A microcyst-overproducing mutant of Polysphondylium pallidum

A mutant, PN6017, of the cellular slime mold Polysphondylium pallidum was selected by cell-surface labeling with a monoclonal antibody, mAb 293, and fluorescence-activated cell sorting. The antibody was directed against an L-fucose-containing epitope on glycoproteins, designated ep 293, and the mutant showed reduced and delayed expression of this epitope. PN6017 was distinguished from other mutants of this kind by extensive microcyst formation on agar plates under conditions where the wild type formed only sparse microcysts. In suspension cultures transformation of cells into microcysts was negligible in the wild type, and close to 100% in the mutant. Under these conditions microcyst formation in the mutant began at 5-7 h of starvation. At the same time expression of ep 293 and also of a developmentally regulated cytoplasmic protein, pallidin, became detectable. This coincidence in time suggests that microcyst formation in PN6017 is coupled to the same control mechanism as the two other developmentally regulated processes.     

Geometry and spatial patterns in Polysphondylium pallidum

The formation of secondary sori in whorls of Polysphondylium pallidum provides an attractive model system for the study of symmetry breaking during morphogenesis. Tip-specific antibodies that permit detection of very early stages in this patterning process are available. We have found that the patterns of tip-specific antigen expression vary considerably depending on the size, shape, and developmental stage of the whorl. All of these patterns, however, are well explained by patterning models that rely on short-range autocatalysis and long-range inhibition, as exemplified by reaction-diffusion theories. In the context of reaction-diffusion, we discuss the possible effects of initial conditions, boundary conditions, and nonlinearities on the selection of patterns in P. pallidum whorls.     

Analysis of developmentally expressed antigens in Polysphondylium pallidum

A series of monoclonal antibodies were previously raised against developing Polysphondylium pallidum cells. In this work, six of these antibodies have been used as probes to identify and characterize antigens regulated during development. Soluble and membrane fractions of P. pallidum cells at six stages of development or three stages of cyclic adenosine monophosphate (cAMP)-induced development were run in sodium dodecyl sulfate (SDS)-polyacrylamide gels and subjected to Western blot analysis. Three of the monoclonals, anti-Tp200, anti-Tp423, and anti-Pg101, stain sorogen tips. Tp423 and Tp200 are membrane-associated antigens; both are stable to urea extraction, and Tp200 remains in the membrane after NaOH extraction. Tp423 is present in starved cells but is more prominent in sorogens and particularly in cAMP-developed cells. In contrast, Tp200 is first detected in early to mid-aggregation and is more abundant late in development. Pg101, which is expressed as a gradient with its highest concentration in tips, first appears in tight aggregates but is much more abundant in sorogens; unlike the Tp antigens, Pg101 is not greatly induced in cAMP-developed cells. All three of these antigens undergo changes in apparent molecular weight at the tight aggregate or sorogen stage: The gel mobilities of Tp200 and Pg101 increase, whereas that of Tp423 decreases. In addition to the tip-specific monoclonals, two monoclonals that stain all but the tips of sorogens have been used for analysis. One of these, anti-3D10Pnk stains most cells within secondary tips, whereas anti-3D10Dif does not. 3D10Dif is membrane associated; it is present very early in development, increasing two- to threefold through the sorogen stage and diminishing in late cAMP-developed cells. 3D10Pnk is a mostly soluble species first detected in late streaming. Anti-1c3, a sixth monoclonal, which stains nuclei uniformly throughout sorogens, is also developmentally expressed. 1c3 is mainly membrane associated and is expressed from late streaming through the sorogen stage.     

Induction of encystment of Polysphondylium pallidum amoeba

The induction of microcyst formation could be triggered in washed amoebae of the cellular slime mold Polysphondylium pallidum (strain-2) by the addition of 2 mM ethionine. Methionine at a ratio of 2: 1 with ethionine would inhibit microcyst induction by ethionine. The involvement of polyamines in morphogenesis was also shown. Putrescine (0.02 to 0.1 M) induced the formation of microcysts, whereas spermidine (2 to 4 mM) was capable of causing a fourfold reduction in 0.05 M putrescine-induced microcysts but incapable of inhibiting microcyst induction by 0.08 M itrescine. Glycerol (0.5 M or 0.4 mM) was also found to be an effective inducer of microcysts.     

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.     

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.     

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.     

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.     

An increase of cAMP-dependent protein kinase during development in Polysphondylium pallidum

Polysphondylium pallidum is a cellular slime mold in which, unlike in Dictyostelium discoideum, cAMP is not the chemotactic agent. The occurrence of a cAMP-dependent protein kinase in D. discoideum was demonstrated earlier and we suggested that it may mediate the intracellular effects of cAMP on the development of the organism, particularly since an increase in the amount of the enzyme during development was noted. In D. discoideum cAMP plays a dual role insofar as it serves both as chemotactic agent and as second messenger; it was of interest therefore, to determine whether a cAMP-dependent protein kinase occurred in P. pallidum. We found a cAMP-dependent protein kinase in P. pallidum using Kemptide as substrate. The regulatory subunit of the enzyme has an apparent molecular weight of 41,000 and seems to be similar in its properties with that isolated earlier from D. discoideum. The cAMP-dependent protein kinase catalytic subunits from the two species are also similar. Furthermore, there is a developmentally regulated, parallel, two- to threefold increase in the two subunits of the cAMP-dependent protein kinase in P. pallidum. The increase occurs before aggregates are formed. These findings are compatible with a role of the intracellular cAMP and of the cAMP-dependent protein kinase in the development of P. pallidum.     

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.     

Tyrosine phosphorylation of actin during microcyst formation and germination in Polysphondylium pallidum

High osmolarity causes amoebae of the cellular slime mould Polysphondylium pallidum to individually encyst, forming microcysts. During microcyst differentiation, actin is tyrosine phosphorylated. Tyrosine phosphorylation of actin is independent of encystment conditions and occurs during the final stages of microcyst formation. During microcyst germination, actin undergoes dephosphorylation prior to amoebal emergence. Renewed phosphorylation of actin in germinating microcysts can be triggered by increasing the osmolarity of the medium which inhibits emergence. Immunofluorescence reveals that actin is dispersed throughout the cytoplasm in dormant microcysts. Following the onset of germination, actin is observed around vesicles where it co-localizes with phosphotyrosine. Prior to emergence, actin localizes to patches near the cell surface. Increasing osmolarity disrupts this localization and causes actin to redistribute throughout the cytoplasm, a situation similar to that observed in dormant microcysts. The tyrosine phosphorylation state of actin does not appear to influence the long-term viability of dormant microcysts. Together, these results indicate an association between actin tyrosine phosphorylation, organization of the actin cytoskeleton, and microcyst dormancy.     

True Divergent Differentiation in a Cellular Slime Mold, Polysphondylium pallidum

Three abstract models of divergent cell differentiation to multiple cell types are presented. These differ primarily in the proportion of developmental events common to the pathways leading to each cell type. Two experimental approaches are outlined to determine which type best describes divergent differentiation occurring in a particular organism. The first technique is to describe and compare changes in labeling of specific polypeptides which characterize development to the several cell types. The second is to observe the ability of mutants which are blocked in one pathway to develop along alternate pathways. These approaches are applied to the case of Polysphondylium pallidum , where amebae develop into stalk cells, spores, or microcysts. It is concluded that cell differentiation in P. pallidum is of the truly divergent type, in which developing cells show identical sequences of events until a branch point, and thereafter very different sequences of events.     

Macrocyst genetics in Polysphondylium pallidum, a cellular slime mould.

The discovery of two mating types in the cellular slime mould Polysphondylium pallidum is reported. Two developmental mutants produced in strains of opposite mating type but which do not proceed past the aggregation stage of development are capable of producing macrocysts. These macrosysts were viable and 5 to 10% germinated after 6 weeks of storage. When the macrocyst progeny were cloned, several classes of non-parental phenotypes were recovered.     

True divergent differentiation in a cellular slime mold, Polysphondylium pallidum.

Three abstract models of divergent cell differentiation to multiple cell types are presented. These differ primarily in the proportion of developmental events common to the pathways leading to each cell type. Two experimental approaches are outlined to determine which type best describes divergent differentiation occurring in a particular organism. The first technique is to describe and compare changes in labeling of specific polypeptides which characterize development to the several cell types. The second is to observe the ability of mutants which are blocked in one pathway to develop along alternate pathways. These approaches are applied to the case of Polysphondylium pallidum, where amebae develop into stalk cells, spores, or microcysts. It is concluded that cell differentiation in P. pallidum is of the truly divergent type in which developing cells show identical sequences of events until a branch point, and thereafter very different sequences of events.     

RNA and protein synthetic patterns during germination of Polysphondylium pallidum microcysts

During microcyst germination in the cellular slime mould Polysphondylium pallidum, an immediate rapid increase in the rate of protein synthesis ([3H]leucine incorporation) is observed within 15 min after the initiation of germination. The data, corrected for amino acid pool changes, reveal that the rate of protein synthesis reaches its peak at 1 1/2 h, after which it decreases. A low level of RNA synthesis ([3H]uridine incorporation) is observed after 1 h and this rate increases markedly after 2 h. Analysis of the RNA species shows a low level of synthesis of all ribosomal RNA's which begins between 1 and 2 h and increases after 2 h. The synthesis of a heterogeneously distributed, poly(A)-containing fraction of RNA (presumptive mRNA) is initiated some time after 2 h and the synthesis of a small molecular weight species in the 4-5S region is observed after 3 h. Thus, it seems that Polysphondylium microcysts show sequentially initiated synthesis of RNA during germination.     

Pattern Formation and Tip Inhibition in the Cellular Slime Mold Polysphondylium pallidum

Abstract. We have shown that the apical tip of the developing sorogen prevents secondary tip formation during normal patterning in Polysphondylium pallidum . In one class of experiments the apical tip was excised and secondary tips soon formed over the surface of the remaining sorogen. In another, very large sorogens formed secondary basal tips spontaneously before regulating to a fixed radius to length ratio of 0.14, the ratio characteristic of control sorogens. These results are discussed in the light of a simple model.     

A killer factor produced by the cellular slime mold Polysphondylium pallidum

A killer strain was discovered in cellular slime molds. The wild isolate CK-8 of Polysphondylium pallidum kills all other strains in Polysphondylium and Dictyostelium, as far as could be determined, except strain CK-8 itself and its complementary mating type strain CK-9. Growth-phase cells of CK-8 excrete a killer factor which is sensitive to heat, above 60°C for 5 min, and trypsin. The apparent molecular mass of the factor was determined at 10 000–12 000.Abbreviations BSS Bonner's salt solution - CM conditioned medium