The axial distribution of plasma membrane molecules in pseudoplasmodia of the cellular slime mold Dictyostelium discoideum

Five plasma membrane molecules were found to be non-uniformly distributed along the axis of the pseudoplasmodium of the cellular slime mold Dictyostelium discoideum and were biochemically characterized. All five of the molecules were distinguished by their ability to bind the lectin wheat germ agglutinin (WGA), and three were recognized by an antibody raised against pseudoplasmodial plasma membranes and exhaustively adsorbed against vegetative cells. The distribution of these molecules corresponded with the pattern of precursors of the terminally differentiated stalk cells and spores before these cells became irreversibly committed to their respective fates. At least four of the molecules appeared in hemispherical aggregates prior to the detection of prestalk or prespore cells, but were not present in undifferentiated vegetative cells.     

The effect of inhibitors of microtubule and microfilament function on the cellular slime mould Dictyostelium discoideum

Colchicine, vinblastine, griseofulvin and isopropyl N-phenyl carbamate (IPC) (agents which inhibit microtubule function) inhibit division of the amoebae of the cellular slime mould Dictyostelium discoideum. These inhibitors also either inhibit or delay the aggregation process characteristic of these amoebae as does cytochalasin B (CB) (an inhibitor of microfilament function). Even when apparently normal fruiting bodies are formed in the presence of these inhibitors the spores may have a diminished heat resistance (CB and IPC) and a volume which is either larger (CB), very much larger (IPC) or smaller (colchicine, vinblastine) than normal. Some of the spores that are formed in the presence of IPC resemble in shape, as well as size, those typical of diploid strains.     

A stage-specific inhibitor of adenylate cyclase in Dictyostelium discoideum

A sudden increase in adenylate cyclase activity occurs during the chemotaxis and aggregation of Dictyostelium discoideum. Preincubation of extracts from the pre-aggregation stage, in which adenylate cyclase activity was low, with post-aggregation stages, in which the increase in activity occurred, resulted in the demonstration of a heat-stable inhibitor of adenylate cyclase (ACI) that was present only during the early stages of development. Cellular fractionation studies showed that ACI was present in both the 100 000 g pellet and supernatant fractions. The inhibitor was not inactivated by proteases or protease inhibitors. A heat-treated preparation of the inhibitor was dialysable. The effect of ACI was dependent upon a pre-incubation treatment, with notable inhibition occurring only after a 20 min pre-incubation period. The apparent inhibition was not artifactual, due to the degradation of the substrate, ATP, or to the loss of the reaction product, cAMP. Additionally, the inhibitor was specific for adenylate cyclase, as it had no effect on the activity of several other enzymes, including cAMP phosphodiesterase.     

Quantitative studies on cell differentiation during morphogenesis of the cellular slime mold Dictyostelium discoideum

Taking advantage of the fact that differentiation of the prespore cell of Dictyostelium discoideum is characterized by synthesis of a prespore specific antigen, the process of its differentiation during the course of morphogenesis was quantitatively studied by determining the proportion of prespore cells and their cellular contents of the antigen, using the method of microfluorometry in combination with immunocytochemistry with antispore serum. The cells synthesizing the antigen became first detectable in the early aggregation center which was about to form a papilla. As the papilla elongated, the number of prespore cells rapidly increased up to the stationary level (70–80% of total cells) before completion of slug formation. During the process antigenic contents of prespore cells were gradually increased and leveled off in the early migration stage. When culmination was induced, antigenic contents were markedly increased to the maximum, which was followed by a sudden decrease immediately before spore formation. On the other hand, the proportions of prespore to total cells were kept constant at the stationary level all through the migration and culmination stages, in spite of a persistent decrease during culmination in the total number of cells due to continuous differentiation of the prestalk into the mature stalk cells. These results were discussed in relation to possible mechanisms of differentiation in this organism.     

Effects of polyunsaturated fatty acids on the growth and differentiation of the cellular slime mould, Dictyostelium discoideum

Cells of Dictyostelium discoideum grown on media containing polyunsaturated fatty acid (PUFA) exhibit impaired differentiation when placed on a solid surface in the absence of all nutrients. Differentiation is inhibited at all temperatures, and although the inhibition is somewhat less pronounced at low temperatures, there is neither a lowering of the optimum temperature of aggregation nor a reversal of inhibition at low temperatures. Furthermore, although the in vitro aggregation of vegetative cells and the reaggregation of dispersed aggregation-phase cells are markedly temperature dependent, PUFA supplementation does not markedly influence this dependence. These data are not consistent with the hypothesis that impaired differentiation is due to increased plasma membrane fluidity. PUFA had no adverse effect on cell growth at temperatures at or below the optimum growth temperature, 22 °C. At 25 °C, however, there was considerable inhibition and at 27 °C growth was completely eliminated in the presence of PUFA.     

Cohesive properties of axenically grown cells of the slime mould Dictyostelium discoideum

It has been found that Dictyostelium discoideum cells from the exponential growth phase of axenically grown cultures are cohesive, whereas those from stationary phase are not. These differences in cohesiveness are seen in phosphate buffer and in axenic medium. Stationary phase medium inhibits the aggregation of log phase cells; stationary phase cells inoculated into freshly prepared medium regain their cohesiveness. Stationary phase medium may contain an inhibitor of cell cohesion. pH differences between the two types of medium are not entirely responsible for loss of cohesiveness.     

Mutants of thermotaxis in Dictyostelium discoideum

Amoebae of Dictyostelium discoideum, strain HL50 were mutagenized with N-methyl-N′-nitro-N-nitrosoguanidine, cloned, allowed to form pseudoplasmodia and screened for aberrant positive and negative thermotaxis. Three types of mutants were found. Mutant HO428 exhibits only positive thermotaxis over the entire temperature range (no negative thermotaxis). HO596 and HO813 exhibit weakened positive thermotaxis and normal negative thermotaxis. The weakened positive thermotactic response results in a shift toward warmer temperatures in the transition temperature from negative to positive thermotaxis. Mutant HO209 exhibits weakened positive and negative thermotactic responses and has a transition temperature similar to the ‘wild type’ (HL50). The two types of mutants represented by HO428, HO596 and HO813 support the model that positive and negative thermotaxis have separate pathways for temperature sensing. The type of mutants which contains HO209 suggests that those two pathways converge at some point before the response.     

Cyclic AMP, pattern formation and movement in the slime mould, Dictyostelium discoideum

It is important to determine whether a gradient of cyclic AMP exists along the migrating slime mould grex because such a gradient might be involved in pattern formation and the polarity of grex movement. Biochemical measurements provided results which could be consistent with Bonner's suggestion that 50% of the cyclic-AMP produced by the grex is produced by the anterior one-tenth. However, chemotactically sensitive cells cannot detect a gradient of cyclic-AMP emission along the grex. Migrating grex cells themselves are not chemotactically sensitive to cyclic-AMP. It seems unlikely that chemotaxis is involved in controlling the polarity of grex movement.     

Microcyst germination in the cellular slime mold, Polysphondylium pallidum: Effects of actinomycin D and cycloheximide on macromolecular synthesis and enzyme accumulation

Germination of microcysts of Polysphondylium pallidum is characterized by an immediate rapid increase in incorporation of [³H]leucine into protein which is cycloheximide-sensitive but unaffected by actinomycin D. Significant RNA synthesis, as measured by [³H]uridine incorporation, does not begin until approx. 2 h after the onset of germination. The increase in [³H]uridine incorporation is prevented by actinomycin D. Germination and the increase in alkaline phosphatase and β-glucosidase enzyme activities are prevented by cycloheximide but unaffected by actinomycin D. The data strongly imply the presence of stable RNA in dormant microcysts and indicate a requirement for a discrete period of protein synthesis for germination of microcysts of P. pallidum.     

Alteration in timing of cell differentiation resulting from cell interactions during development of the cellular slime mold, Dictyostelium discoideum

The effect of cell interactions on the timing of cell differentiation of Dictyostelium discoideum was studied by monitoring spore differentiation in chimeric aggregates composed of two cell populations, one of which was cycloheximide resistant.Chimeras were constructed with cells of a rapidly developing strain, FR-1, and cells of a normally developing cycloheximide resistant strain, CR-2. CR-2 cells formed spores 4 to 5 hr prematurely while spore formation by FS-1 cells was delayed by 1 hr. The two cell populations formed spores 3 hr apart from each other. FS-1 spores were localized to the enlarged bases of the fruiting bodies, whereas CR-2 spores were found in the apical structures.Chimeras were also constructed with cells of two normally developing strains, CR-2 and A3, one population of which had been dissociated at 8.5 hr and the other at 19 hr of development. The 19-hr cells formed spores 2 hr late and 8.5-hr cells 2 to 4 hr prematurely. The two cell populations formed spores within 1 hr of each other although they were asynchronous by 10.5 hr at the time of mixing. These chimeras formed normal fruiting bodies. The results indicate that the timing of cell differentiation is altered by the presence of cells developing at a different rate.     

Cytochrome b of the Dictyostelium discoideum plasma membrane

Cytochrome b of the plasma membrane of Dictyostelium discoideum was investigated in purified plasma membranes and in solubilized form. The membrane-bound cytochrome b can be reduced by NADH. This reduction is inhibited by p-hydroxymercuribenzoate. The reduced cytochrome b does not react with carbon monoxide. Its apparent molecular weight lies between 13000 and 16000. Tryptic digestion yields a large, heme-containing peptide with an apparent molecular weight between 12000 and 15000. After solubilization with cholate, cytochrome b can be enriched by reversed-phase HPLC, indicating that it contains also a hydrophobic component. With these properties, cytochrome b of the D. discoideum plasma membrane resembles microsomal cytochrome b₅.     

Gene regulation during dedifferentiation in Dictyostelium discoideum

During development of Dictyostelium discoideum, cells acquire the capacity to rapidly recapitulate morphogenesis. Therefore, when cells at the loose aggregate stage are disaggregated and challenged to reaggregate, they do so in a tenth of the original time. If loose aggregate cells are disaggregated and resuspended in buffered dextrose solution (erasure medium), they retain the capacity of rapid recapitulation for 80 min, then completely lose this capacity in a single, synchronous step referred to as the "erasure event." The erasure event sets in motion a program of dedifferentiation during which cells lose developmentally acquired characteristics at different times. The erasure event is inhibited by the addition of 10(-4) M cAMP to erasure medium. The synthesis of 33 growth-associated polypeptides, the synthesis of 53 development-associated polypeptides, and the level of 2 development-associated RNAs have been monitored during the erasure program and in cultures inhibited from erasing by the addition of 10(-4) M cAMP. Growth-associated polypeptides begin to be resynthesized and development-associated polypeptides exhibit dramatic decreases in rate of synthesis at different times throughout the first 240 min in erasure medium. Inhibiting the erasure event with cAMP has no major effect in the resynthesis of the majority of growth-associated polypeptides. Only one growth-associated polypeptide, V28, is completely inhibited by cAMP, suggesting that it may play a role in the erasure process. In contrast, inhibiting the erasure event with cAMP has a marked effect on the synthesis of development-associated polypeptides, causing a dramatic reduction in the rate at which synthesis decreases for 6 polypeptides, and completely inhibits the decrease in the synthetic rate of 8 polypeptides. The two development-associated RNAs, 16G1 and 10C3, exhibit two distinctly different patterns of loss during erasure, but in both cases cAMP added at time zero of the erasure process dramatically retards or inhibits loss. In addition, when cAMP is added just prior to the erasure event, it inhibits the erasure event and stimulates a rapid increase in the level of 16G1 RNA back to the developmental level. The level of 16G1 RNA then remains at this level for at least 400 min. When cAMP is added after the erasure event, it causes a low, transient increase in the level of 16G1 RNA. These results are considered both in relation to the program of erasure, and in relation to the role of cAMP in the expression of developmental genes during the forward program of development.     

A phototaxis signalling complex in Dictyostelium discoideum

Phototaxis has been studied in a variety of organisms belonging to all three major taxonomic domains – the bacteria, the archaea and the eukarya. Dictyostelium discoideum is one of a small number of eukaryotic organisms which are amenable to studying the signalling pathways involved in phototaxis. In this study we provide evidence based on protein coimmunoprecipitation for a phototaxis signalling complex in Dictyostelium that includes the proteins RasD, filamin, ErkB, GRP125 and PKB.     

Genes involved in Dictyostelium discoideum sexual reproduction

Macrocyst formation in the cellular slime moulds is a sexual process induced under dark and humid conditions. Normal development life cycle in these organisms involves proliferation by cell division and, upon starvation, formation of multicellular aggregates and fruiting bodies, consisting of spores and stalk cells. Macrocyst formation, cell division by binary fission and spore formation are thus three alternative modes of reproduction, for which it is of interest to understand how a choice is made. The genetic basis of asexual development and fruiting body formation is well known, by contrast information on the genetic control of sexual reproduction during macrocyst formation is scarce. In Dictyostelium discoideum, the most widely used species, several cell-surface proteins relevant to sexual cell fusion have been identified using cell fusion-blocking antibodies, but isolation of the relevant genes has been unsuccessful. Analysis of sexually deficient mutants, some of which are normal for asexual development, has shown that sexual reproduction is regulated by both specific genes and genes that are also involved in asexual development. Reverse genetic analysis of 24 genes highly enriched in a gamete-specific subtraction library has revealed four genes involved in the regulation of sexual cell interactions. One of them was found to be a novel regulator of the cAMP signalling pathway specific to sexual development. Studies on the molecular genetic control of the sexual cycle will be reviewed and their contribution to our understanding of the organization and function of the D. discoideum genome as a whole discussed.     

The endocytosis of concanavalin A by Dictyostelium discoideum cells

Differentiating cells of D. discoideum in suspension bind ConA. The proportion of the bound lectin which is competitively removed by methyl-α- mannopyranoside decreases with the time of exposure. Ferritin conjugated ConA is seen to bind both to the cell surface and to be taken into the cells, the proportion of the ConA inside the cells increasing with time. The surface bound ConA is removed by washing with methyl-α- mannopyranoside while the endocytosed ConA appears unaffected. It is suggested that much of the [¹²⁵I]ConA, uncompetable by methyl-α- mannopyranoside in our and other binding studies, may be this intracellular ConA.     

The activation of cell aggregation by phosphorylation in Dictyostelium discoideum

Single amoebae of D. discoideum are phosphorylated in the presence of external ATP. Phosphorylation is catalyzed by a cAMP independent cell membrane bound protein kinase. As a result of phosphorylation cell aggregation is induced and the chemotactic sensitivity of the amoebae to a cAMP gradient decreased. Cell membrane phosphorylation may be involved in triggering cell aggregation in vivo. The fact that the number of free phosphorylable sites per cell decreases at the onset of aggregation gives support to this hypothesis. The existence of a plasma membrane bound phosphoprotein phosphatase suggests a possible regulator role for this enzyme on the phosphorylation of the amoebae. Finally, ATP inhibits intercellular contact sites outside the aggregation center. Despite this inhibiting effect on cell adhesiveness, amoebal movement toward an aggregation center maintains its normal periodicity.     

Nuclear plasmids in the simple eukaryotes Saccharomyces cerevisiae and Dictyostelium discoideum

High copy number nuclear plasmids are becoming recognized as common genetic components of simple eukaryotes. Like bacterial plasmids, eukaryotic plasmids ensure their persistence in dividing cells by having a partitioning system and a regulated means of amplifying copy number to correct inherent fluctuations in partitioning. By virtue of their small size and autonomy from the chromosomes, eukaryotic plasmids are useful for studying not only features of eukaryotic replicons but many aspects of gene regulation and DNA organization in nucleated cells.     

The spatial pattern of DNA synthesis in Dictyostelium discoideum slugs

We have used [³H]DNA labelling and autoradiography to investigate the localisation of cells in S phase of the cell cycle during the aggregation and slug stages of Dictyostelium discoideum development. Our results indicate that S phase cells occur behind a sharp transverse boundary, which falls just below (or behind) the anterior tip of the aggregate or slug. PAS staining indicates that this is the boundary between the prestalk and prespore regions.