Cyclic GMP regulation and responses of Polysphondylium violaceum 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.     

Negative chemotaxis in cellular slime molds.

This study confirms the suggestion of earlier workers that the vegetative amoebae of Dictyostelium repel each other while those of Polysphondylium violaceum do not. When Dictyostelium amoebae were placed in drops on thin and thick agar, the cells moved out faster on the thin agar, presumably because the repellent was more concentrated. This did not occur with Polysphondylium amoebae. Also, if 2 drops of cells were placed side by side, or a single drop was placed near an edge, in Dictyostelium there were fewer cells emerging between the drops (or near an edge) than on the far side. Polysphondylium showed no such difference. However, Polysphondylium amoebae were repelled by Dictyostelium cells (but not vice versa) when drops of each were placed beside one another. Finally, if Dictyostelium discoideum cells were placed in drops over thick and thin agar, but separated from the agar by a dialysis membrane, the cells again spread farther on the thin agar, indicating that the repellent is a dialyzable molecule.     

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.     

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.     

Element analysis of the Polysphondylium pallidum gp64 promoter

gp64 mRNA in Polysphondylium pallidum is expressed extensively during vegetative growth, and begins to rapidly decrease at the onset of development. To examine this unique regulation, 5' deletion analysis of the gp64 promoter was undertaken, and two growth-phase activated elements have been found: a food-dependent, upstream regulatory region (FUR, -222 to -170) and a vegetatively activated, downstream region (VAD, -110 to -63). Here we concentrate our analysis on an A1 and A2 sequences in the FUR region: A1 consists of a GATTTTTTTA sequence called a corresponding sequence and A2 consists of the direct repeat TTTGTTGTG. The cells carrying a combined construct of A1 and A2 acted synergistically in a reporter activity. A point mutation analysis in A1 indicates that a G residue is required for the activation of A1. From analyses of promoter regulation in a liquid or a solid medium, the promoter activity of the cells fed on bacteria in A-medium (axenic medium for Polysphondylium) or grown in A-medium alone was only one fourth of that of the cells fed on bacteria. By the gel retardation, we detected a protein bound to the A1 sequence.     

cAMP-dependent protein kinase regulates Polysphondylium pallidum development

In eukaryotic cells, the universal second messenger cAMP regulates various aspects of development and differentiation. The primary target for cAMP is the regulatory subunit of cAMP-dependent protein kinase A (PKA), which, upon cAMP binding, dissociates from the catalytic subunit and thus activates it. In the soil amoeba Dictyostelium discoideum, the function of PKA in growth, development and cell differentiation has been thoroughly investigated and substantial information is available. To obtain a more general view, we investigated the influence of PKA on development of the related species Polysphondylium pallidum. Cells were transformed to overexpress either a dominant negative mutant of the regulatory subunit (Rm) from Dictyostelium that cannot bind cAMP, or the catalytic subunit (PKA-C) from Dictyostelium. Cells overexpressing Rm rarely aggregated and the few multicellular structures developed slowly into very small fruiting bodies without branching of secondary sorogens, the prominent feature of Polysphondylium. Few round spores with reduced viability were formed. When mixed with wild-type cells and allowed to develop, the Rm cells were randomly distributed in aggregation streams, but were later found in the posterior region of the culminating slug or were left behind on the surface of the substratum. The PKA-C overexpressing cells exhibited precocious development and formed more aggregates of smaller size. Moreover, expression of PKA-C under the control of the prestalk-specific ecmB promoter of Dictyostelium leads to protrusions from aggregation streams. We conclude that Dictyostelium PKA subunits introduced into Polysphondylium cells are functional as signal components, indicating that a biochemically similar PKA mechanism works in Polysphondylium.     

Diphospho-myo-inositol phosphates during the life cycle of Dictyostelium and Polysphondylium.

The intracellular amounts of diphospho-myo-inositol phosphates and InsP6 were determined in Dictyostelium discoideum AX2 throughout the life cycle, including exponential growth, starvation, differentiation, sporulation and spore germination. Similar experiments were performed with the closely related species Polysphondylium pallidum under conditions resulting in microcyst formation. A distinct accumulation of these compounds is observed during the early starvation phase of the cell population before the onset of the actual differentiation program. When exponentially growing D. discoideum cells were shifted to starvation conditions, a 25-fold accumulation of 5,6-bis-PP-InsP4 within 3 h was observed. In P. pallidum, the 5,6-bis-PP-InsP4 pool rises around 20-fold within 8 h during the formation of microcysts from vegetative cells. Finally, the diphosphoinositol phosphates are deposited in spores or microcysts and are degraded when spores or microcysts germinate at low cell density.     

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.     

Synthesis of macromolecules during microcyst germination in the cellular slime mold Polysphondylium pallidum

Microcyst germination in the cellular slime mold Polysphondylium pallidum is a useful model for studying macromolecular changes necessary for or coincident with the transition from one cell type (cyst) to another (amoebae). Protein synthesis starts soon after cysts are incubated under permissive conditions, as evidenced by the incorporation of precursors and the appearance of polysomes. Sodium dodecyl sulfate-polyacrylamide gel analysis of proteins made at intervals during germination shows that protein synthesis is developmentally regulated during this process. RNA synthesis also begins early during germination. Cysts contain polyadenylated RNA that can stimulate the incorporation of radioactive amino acids into protein in an in vitro wheat germ protein synthesizing system. The concentration of poly(A)-containing RNA increases during germination and during inhibition of protein synthesis by cycloheximide.     

Heat shock response in a cellular slime mold,Polysphondylium pallidum

Cells of Polysphondylium pallidum were exposed to a heat shock by raising the temperature from 25 to 31°C. A set of four major polypeptides of approximate molecular weights 105,000, 87,000, 74,000, and 33,000 incorporated [1-¹⁴C]acetate when pulse labeled during the first hour after heat shock. The response resembles the heat shock response of Drosophila in occurring in cells at different stages of development (early in aggregation, late in aggregation, and during microcyst formation) and in being triggered by a threshold high temperature rather than a minimal change in temperature.     

Acid proteinases in various species of cellular slime mold

The proteolytic activities of the cellular slime moldsDictyostelium mucoroides, Dictyostelium purpureum, Polysphondylium pallidum, andPolysphondylium violaceum have been examined. Myxamoebae possessed activity against Hide Powder Azure at pH 2–5 which was enhanced by dithiothreitol: this enhancement was small inDictyostelium species but three- to four-fold in thePolysphondylium 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 HgCl₂ and to a lesser extent by other thiol proteinase inhibitors such asN-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 HgCl₂, 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 inD. mucoroides; there was a relative increase in one proteinase inP. violaceum but three proteinases were lost during fruiting body formation inP. pallidum. During microcyst formation inP. 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.     

Intercellular Adhesion in the Cellular Slime Mold Polysphondylium pallidum

Evidence is reviewed implicating a cell surface carbohydrate-bindingprotein (lectin) named pallidin as the mediator of intercellularadhesion in the cellular slime mold Polysphondylium pallidum.Three isolectin forms of pallidin have now been purified andcharacterized. Both lectin and receptor to which lectin canbind are present on the cell surface of adhesive amoebae. Sincepallidin antagonists such as specific sugars, asialofetuin,or specific univalent antibody interfere with intercellularadhesion, cell-cell binding may be based on complementary interactionsbetween pallidin and specific receptors on adjoining cells.     

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.     

Monoclonal antibodies against the mercaptoethanol-sensitive structure of a cell-cell adhesion protein of Polysphondylium pallidum

Monoclonal antibodies were prepared against a putative cell-cell adhesion glycoprotein, with an apparent molecular mass of 64,000 (gp64), of the cellular slime mold, Polysphondylium pallidum. Five monoclonal antibodies obtained by means of an enzyme-linked immunoadsorbent assay did not bind to the antigens which were subjected to gel electrophoresis and blotting method in the presence of a reducing agent, but they did bind specifically to the antigens prepared in unreducing conditions of samples and then processed by the same blotting method. To solubilize gp64 in a sodium dodecyl sulfate (SDS)-sample buffer without mercaptoethanol (heated) or SDS-sample buffer with 2-mercaptoethanol (nonheated) was critical for the antibody binding onto gp64 on a membrane. Hence the antibodies seem to bind to a surface portion(s) of the localized protein structure folded up by disulfide cross-linkages. One of the antibodies obtained blocked cell-cell adhesion by about 20%.     

Acid phosphatase activity and microcyst differentiation in the cellular slime mold Polysphondylium pallidum

The enzyme acid phosphatase (E.G. 3.1.3.2) is present in Polysphondylium pallidum and increases during microcyst differentiation. The increase of enzyme activity occurs during the period of morphological differentiation of microcysts. The use of the inhibitor cycloheximide provides evidence that the increase in enzyme activity requires coincident protein synthesis. Acid phosphatase activity also accumulates extracellularly but the extracellular release of this activity is not stopped by cycloheximide. A cystless mutant (strain NG-6) shows a high but essentially unchanging intracellular level of acid phosphatase and a slightly delayed extracellular pattern of accumulation. A comparison of other enzyme patterns in strain NG-6 indicates that different control mechanisms in addition to the regulation of translation may mediate the appearance of. different enzymes during microcyst differentiation.     

Polyadenylation of maternal mRNA during oocyte maturation: poly(A) addition in vitro requires a regulated RNA binding activity and a poly(A) polymerase.

Specific maternal mRNAs receive poly(A) during early development as a means of translational regulation. In this report, we investigated the mechanism and control of poly(A) addition during frog oocyte maturation, in which oocytes advance from first to second meiosis becoming eggs. We analyzed polyadenylation in vitro in oocyte and egg extracts. In vivo, polyadenylation during maturation requires AAUAAA and a U-rich element. The same sequences are required for polyadenylation in egg extracts in vitro. The in vitro reaction requires at least two separable components: a poly(A) polymerase and an RNA binding activity with specificity for AAUAAA and the U-rich element. The poly(A) polymerase is similar to nuclear poly(A) polymerases in mammalian cells. Through a 2000-fold partial purification, the frog egg and mammalian enzymes were found to be very similar. More importantly, a purified calf thymus poly(A) polymerase acquired the sequence specificity seen during frog oocyte maturation when mixed with the frog egg RNA binding fraction, demonstrating the interchangeability of the two enzymes. To determine how polyadenylation is activated during maturation, we compared polymerase and RNA binding activities in oocyte and egg extracts. Although oocyte extracts were much less active in maturation-specific polyadenylation, they contained nearly as much poly(A) polymerase activity. In contrast, the RNA binding activity differed dramatically in oocyte and egg extracts: oocyte extracts contained less binding activity and the activity that was present exhibited an altered mobility in gel retardation assays. Finally, we demonstrate that components present in the RNA binding fraction are rate-limiting in the oocyte extract, suggesting that fraction contains the target that is activated by progesterone treatment. This target may be the RNA binding activity itself. We propose that in spite of the many biological differences between them, nuclear polyadenylation and cytoplasmic polyadenylation during early development may be catalyzed by similar, or even identical, components.     

Cell-division inhibitor produced by a killer strain of cellular slime mold Polysphondylium pallidum

The killer strain CK-8 of cellular slime mold Polysphondylium pallidum produces a cell-division inhibitor, in addition to a killer factor. This inhibitor represses cell division of many strains and species of cellular slime molds, except CK-8 itself and its complementary mating-type strain. It is sensitive to both heat and trypsin, and capable of binding to Con A. Its apparent molecular mass is more than 100 kDa. Repression of cell division by this inhibitor is reversed by trypsin treatment of the cells.     

Developmental regulation of protein synthesis during Polysphondylium pallidum cyst germination: A two-dimensional gel electrophoresis study

Microcyst germination in Polysphondylium pallidum can be used as a model for studying gene expression because temporally regulated modulations in protein synthesis occur in this developmental pathway. Germinating cysts were labeled with [³⁵S]methionine for half-hourly periods during the synchronous germination sequence, and the proteins labeled in each period were resolved by two-dimensional polyacrylamide gel electrophoresis. Three major classes of proteins observed were distinguished by the time of onset and duration of their synthesis: (a) proteins made throughout germination; (b) proteins synthesized only during a portion of the germination pathway; and (c) polypeptides whose synthesis started at 1 or 1.5 h and then continued throughout germination.     

Cysteine proteinase changes during microcyst formation and germination inPolysphondylium pallidum

Microcyst formation inPolysphondylium pallidum WS320 was accompanied by a decrease in intracellular cysteine proteinase activity measured with the peptide nitroanilides Z-Arg-Arg-Nan and Bz-Pro-Phe-Arg-Nan. Some activity was released into the buffer, and secretion of that towards Z-Arg-Arg-Nan continued until encystment occurred. Cells grown in association withEscherichia coli had an electrophoretic proteinase pattern different from cells grown axenically. Microcysts formed from the two cell populations also had distinct proteinase patterns; those from bacterially grown cells retained significant quantities of proteinase ppCP22, whereas those derived from axenic cells were devoid of detectable proteinases. No significant changes in cysteine proteinase activities were observed during microcyst germination, although some changes in activity occurred subsequent to emergence. The results indicate that there is not a close correlation between particular cysteine proteinases and specific stages of microcyst formation. Intracellular proteinase loss and concomitant secretion are, however, processes typical of cellular slime molds developing in response to starvation.     

Increase in proteinase activity in the cellular slime mould Polysphondylium pallidum induced by bacteria

Abstract Polysphondylium pallidum strain PPHU8 grown in association with bacteria contains aspartic and cysteine proteinases. When myxamoebae were grown in axenic medium the contribution of cysteine proteinases was much lower. The proteinase activity could be altered by addition of heat-killed bacteria to axenically growing cells. This was detected as an increase in the specific activity towards N -benzoyl-L-prolyl-L-phenylalanyl-L-arginine- p -nitroanilide, a cysteine proteinase substrate, and by the appearance of cysteine proteinase bands after electrophoretic analysis. The changes were inhibited by cycloheximide, azide and dinitrophenol. All the available evidence suggests that they are due to the de novo synthesis of cysteine proteinases.