PHAGOCYTOSIS BY THE CELLULAR SLIME MOLD POLYSPHONDYLIUM PALLIDUM DURING GROWTH AND DEVELOPMENT

The phagocytic ability of amoebae of the cellular slime mold Polysphondylium pallidum, grown in shaken suspension, was examined. An established quantitative assay of the uptake of polystyrene (PS) beads was shown to be valid for this organism. The kinetics of phagocytosis were determined, and estimates of the concentration of PS beads necessary to achieve half-maximal phagocytic velocity (K_p), as well as the maximal velocity itself (V_p^max), were made. Comparison with previously published data on Acanthamoeba and guinea pig leukocytes suggested that the P. pallidum amoebae had the lowest K_p, while the leukocytes had the highest V_p^max. Beads approximately 1 µm in diameter appeared to be the optimal size for ingestion. Simultaneously with phagocytosis, comparable numbers of beads accumulated at the cell surface; this accumulation did not occur when phagocytosis was inhibited. Phagocytosis was depressed by protein in the medium, by increased osmolarity, and by inhibitors of aerobic metabolism. Starvation-initiated development, leading to encystment, was shown to affect the capacity of the cells to phagocytize, mainly by progressively decreasing the time span over which the cells ingested particles at a constant initial rate.     

DEDIFFERENTIATION OF THE DISAGGREGATED SLUG CELL OF THE CELLULAR SLIME MOLD DICTYOSTELIUM DISCOIDEUM

It was previously shown that differentiation of the prespore cell in the pseudoplasmodium (slug) of the cellular slime molds is characterized by the synthesis of a specific substance which is detectable by a heteroplastic antispore serum (T akeuchi , 1963). When a prespore cell which was already differentiated was disaggregated from a slug of Dictyostelium discoideum and was incubated in salt solution under a sparsely populated condition, it gradually lost its specific substance and dedifferentiated. The dedifferentiation proceeded without accompanying cell growth and was completed within 5 hr of incubation. This process was inhibited at a low temperature and also in the presence of cyclohexamide, actinomycin D, and cyclic AMP. The dedifferentiation was induced and proceeded at a normal rate in the absence of bacteria. When a disaggregated slug cell was incubated in the presence of bacteria, however, every prestalk and prespore cell was able to grow and underwent its first cell division after about 9–10 hr of incubation, and then multiplied with the generation time of 3 hr. The relationship between the dedifferentiation and the growth of a disaggregated slug cell was discussed.     

FORMATION OF A PRESPORE SPECIFIC STRUCTURE FROM A MITOCHONDRION DURING DEVELOPMENT OF THE CELLULAR SLIME MOLD DICTYOSTELIUM DISCOIDEUM

The origin of a unique vacuole (PSV), which was specifically present in the prespore cell of the cellular slime mold Dictyostelium discoideum. was investigated electronmicroscopically. A considerable number of PSV-mitochondrion complexes was found in the intermediate fraction between a pure PSV and a pure mitochondria fractions, which were obtained by isopicnic centrifugation of cellular components of the prespore cell. Similar complexes were also observed in the differentiating prespore cells. Furthermore, the activity of succinic dehydrogenase, a typical mitochondrial enzyme was found cytochemically to be localized in the PSV as well as in mitochondria. From these results, it was concluded that the PSV was formed from the mitochondrion through some intermediate steps.     

SOME ASPECTS OF BEHAVIOR OF THE MIGRATING SLUG OF THE CELLULAR SLIME MOLD DICTYOSTELIUM DISCOIDEUM

The characteristic motile responses of the pseudoplasmodium (slug) of the cellular slime mold, Dictyostelium discoideum , to such experimental conditions as dissection, fusion, grafting, transplantation, insertion of a mica platelet and introduction into a blind tube were studied. The results indicate that the slug tip plays a kind of "leadership" role and directs the cells in the rear part of the slug forward.
By combining an improved transplantation technique to introduce a slug into an agar tube with staining with vital dyes and fluorescent antispore serum, it has been shown that anterior cells transplanted into the posterior region of the intact slug migrate toward the anterior, thus confirming the results obtained by B onner (3). The experiment verified that vital staining by Nile blue sulfate is a useful way to mark the transplanted cells.     

CHANGES IN CHARGED GROUPS ON THE CELL SURFACE DURING DEVELOPMENT OF THE CELLULAR SLIME MOLD DICTYOSTELIUM DISCOIDEUM: AN ELECTRON MICROSCOPIC STUDY

The distribution of charged groups on the surface of Dictyostelium cells and their change during development were examined by electronmicroscopy using cationic and anionic ferritins. The number of anionic sites on the cell surface decreased greatly during the course of development. The whole surface of vegetative cells stained strongly with cationic ferritin (CF). On the other hand, the surface of aggregation-competent cells had fewer negative charges and these were unequally distributed, the surface of the advancing area (lobopodial region) being devoid of anionic sites. The number of anionic sites on the cell surface decreased progressively during further development, and the suface of slug cells did not stain at all with CF. The cell surface did not stain with anionic ferritin at any developmental stage, indicating the absence of detectable cationic sites. The biological significance of these findings is discussed in connection with cell adhesiveness and movement.     

A "CELL-CONTACT TEMPERATURE-SENSITIVE" MUTANT OF THE CELLULAR SLIME MOLD DICTYOSTELIUM MUCOROIDES

A mutant (TS-2) that is temperature-sensitive with respect to cell contact was isolated from the cellular slime mold, Dictyostelium mucoroides. The TS-2 were able to grow and develop normally at 21°C, but unable to grow at 31.5°C. When TS-2 were allowed to develop until the aggregation stage at 21°C and then shifted to 31.5°C, they instantly lost cell-to-cell contact, resulting in disintegration of the aggregation stream and flattening of the aggregation center. Although a slug transferred to 31.5°C retained its original shape, loss of cell-to-cell contact within the cell mass was evidenced by several facts. The TS-2 interphase amebas, at 31.5°C, also lost cell-to-substratum contact, and the loss of contact was followed by the production of cell-wall substance on their surface. The production of the same substance at 31.5°C was also observed in cells at aggregation and migration stages, but not in those at the vegetative stage. When TS-2 cells at various developmental stages were kept at 31.5°C for various periods of time and returned to 21°C they lost morphogenetic capacity in proportion to the production of the cell-wall substance.     

BIOCHEMICAL CHANGES DURING GROWTH AND ENCYSTMENT OF THE CELLULAR SLIME MOLD POLYSPHONDYLIUM PALLIDUM

The growth of the cellular slime mold, Polysphondylium pallidum, was studied on a semidefined medium in shaken suspension. When the medium contained large quantities of particulate material, growth was more rapid and the cellular size and protein content were smaller than when growth occurred on a medium containing less particulate material. The cellular levels of DNA, RNA, and protein; of lysosomal enzymes (acid phosphatase, acid proteinase); and of peroxisomal enzymes (catalase) were assayed during growth and the subsequent stationary phase that led eventually to encystment. Only DNA remained at a constant cellular level. Encystment of exponentially growing cells could also be initiated by washing them and introducing them into a soluble peptone medium. The rate of encystment was proportional to the osmolarity of this medium. The encystment process was followed with respect to the cellular levels of DNA, RNA, protein, carbohydrates, acid phosphatase, acid β-N-Ac-glucosaminidase, and catalase. The most dramatic change occurred in the cellular cellulose content, which increased by at least an order of magnitude by the time encystment was morphologically complete. It was concluded that the encystment of this slime mold in suspension exhibits a number of biochemical similarities to the development of this and other cellular slime molds on a surface.     

RNA IN CYTOPLASMIC AND NUCLEAR FRACTIONS OF CELLULAR SLIME MOLD AMEBAS

A method is described for the rapid separation of cellular slime mold (Dictyostelium discoideum) cells into nuclear and cytoplasmic fractions. Sucrose density sedimentation profiles of radioactivity from cells that had been grown for long or short periods in the presence of uridine-³H indicate very low levels of cross-contamination between the fractions. The nuclear fraction contains few, if any, ribosomes. In exponentially growing cells, at least 80% of the ribosomes were associated in polysomal complexes. No loss of counts from pre-labeled rRNA was observed during 2 generations (24 hr) of logarithmic growth and, within the polysomal complexes, the distributions of the preformed material and of rRNA synthesized during the 2 generations were identical. In stationary phase cells that had entered the developmental program leading to fruiting body construction, the rRNA turned over rapidly so that by the end of development at least 75% of the ribosomes fabricated during exponential growth had disappeared and had been replaced by new ones synthesized during the morphogenetic sequence. The preformed ribosomes disappeared preferentially from the monosomal contingent; the newly synthesized ribosomes appeared exclusively in the polysomal contingent and did not appear as monosomes in appreciable numbers for at least 6 hr. The possible significance of this wholesale replacement of ribosomes is discussed.     

ISOLATION AND CHARACTERIZATION OF A PRESPORE SPECIFIC STRUCTURE OF THE CELLULAR SLIME MOLD, DICTYOSTELIUM DISCOIDEUM

A method was described for isolation of the prespore specific vacuole (PSV) from slugs of the cellular slime mold, D. discoideum . A cellular component, which was fractionated in accordance with immunohistochemical staining using heteroplastic antispore serum, was found to consist of only the PSV. It was thus concluded that the PSV is identical with the cytoplasmic granule which has been shown by the antiserum to be specifically present in the prespore cell, and hence that the PSV is the only structure which contains the prespore specific substance (antigenic mucopolysaccharide). The isolated PSV contained polysaccharide equivalent to 14% of its protein content, and antigenic mucopolysaccharide constitutes about 60% of the total polysaccharide.     

THE EFFECT OF ACTIDIONE ON MITOSIS IN THE SLIME MOLD PHYSARUM POLYCEPHALUM

Actidione, reportedly a specific inhibitor of protein synthesis, was found to reduce the incorporation of labeled amino acids into proteins of the slime mold Physarum polycephalum without drastically inhibiting the incorporation of nucleic acid precursors into RNA. This inhibitor was found to completely block the ensuing mitosis if it was added at any time between telophase and late prophase. Plasmodia given Actidione in late prophase (about the time of nucleolar dissolution) went on through telophase to reconstruction even though nuclear amino acid incorporation was drastically reduced during that period.     

EFFECTS OF LIGHT ON THE DEOXYRIBONUCLEIC ACID FORMATION AND CELLULAR DIVISION IN CHLORELLA PROTOTHECOIDES

1. It has been demonstrated previously that when Chlorella protothecoidesis grown in a medium rich in glucose and poor in nitrogen source(urea), chlorophyll-less cells with markedly degenerated plastids—called "glucose-bleached" cells—are produced eitherin the light or in darkness. When the glucose-bleached cellsare incubated in a medium enriched with the nitrogen sourcebut without added glucose, normal green cells with fully organizedchloroplasts are obtained in the light, and pale green cellswith partially organized chloroplasts in darkness. During theseprocesses of chloroplast development in the glucose-bleachedcells, there occurs, after a certain lag period, an active DNAformation followed by a more or less synchronous cellular division.In the present study the effects of light on the DNA formationand cellular division were investigated in the presence of CMUor under aeration of CO²-free air to exclude the interveninginfluence of photosynthetic process.
2. It was revealed thatlight severely suppresses the DNA formationand cellular divisionof the glucose-bleached cells while enhancingremarkably theirgreening. The suppression was saturated atthe light intensityof about 1,000 lux. Blue light was mosteffective, being followedby green, yellow and red light inthe order of decreasing effectiveness.
3. Further experiments unveiled that light exerts two apparentlyopposing effects on the DNA formation depending upon the timeof application during the incubation of algal cells. When thealgal cells were illuminated only during the lag period beforethe active DNA synthesis, there occurred an enhancement of theDNA synthesis occurring during the subsequent dark incubation.When, on the other hand, the cells were transferred to the lightfrom darkness at or after the start of the DNA synthesis, itcaused an almost complete abolition of the subsequent synthesisof DNA in the algal cells. No such effects of light were observedwith RNA and protein (total)
4. These findings were discussedin relation to the process ofchlorophyll formation occurringconcurrently in the algal cells.

(Received August 10, 1967; )     

AGGREGATELESS MUTANT DEFECTIVE IN SIGNALING AND RELAYING IN THE CELLULAR SLIME MOLD, DICTYOSTELIUM DISCOIDEUM

An aggregateless mutant (HT41), isolated from D. discoideum NP14, was examined for the parameters involved in cell aggregation. HT41 cells were induced to express some of these parameters by cyclic AMP pulses. Spontaneous oscillations in light scattering were not observed in the suspension of the mutant cells. A pulse of cyclic AMP caused a monophasic decrease in light scattering, which was similar to the response of pre-aggregation cells of the wild-type. When cyclic AMP pulses were applied on a cell layer, HT41 cells aggregated without forming the streams. These results suggest that HT41 is a mutant defective in signal emission and relay.     

INFLUENCE OF IONIC CONDITIONS ON CELL DIFFERENTIATION AND MORPHOGENESIS OF THE CELLULAR SLIME MOLDS

Effects of various ionic conditions on the development of the cellular slime molds D. discoideum and D. mucoroides were studied. A certain concentration of lithium ions (7 mM) promoted differentiation of the stalk cells and conversely inhibited formation of the spores. The presence of calcium and magnesium ions was needed for Li to manifest its specific effect. A high concentration of Ca (100-120 mM) also facilitated differentiation of the stalk cells. On the other hand, fluoride ions stimulated considerable formation of spores at 15 mM. In the absence of divalent cations, sodium ions inhibited morphogenesis and cell differentiation proportionately with its concentration, and complete inhibition was obtained at 20 mM. The inhibitory effect of Na was nullified by addition of small amounts of Ca. Possible mechanisms by which these ions exert their influences on development of this organism were discussed.     

THE EFFECTS OF LIGHT ON LUMINOUS BACTERIA

A conservative statement would therefore be that luminous bacteria show no changes in luminescence as a result of illumination by 625 foot candles for 1.5 minutes when examined 1/200 of a second after exposure, and none as the result of illumination by 15,000 foot candles for 6 minutes when examined ⅙ of a second after exposure.     

FIBRILLAR DIFFERENTIATION IN A MICROPLASMODIUM OF THE SLIME MOLD PHYSARUM POLYCEPHALUM

The motility of Physarum polycephalum microplasmodia depends upon the conditions under which they are cultured. To investigate the relation between protoplasmic streaming and filamentous structures observed in the cytoplasm, microplasmodia were collected from shaken cultures, agar plates and shaken cultures of the organism which had previously been plate-cultured.
No sign of streaming could be found in materials in shaken culture, even in those which were shaken after they had once been motile on an agar plate. The immotile microplasmodia in both cases failed to contain any filamentous structures.
Microplasmodia on agar plates were motile, showing vigorous peripheral movements (projection of pseudopods) and inner protoplasmic streaming. In the motile organisms two types of filamentous structures were observed: loose networks just inside the plasma membrane of rounded pseudopods with smooth surfaces; and compact, straight bundles beneath the pseudopods or in much deeper locations.