Cell patterning in branched and unbranched fruiting bodies of the cellular slime mold Polysphondylium pallidum

Cell patterning, the percentage of spores and stalk cells, was measured in branched and unbranched asexual fruiting bodies of Polysphondylium pallidum. Unlike D. discoideum, where small and large fruiting bodies are more stalky than average-sized fruiting bodies, the overall cell patterning was the same in branched and unbranched fruiting bodies of all sizes in P. pallidum. Light greatly increased the numbers of fruiting bodies in P. pallidum per unit area (or decreased aggregation territory size) so that most fruiting bodies formed in the light were small and unbranched. By contrast, light had little effect on the cell patterning of P. pallidum, although there was a slight increase in the percentage of stalk cells in the light compared to the dark. This indicates that the mechanisms governing light sensitivity of aggregation territory size and cell patterning have different components in P. pallidum. The accuracy of cell patterning of individual branches of branched fruiting bodies was so imprecise as to leave doubt that patterning is occurring at the branch level. Individual whorls of branched fruiting bodies had a greater percentage spores (90%) than whole fruiting bodies (78%) and the cell patterning was relatively imprecise. Only in whole fruiting bodies was the spore:stalk ratio highly correlated. These findings are consistent with cell pattern determination operating at the whole aggregate level, rather than at the individual whorl or branch level in P. pallidum.     

Intracellular and extracellular enzyme patterns during microcyst germination in the cellular slime mold Polysphondylium pallidum

Microcysts of Polysphondylium pallidum, formed in modified Sussman's (1963) medium, germinate synchronously without activation in 4 hr in a sterile nonnutrient medium. Germination is cycloheximide-sensitive only within the first 2 hr. In this early period, cycloheximide stops emergence of amoebae but does not prevent the initial swelling phase. Actinomycin D and nucleic acid analogs at high concentrations have no evident effects on the germination process.Of nine hydrolytic enzymes studied, only alkaline phosphatase shows a large intracellular increase in activity. The increase in activity is cycloheximide-sensitive, indicating a requirement for coincident protein synthesis. Additional experiments show the lack of activators or inhibitors of alkaline phosphatase activity at different stages of the germination process. Changes in the phosphatase isozyme population could not be detected and the enzyme is not excreted.Several lysosomal glycosidases show little change in intracellular activity but their activity increases dramatically in the extracellular medium. The extracellular increases in these enzymes correlate well with the morphological loss of the microcyst wall, suggesting a possible role for certain of these enzymes in wall digestion.     

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.     

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     

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.     

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.     

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.     

Two mating groups of Polysphondylium pallidum, a dictyostelid cellular slime mold

 Seven strains representative of Polysphondylium pallidum were examined in terms of mating relationship and morphology. By mating tests, two heterothallic mating groups were detected. By morphological comparisons, they were clearly different in number of branches and in shape of bases and tip cells of sorophores. These results suggested that these two mating groups were distinct taxa. Received: January 23, 2002 / Accepted: August 30, 2002 Present address:Kanagawa Prefectural Museum of Natural History, Odawara, Kanagawa, Japan (visiting researcher) Acknowledgments I thank Dr. James C. Cavender, Department of Environmental and Plant Biology, Ohio University, Athens, OH, USA, for providing the cultures of three strains, V-1, WS320, and WS543. Correspondence to:H. Hagiwara     

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.     

Mitosis in the cellular slime mold Polysphondylium violaceum

Myxamebas of Polysphondylium violaceum were grown in liquid medium and processed for electron microscopy. Mitosis is characterized by a persistent nuclear envelope, ring-shaped extranuclear spindle pole bodies (SPBs), a central spindle spatially separated from the chromosomal microtubules, well-differentiated kinetochores, and dispersion of the nucleoli. SPBs originate from the division, during prophase, of an electron-opaque body associated with the interphase nucleus. The nuclear nevelope becomes fenestrated in their vicinity, allowing the build-up of the intranuclear, central spindle and chromosomal microtubules as the SPBs migrate to opposite poles. At metaphase the chromosomes are in amphitelic orientation, each sister chromatid being directly connected to the corresponding SPB by a single microtubule. During ana- and telophase the central spindle elongates, the daughter chromosomes approach the SPBs, and the nucleus constricts in the equatorial region. The cytoplasm cleaves by furrowing in late telophase, which is in other respects characterized by a re- establishment of the interphase condition. Spindle elongation and poleward movement of chromosomes are discussed in relation to hypotheses of the mechanism of mitosis.     

Acid protease activity during germination of microcysts of the cellular slime mold Polysphondylium pallidum.

Extracts of dormant microcysts of Polysphondylium pallidum demonstrate pH optima for the hydrolysis of casein at 3.5 and 6.0. During germination the intracellular pH 6.0 caseinolytic specific activity does not change significantly. The pH 6.0 protease is also active on azo-albumin, revealing the same developmental pattern with this substrate. Both acid protease activities are excreted during the germination process. Addition of purified nonspecific protease to cultures speeds up germination, suggesting that the excreted protease may play a role in removal of the microcyst wall. When cycloheximide is added to cultures, complete germination (emergence) is stopped whereas the pH 6.0 protease activity still accumulates to between 50 and 60% of the maximum control activity. Although this suggests that post-translational controls might mediate the accumulation of a portion of the pH 6.0 protease increase, mixing and dilution experiments with cell extracts do not reveal the differential presence of soluble activators or inhibitors of this activity at different developmental stages. The presence of tightly bound enzyme-inhibitor complexes for protease B in dormant microcysts has not been ruled out and is currently under study.     

Induction of cell fusion by a factor released by the cellular slime mold Polysphondylium pallidum

Heterokaryons and hybrid cells, which are extremely useful for research in cell biology, can be produced artificially by treating cells with either polyethylene glycol or certain inactivated viruses that alter the plasma membrane. We report here a novel cell-fusion inducing factor secreted by CK-8 strain cells of cellular slime mold Polysphondylium pallidum. Treatment of other strains or other species of cellular slime molds, such as NC-4 of Dictyostelium discoideum with the diluted fraction, containing molecules larger than 50 kDa, of the conditioned medium of CK-8 cell culture induces cell fusion at high frequency and produces multinucleated large cells. This cell fusion is inducible between cells of either a single strain or of two different strains of cellular slime molds.Abbreviations BSS Bonner's salt solution - CM conditioned medium - EDTA ethylenediaminetetraacetic acid - F2 fraction containing cell-fusion induction factor - Mr molecular mass     

Changes in protein synthesis during alternate pathways of differentiation in the cellular slime mold Polysphondylium pallidum.

Acrylamide gel electrophoresis in the presence of sodium lauryl sulfate and autoradiography of dried gels were used to detect incorporation of [¹⁴C]acetate into specific polypeptides of developing cells. Twenty-four polypeptides showed changes in net rate of synthesis during light-induced aggregation and sorogenesis, and 15 bands changed during KCl-induced encystment. Most of the changes during aggregation were common to three strains of P. pallidum, but different from those exhibited by D. discoideum. They failed to occur if the cells were maintained in darkness, and the pattern of changes was altered by sublethal high temperature. A few changes were common to both aggregating and encysting cells, and to stalkforming cells induced by cyclic AMP. A pulse-chase experiment indicated that all detectable increases in net synthesis were caused by alterations in actual synthesis rather than in degradation. It is suggested that the observed changes in synthesis may be essential to the correlated pathways of cellular differentiation.     

Changes in glucan synthetase activity and plasma membrane proteins during encystment of the cellular slime mold Polysphondylium pallidum

The activity of glucan synthetase increased dramatically during encystment of Polysphondylium pallidum cells. The majority of activity was present in purified plasma membranes. Activity, measured as glucose incorporation from UDPG into NaOH-insoluble glucan, increased 30–40 fold in the membranes. Increases in activity within the cells preceded plasma membrane increases and the enzyme appeared to be rapidly transported to the plasma membrane. Intracellular activity was relatively low. When cells were incubated with UDPG and when phloretin was included to inhibit glucose uptake, no NaOH-insoluble glucan was synthesized. Hence, the UDPG-binding site was not exposed at the cell-surface. When the NaOH-insoluble glucan was digested with endo--1,4-glucanase the products were cellobiose and glucose. The glucan could also be precipitated from Schweizer's reagent with acetic acid. These results suggest that the glucan contained predominantly -1,4-linkages and may be cellulose. Experiments with cycloheximide confirmed that protein synthesis was required for encystment. Labeling of cells with [1-¹⁴C]-acetate showed that the synthesis of certain plasma membrane proteins was developmentally regulated. A number of proteins (e.g., myosin heavy chains and actin) were synthesized during the lag phase and their synthesis was subsequently reduced or ceased altogether. Immediately prior to the commencement of cyst wall formation seven new plasma membrane proteins were synthesized. These proteins were not detected intracellularly, indicating rapid transfer to the plasma membrane. The possible relationship between the seven developmentally regulated proteins and a postulated multi-enzyme-complex involved in cellulose synthesis is discussed. Their synthesis may be related to the increase in particles in the outer leaflet of the plasma membrane observed during encystment with freeze-etching (G.W. Erdos and H.R. Hohl, 1980, Cytobios, 29, 7–16).     

Ammonia and the induction of microcyst differentiation in wild-type and mutant strains of the cellular slime mold Polysphondylium pallidum

Although low levels ($\text{0.16 μ}\text{mole}\text{10}{}^7\text{cells, ml}$) of ammonia are present in vegetative, germinating, and young encystment cultures of wild-type (WS-320) cells, a 12-fold increase in extracellular ammonia occurs during microcyst differentiation in the cellular slime mold Polysphondylium pallidum. When WS-320 amoebae were placed in conditioned medium from other wild-type encystment cultures, microcyst formation was initiated earlier than in control (120 mM KCl) cultures. Isoosmotic solutions containing NH₄Cl and KCl also caused WS-320 cells to encyst earlier. Similar results were obtained with mutant strains. In 120 mM KCl, strains mic-1 and mic-2 produce 21 and 64% microcysts, respectively, while strains PN582 and PN651 do not encyst. The mutant mic-1 secretes wild-type levels of ammonia, while mic-2, PN582, and PN651 all secrete much lower amounts. Conditioned medium and solutions containing NH₄Cl and KCl increased the rate of development and the number of microcysts produced by mic-2 and induced microcyst development in the cystless mutant PN582. Taken together these data indicate that ammonia acts as an inducer of microcyst differentiation.