Migration in Dictyostelium polycephalum.

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.

Behavior of cellular slime molds in the soil.

Cellular slime molds are soil organisms, yet since they were discovered in 1869 they have been studied on agar surfaces. Here the behavior of a number of species is examined and it is evident that they have different responses to directional light and they all thrive in the presence of soil. While phototaxis clearly plays a significant role in their ability to come to the soil surface for dispersal, even more important are gradients in the soil: both temperature gradients known from earlier studies, and as we show here gas gradients, presumably ammonia as a repellent and oxygen as an attractant. There are numerous differences in both morphology and behavior among slime mold species, some of which are likely to be the result of natural selection to particular habitats, while others could be explained more easily by neutral phenotypic variation.

Perspective: the size-complexity rule.

It is widely accepted that bigger entities have a greater division of labor than smaller ones and this is reflected in the fact that larger multicellular organisms have a corresponding increase in the number of their cell types. This rule is examined in some detail from very small organisms to large animals, and plants, and societies. Compared to other size-related rules, the size-complexity rule is relatively rough and approximate, yet clearly it holds throughout the whole range of living organisms, as well as for societies. The relationship between size and complexity is analyzed by examining the effects of size increase and decrease: size increase requires an increase in complexity, whereas size decrease permits, and sometimes requires, a decrease in complexity. Conversely, an increase or decrease in complexity permits, but does not require changes in size. An especially compelling argument for the close relation between size and complexity can be found in size quorum sensing in very small multicellular organisms.

On the origin of differentiation.

Following the origin of multicellularity in many groups of primitive organisms there evolved more than one cell type. It has been assumed that this early differentiation is related to size the larger the organism the more cell types. Here two very different kinds of organisms are considered: the volvocine algae that become multicellular by growth, and the cellular slime moulds that become multicellular by aggregation. In both cases there are species that have only one cell type and others that have two. It has been possible to show that there is a perfect correlation with size: the forms with two cell types are significantly larger than those with one. Also in both groups there are forms of intermediate size that will vary from one to two cell types depending on the size of the individuals, suggesting a form of quorum sensing. These observations reinforce the view that size plays a critical role in influencing the degree of differentiation.

Expression of prestalk and prespore proteins in minute, two-dimensional Dictyostelium slugs.

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.

A way of following individual cells in the migrating slugs of Dictyostelium discoideum.

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.

Development in one dimension: the rapid differentiation of Dictyostelium discoideum in glass capillaries.

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.

Proteolysis and orientation in Dictyostelium slugs.

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.

Regulation of the anterior-like cell state by ammonia in Dictyostelium discoideum.

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.

Ammonia and thermotaxis: Further evidence for a central role of ammonia in the directed cell mass movements of Dictyostelium discoideum.

Evidence is presented to support the hypothesis that, in addition to its possible role in mediating chemo- and phototaxis, ammonia (NH(3)) is also the key substance responsible for directing thermotaxis of the migrating slugs of Dictyostelium discoideum. NH(3) is produced by the cells of the slug and we show that high and low concentrations of NH(3) decrease the speed of the amoebae while intermediate concentrations increase their speed. NH(3) production by amoebae is affected by temperature: the greater the temperature, the more NH(3) is produced. From these facts we speculate that both the positive and the negative thermotaxis found in slugs can be explained by temperature gradients stimulating regional differences in NH(3) production, and depending upon the temperature, the amount of NH(3) will either be in the range that stimulates or inhibits the rate of movement. If this explanation is correct, then minute localized differences in the production of NH(3) and their differential effect on cell speed could account for all the directed movements of the cell masses of these slime molds.

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.

The possible role of ammonia in phototaxis of migrating slugs of Dictyostelium discoideum.

Previously we showed that the rising cell masses of cellular slime molds orient away from high concentrations of ammonia gas, presumably by speeding up the cells on one side. Here we show that in the same way NH(3) could also be involved in the highly sensitive phototaxis found in the migrating slugs of Dictyostelium discoideum. We have evidence that light increases their speed of migration and their production of NH(3). Since unilateral light is concentrated on the distal side of a cell mass by the "lens effect," this leads to the obvious hypothesis that the light stimulates the local production of NH(3), which, in turn, stimulates the cells in the illuminated region to move faster.

Successive asexual life cycles of starved amoebae in the cellular slime mould, Dictyostelium mucoroides var. stoloniferum.

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.

pH affects fruiting and slug orientation in Dictyostelium discoideum.

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.

Chemical identity of the acrasin of the cellular slime mold Polysphondylium violaceum.

The aggregation chemoattractant (or acrasin) of Polysphondylium violaceum, a species of cellular slime mold that does not respond chemotactically to cAMP, has been identified. It was extracted and purified from aggregating amoebae, then analyzed for amino acid composition and by IR and mass spectrometry. The active molecule is N-propionyl-gamma-L-glutamyl-L-ornithine-delta-lactam ethyl ester (Mr, 327), which we have named glorin. The compound has been synthesized and shows normal chemotactic activity with the amoebae of P. violaceum.