Search

By comparing two species of cellular slime molds that have stalkless migration stages it is possible to gain interesting insights into how the cells move. In contrast to the familiar behavior of Dictyostelium discoideum, Dictyostelium polycephalum slugs can travel greater distances through soil and even can migrate through agar. In addition to the interest in the differences, these differences shed light on the mechanism of slug movement. Unlike D. discoideum, D. polycephalum does not have prestalk and prespore zones and severed sections of any part of these slugs move at a rate proportional to their length. This leads to the hypothesis that longer slugs move faster because the amoebae aligned along the inside of the slime sheath each contribute a forward push and the more extended the amoebae line is the faster the slug moves.

We show that exceedingly small two-dimensional slugs of Dictyostelium differentiate normally and have an anterior prestalk zone and a posterior prespore zone. Using GFP as a marker attached to the appropriate promoter, prestalk expression is concentrated in the anterior, while prespore expression is produced in the posterior, closely resembling what is found in normal, large slugs.

In the development of the cellular slime mold Dictyostelium discoideum there is a stage in which the aggregated amoebae form a migrating slug that moves forward in a polar fashion, showing sensitive orientation to environmental cues, as well as early signs of differentiation into anterior prestalk and posterior prespore cells. Heretofore it has been difficult to follow the movement of the individual cells within the slug, but a new method is described in which small, flat (one cell thick) slugs are produced in a glass-mineral oil interface where one can follow the movement of all the cells. Observations of time-lapse videos reveal the following facts about slug migration: (i) While the posterior cells move straight forward, the anterior cells swirl about rapidly in a chaotic fashion. (ii) Turning involves shifting the high point of these hyperactive cells. (iii) Both the anterior and the posterior cells move forward on their own power as the slug moves forward. (iv) There are no visible regular oscillations within the slug. (v) The number of prestalk and prespore cells is proportional for a range of sizes of these mini-slugs. All of these observations on thin slugs are consistent with what one finds in normal, three-dimensional slugs.

When Dictyostelium discoideum cells are drawn into a fine glass capillary, they rapidly begin the first steps toward the formation of prestalk and prespore zones. Some of the events occur within a minute or two, whereas others follow later. The cells in the front segment are actively motile and those in the hind segment are passive. The volumes of the segments are proportional for different-sized cell masses, and those proportions are the same as those found in normal slugs. When the cells are stained with the vital dye neutral red, the anterior zone becomes darker simultaneously with the formation of the division line. Green fluorescent protein expressed from a stalk-specific promoter is synthesized mostly in the anterior end. Later, this capillary prestalk zone shows a sharp increase in alkaline phosphatase activity, which is known to be characteristic of prestalk cells.

Dictyostelium slugs expressing a dominant inhibitor of the cAMP-dependent protein kinase (PKA) selectively in prestalk cells are insensitive to the environmental signals that normally induce culmination (Harwood et al., 1992a). When such slugs do, eventually, attempt to culminate they are unable to activate stalk-specific gene expression and they become arrested in their development. We show here that they are also about half the size of normal slugs and that they move at about half normal speed, even after their size difference has been taken into account. They are also less accurate in orienting their direction of movement towards the light. Thus, in addition to its role in cellular differentiation, PKA is required for several important aspects of slug behaviour.

It has been long known that the migrating slugs of the cellular slime moulds are highly sensitive to their environment and orient towards light and in temperature and chemical gradients. There is considerable evidence from past work that these orientations are governed by NH3 which affects the rate of movement of cells within the slug with such precision that orientation to the external stimuli is achieved. In order to test this hypothesis further, various ways to alter the internal NH3 concentration were devised. Substances that either increased or decreased proteolysis were applied to one side of the tip of a slug, thereby affecting its orientation. Some of the treatments strongly support the role of internally produced NH3 in orientation, and all the treatments produce results that are consistent with the hypothesis.

Ammonia appears to be an important regulatory signal for several aspects of the Dictyostelium life cycle. The postulated role of ammonia in the determination of the prespore pathway in cells of the slug stage has led us to examine the effect of ammonia on the prestalk/prespore ratio of migrating slugs. In the presence of 10(-3) M ammonium chloride, the volume of the prestalk region decreases by 40.8%. The kinetics of the process make it unlikely that this is due to a shift in the differentiation pathway. A test of the hypothesis that the decrease in volume of the prestalk region is due to the conversion of prestalk cells to anterior-like cells shows that the percent of anterior-like cells in the posterior region increases by the amount predicted by the hypothesis. This suggests that ammonia may be the molecular signal, produced by the tip, that prevents anterior-like cells from chemotactically migrating to the tip and thereby becoming anterior cells. The effect of enzymatic removal of ammonia from vitally stained migrating slugs is the appearance of a series of dark stripes beginning at the posterior end and progressing forward. We interpret this as a result of progressive removal of anterior-like cells from tip dominance and essentially as the formation of new potential tips. Indeed, in a few cases one or even two of the stripes separate from the posterior of the cell mass and form small fruiting bodies. We consider the phenomenon of stripe formation further evidence that the tip acts on anterior-like cells through ammonia.

Dictyostelium chromatin has been purified from nuclei in high yield by differential centrifugation and nuclease cleaving. Its chemical composition has been assayed, and its histones have been analyzed by gel electrophoresis, peptide fingerprints, amino acid composition, and ion-exchange chromatography. The mass ratios of DNA/RNA/histone/nonhistone are 1.0:0.18:0.98:1.02. There are four histones including one unusual histone, H7, which is the most abundant histone in the slime mold. The H4-like protein is the most conserved protein, while the other histones show both similarities and differences with mammalian histones.

Antibodies, shown to be specific for DNA/RNA hybrids, have been covalently attached to CNBr-activated Sepharose. The resultant affinity resin specifically binds DNA/RNA hybrids and has been used to enrich for the DNA which codes for rRNA in the slime mold Dictyostelium discoideum. By utilizing the technique of R-loop formation, DNA molecules containing the rRNA genes were isolated from total nuclear DNA in a double-stranded form. These rDNA molecules, which were recovered by high salt elution from the affinity resin, were typically 15-40 kbp in length, and thus contained DNA sequences adjacent to the selected sequences coding for the 17S and 26S rRNAs. In addition, evidence has been obtained concerning the structure of Dictyostelium rDNA which agrees with the finding (Taylor et al. (1977) ICN-UCLA Symp. Mol. Cell. Biol. 8, 309-313) that the rDNA molecules are not covalently attached to the chromosomes of this organism.

Protein degradation appears to be essential for normal differentiation in the cellular slime mold Dictyostelium discoideum. Several protease inhibitors block normal differentiation, and in most cases this inhibition can be reversed by addition of amino acids. For example, chloroquine, which inhibits slime mold cathepsin B activity, interferred with development by blocking sorocarp formation, and this inhibition was reversed by the addition of amino acids. Tosyllysyl chloromethyl ketone also blocked development, and this inhibition was reversed by simultaneous additions of amino acids and glutathione. Moreover, the addition of antipain and leupeptin delayed sorocarp formation. These results, together with the finding reported earlier that cathepsin B activity is differentially localized in the prestalk-prespore zones of the migrating slugs, suggest that proteolysis might play a regulatory role in cellular slime mold differentiation.

A dialyzable sex hormone is produced by one strain of Dictyostelium discoideum (NC-4) that induces macrocyst formation (sexual stage) in its opposite mating type (V-12).

Hitherto it has not been possible to obtain spore and stalk cell differentiation of the cellular slime molds in submerged cultures. It is shown here that cells, when placed in roller tubes under an atmosphere of oxygen, will form clumps and differentiate in 48-72 hr into mature spores and stalk cells. Although differentiation occurs without the normal morphogenetic movements, there is the appearance of an anteroposterior polarity of the cells in the clump. In addition to oxygen we examined a number of other factors that affect differentiation.

In cellular slime molds the slugs become divided into two regions with different properties, and anterior prestalk-zone and a posterior prespore zone. Although the cells in these zones are normally destined to form the stalk cells and spores of the fruiting body, respectively, they are not irreversibly committed to one sort of differentiation or the other during the slug stage. The volume ratio of the two zones remains almost constant over a wide range of slug sizes. If the prestalk-prespore pattern is distrubed by removing tissue from the slug, conversion of tissue from prestalk to prespore or vice versa occurs as necessary to restore a normal pattern with normal proportions. Conversions also occur in both directions during normal development. The initial formation of the prestalk-prespore pattern may well involve sorting-out, but other mechanisms must be invoked to account for regulation. We describe three different models of the generation of the prestalk-prespore pattern, the'cell-contact model' of McMahon, in which pattern is created by interactions of cells with their immediate neighbors, the 'positional-information model' of various authors, in which pattern formation involves an overall gradient and a gradient-reading mechanism, and the 'activator-inhibitor model' of Gierer and Meinhardt, in which the prestalk-prespore pattern is formed by a system of diffusible substances that affect one another's production. The activator-inhibitor model is the most successful of the models at describing the known features of the prestalk-prespore pattern. The various models lead to a number of distinctive predictions. According to the cell-contact model, small transplants may cause gross changes in the prestalk-prespore pattern, and mutants may exist which severely disrupt pattern formation even if diluted with a large excess of wild-type cells. Positional-information models predict the existence of 'gradient-reading mutants'; slugs that are a mixture of such mutants and wild-type cells would show two prestalk-prespore boundaries, one at the mutant and one at the normal position. Both the activator-inhibitor model and some versions of the positional-information model predict that small transplants will sometimes induce accessory prestalk or prespore zones; the quantitative characteristics of these effects may allow one to make a case in favor of one or other of the two models. Finally, the activator-inhibitor model leads one to expect that mutants may be isolated which normally show accessory prestalk or prespore zones. A search for these phenomena may help determine whether the activator-inhibitor model will continue to enjoy its present preeminent position.

We have demonstrated two interesting facts about the transition from the migration stage to the final fruiting stage of Dictyostelium discoideum. One is that fruiting is favoured on acid substrata, and secondly, migrating slugs tend to migrate towards the acid side of a pH gradient. The suggestion is offered that these results can be interpreted in terms of the effects of NH3. It appears to be an additional mechanism (besides phototaxis and thermotaxis) to assure that the final fruiting takes place in a favourable environment.

Dense masses of spores of Dictyostelium mucoroides var. stoloniferum have the ability to germinate and aggregate rapidly in the absence of food. This is made possible by the presence of a dominant, self-produced spore germination activator. The germination-aggregation cycle can be repeated in as many as six successive generations. In each generation the spore size is reduced so that ultimately they are only a fraction of the size of those produced by the parental, bacteria-fed amoebae.