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.     

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.     

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.     

The Breeding System of Polysphondylium pallidum,a Cellular Slime Mold*

SYNOPSIS. A mating type analysis was performed on 231 isolates of the cellular slime mold, Polysphondylium pallidum found in 61 samples collected in eastern North America between northern Florida and southern Canada. Seventy-eight percent of the isolates belonged to one of 2 mating types; 18% were incapable of mating with any partner; 3% were homothallic; and 1%, consisting of 2 isolates from a Florida sample, belonged to a separate breeding group. It is suggested that the majority of isolates represent a species capable of local genetic adaptation to a niche, the parameters of which undergo considerable variation over space and time.     

Microcysts of the Cellular Slime Mold Polysphondylium pallidum I. Factors Influencing Microcyst Formation

Microcyst formation can be induced by increasing the osmotic pressure of the surrounding medium. Certain ions such as K(+), Ca(++), or Mg(++) may be needed in the encystment process, and the presence of divalent cations increases the rate of encystment and cyst maturation. Chloride of potassium is optimal for encystment, but other anions of potassium are either less effective or toxic. The optimal pH for encystment was found to be pH 6.0. The use of agar plates containing KCl revealed the importance to the encystment process of inhibiting cell aggregation. When myxamoebae of Polysphondylium pallidum strain Pan-17 are deposited on KCl-agar plates, approximately 20% of the population proceeds through aggregation to sorocarp formation at the concentration of KCl optimal for microcyst formation. However, the same proportion of myxamoebae remains unaligned, or forms defective aggregation centers, if synergistic inhibitors (such as incubation in darkness or at low temperature) are employed in addition to KCl. The possibility that this is due to heterocytosis has been excluded. Accordingly, it is suggested that during the stationary phase approximately 20% of the population becomes committed to forming component cells of fruiting bodies, and that these myxamoebae cannot be induced to form microcysts by exposure to KCl. In P. pallidum strains WS-320 on the other hand, the imposition of synergistic inhibitors leads to the total encystment of the cell population. This suggests that, in contrast to Pan-17, the myxamoebae of the latter strain remain potentially equal and exhibit minimal presumptive specialization.     

Microcysts of the Cellular Slime Mold Polysphondylium pallidum II. Chemistry of the Microcyst Walls

Methods are described for obtaining large masses of myxamoebae, for inducing these to form microcysts, and for the isolation of the microcyst walls from other cell components. The walls were fractionated into two parts, one alkali-soluble, the other alkali insoluble. The alkali-insoluble fraction is a type of cellulose and constitutes 28% of the microcyst wall by weight. The alkali-soluble fraction contains a glycogenlike material, lipids, and proteins. A possible mechanism of microcyst wall synthesis is discussed.     

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.     

α-Mannosidase and Microcyst Differentiation in the Cellular Slime Mold Polysphondylium pallidum

The intracellular and extracellular pattern of α-mannosidase (EC 3.2.1.24) activity was studied during microcyst differentiation in the cellular slime mold, Polysphondylium pallidum. The evidence suggests that microcyst differentiation requires continuous protein synthesis. α-Mannosidase activity is present in amoebae and increases with differentiation, and the data indicate that this increase in activity requires concurrent protein synthesis. The enzyme is excreted during the differentiation process, and the release of the enzyme is not stopped by cycloheximide. A cystless mutant does not show the normal intracellular pattern of α-mannosidase but does excrete the enzyme. Microcyst differentiation is proposed as an alternative system to multicellular slime mold development for the biochemical analysis of certain aspects of cellular differentiation.     

Syngenic Divisions of the Cellular Slime Mold Polysphondylium violaceum*

SYNOPSIS. Mating type analysis of new isolates of Polysphondylium violaceum supports the subdivision of this morphological species into two reproductively isolated breeding groups or syngens. Representatives of both syngens have been identified in soil samples taken from widely separated geographical locations. Intersyngenic cross reactions have been observed in some stocks.     

Ultrastructure of Acrasis rosea,a Cellular Slime Mold,During Development*

SYNOPSIS. An ultrastructural study of the myxamoebae of Acrasis rosea in the vegetative, aggregative and culminative stages was made. An intracytoplasmic system of microfibrillar bundles develops as the cells enter the aggregative stage and commence the morphogenetic sequence leading to the construction of a fruiting body. The fibrillar bundles disappear in the cells of the mature fruiting body. No relevant ultrastructural differences were observed between spores, stalk cells and microcysts. Each of these cells is surrounded by a single-layered coat of fibrillar material that is oriented parallel to the cell surface. Tubular structures were observed between the plasma membrane and the cell coat. The tubules may be layered along the cell periphery or they may be recessed in pockets formed by the plasma membrane. They resemble lomasomes typical of fungal cells. The myxamoebae of A. rosea clearly differ from the Dictyostelium-type myxamoebae in mitochondrial structure, the presence of lamellate structures in the nucleolus and the absence of prespore vacuoles.     

Natural Variants of the Cellular Slime Mold Acrasis rosea

SYNOPSIS. Twenty different isolates of the cellular slime mold Acrasis rosea, obtained from diverse sources and geographic regions, were studied to determine similarities and differences in their development and structure in culture and their sensitivity or resistance to selected chemicals incorporated into the culture media. Six different classes of fruiting were defined based on the size, distribution, and type of sorocarps formed on the yeast, Rhodotorula, streaked on agar. In the course of these studies a significant mutant, NC-18V (variant), developed spontaneously from the wild type, normal parent strain NC-18N. The mutant differed considerably from all other Acrasis isolates, appeared several times in purified parental cultures, and represents the first laboratory derived variant of A. rosea to be described. Purified strains of the variant (V) and normal (N) cultures were obtained by single-spore isolation. Normal and variant amebae were mixed in ratios of 10:1 and 100:1 (N:V) and spore and stalk cells were selected from different sorocarps in various regions of the culture plate for analysis. The results of these selection experiments clearly indicate that the individual variant amebae have increased migratory ability and that they develop smaller, morphologically different, and more numerous sorocarps that form at distances further from the food source than NC-18N. Some isolates of Acrasis no longer were able to fruit and were classified as “non-fruiters” and a few other isolates formed only a few, small sorocarps on rare occasions. These isolates were mixed together in various combinations of 2 and 3 to screen for cell interaction, but no synergism contributing to fruiting was found. Although fruiting of many A. rosea isolates was inhibited by exposure to continuous light or constant darkness, some “escape”fruiting was noted in certain isolates even when small inocula were used. Single spore isolates of these escape fruiters still fruited in continuous light or dark, but fruiting was always greatly enhanced by a routine 12 hr light : 12 hr dark incubation cycle. It was shown by biochemical studies that actidione, crystal violet, malachite green, ethyl violet, and 5-fluorodeoxyuridine selectively killed some isolates and permitted a classification of isolates as either sensitive or resistant. In a further study of cell interaction between 2 different sets of Acrasis isolates with contrasting biochemical and morphologic markers the formation of neotypes or recombinants could not be demonstrated. The results of this study clearly indicate the existence of significant variation in A. rosea and the potential for application of these differences to developmental studies.     

Growth of the Cellular Slime Mold, Dictyostelium discoideum, Is Gravity Dependent

The effect of artificial gravity on the growth of a microorganism, Dictyostelium discoideum, was studied and the following results were obtained: (a) Germination efficiency increased as gravity increased up to 3 gravities. (b) Cell differentiation was influenced by gravity. Retardation of spore formation or reduction in the spore fraction was observed at hypergravity. (c) Fruiting bodies were taller at hypergravity and smaller at simulated microgravity when compared at 1 gravity. It is suggested that modulation of gravity provides useful information on the mechanisms of life.     

Evidence for a Second Chemotactic System in the Cellular Slime Mold, Dictyostelium discoideum

An unknown substance found in bacteria (Escherichia coli) is especially effective in attracting the vegetative amoebae of the cellular slime mold, Dictyostelium discoideum. However, the aggregating amoebae are not attracted to it at all. On the other hand, the vegetative amoebae show very little chemotactic response to cyclic adenosine monophosphate (cyclic AMP), whereas the aggregating amoebae are exceptionally responsive to it. It is suggested that the new factor may be used in food seeking, whereas cyclic AMP, the chemotactic substance responsible for aggregation, is the acrasin of this species. The important point is that the amoebae are differentially stage-specific in their responses to these two chemotactic agents.     

Isolation of a Spore Germination Inhibitor from a Cellular Slime Mold Dictyostelium discoideum

The functional properties of gluten obtained by treating with chymotrypsin at alkali pH were investigated. The gluten was treated by chymotrypsin at pH 10.0 and 20°C, and was found to be deamidated to a state that was scarcely subject to proteolysis by chymotrypsin. The degree of deamidation of the gluten reached about 25% by this treatment for 2 hr. The functional properties of the gluten thus obtained were investigated in regard to deamidation. The enzymatically deamidated gluten greatly improved such functional properties as solubility and emulsifying ability. In particular, the solubility of the treated gluten was remarkably high in the pH range of 5 to 8, in which native gluten is insoluble. It was apparent that the improvement in functional properties of gluten was mainly due to the deamidation induced by treating with chymotrypsin at pH 10.0 and 20°C.     

Evidence for Mitochondrial DNA Polymorphism and Uniparental Inheritance in the Cellular Slime Mold Polysphondylium Pallidum: Effect of Intraspecies Mating on Mitochondrial DNA Transmission

Restriction fragment length polymorphisms (RFLPs) were used as markers to monitor mitochondrial inheritance in the cellular slime mold, Polysphondylium pallidum. When two opposite mating types (mat1 and mat2) of closely related strains were crossed, all the haploid progeny regardless of mating type inherited their mitochondrial DNA from the mat2 parent only. When opposite mating types from more distantly related strains were crossed, most of the progeny also inherited their mitochondrial DNA from the mat2 parent, but some inherited their mitochondrial DNA from the mat1 parent. In both cases however, the transmission of mitochondrial DNA was uniparental, since in every individual progeny only one type of mitochondrial DNA exists. Moreover, in crosses involving more distantly related strains all the progeny of a single macrocyst were shown to contain the same type of mitochondrial DNA. These findings are discussed in regard to mechanisms of transmission and the possible involvement of nuclear genes in the control of transmission of mitochondrial DNA in Polysphondylium.     

Silica Gel as a Preserving Agent for the Cellular Slime Mold Acrasis rosea

SYNOPSIS. Modifications of the silica gel technic of Perkins and Ogata have been successfully used to preserve Acrasis rosea for 6–13 months and Dictyostelium purpureum for 31 months. The technic is simple, reliable and inexpensive and is recommended for its possible application for the general preservation of certain fungi and protozoa.     

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.