebas show uniform fluorescence throughout. The reaction is specific for actin since the fluorescence observed is blocked when the antibody is absorbed by actin purified from avian skeletal muscle, human platelets, and Dictyostelium. These results, in addition to describing the distribution of actin in D. discoideum, demonstrate that actins from these diverse sources share at least one common antigenic determinant.

Isolation and characterization of the RNA of membrane-bound ribosomes in Dictyostelium discoideum.

The RNA of membrane-bound ribosomes, isolated from Dictyostelium discoideum, represented 13 to 16% of the total ribosomal RNA (rRNA) present throughout growth and development. Membrane-bound ribosomes were released by treatment with sodium deoxycholate and Brij 58. There were no obvious differences in size and base composition between RNAs derived from membrane-bound or free ribosomes. The 17S membrane-bound rRNA and free rRNAs appeared to have similar methyl contents. However, the 25S membrane-bound rRNA contained about 16 to 20% fewer methyl groups than the 17S membrane-bound rRNA and free rRNAs. Free rRNAs turned over rapidly during early development but not during the disaggregation and reaggregation processes. Membrane-bound rRNAs showed very little turnover during the early stages of morphogenesis, but showed rapid turnover during the late stages of development; this class of rRNAs did not turn over during early stages of reaggregation but turned over rapidly during later stages of reaggregation.

Chemical analysis of stalk components of Dictostelium discoideum.

Structural components of the stalks of mature fruiting bodies of Dictyostelium discoideum have been isolated and characterized after solubilizing non-structural components with urea and sodium dodecyl sulfate. The urea/sodium dodecyl sulfate-insoluble stalks are composed of about 52% cellulose, 15% protein and 3% of a non-cellulosic heteropolymer in a covalently bound matrix. Non-covalently bound fatty acid containing material was also found. The composition and structural interrelationships of these components are essentially identical to that of the urea/sodium dodecyl sulfate-insoluble surface sheath which is produced earlier in development before culmination. These results suggest that the same components are involved in making structural elements which differ substantially in their functional role in the developmental sequence as well as in their spatial and temporal localization and morphological appearance.

Characterization of colchicine-binding activity in Dictyostelium discoideum.

A colchicine-binding component was detected in vegetative amoebae of Dictyostelium discoideum by using a Millipore-filter assay. The colchicine-binding activity is temperature-and time-dependent, maximum binding occurring at 22-35 degrees C after 60 min incubation. Further increases in temperature are without effect on the extent of binding, but bound colchicine is released with increased time of incubation. Furthermore, colchicine-binding activity itself decreased in the high-speed supernatant from D. discoideum, with half the activity being lost in approx. 2.5h. Several lines of evidence, including the saturation kinetics of colchicine binding, enhancement of colchicine binding by tartrate, insensitivity to lumicolchicine, precipitation of the binding protein by vinblastine and behaviour of the binding protein on DEAE-cellulose and Sephadex resins, suggest that the colchicine-binding protein may be tubulin.