Changes in cellular adhesiveness during the development of the slime mold Dictyostelium discoideum

A method was devised to measure the adhesiveness to the substratum of the amoebae of the cellular slime mold, Dictyostelium discoideum, and measurements were conducted with the cells at various stages of development. The adhesiveness of the vegetative amoebae was low, and remained unchanged as long as they fed on bacteria. During the transition from the vegetative stage to the interphase (due to the cessation of feeding), the adhesiveness increased rapidly, and afterwards continued to rise, as development proceeded. The adhesiveness of the interphase amoebae was greatly decreased by the treatment with proteolytic enzymes, lipase, and acid phosphatase. These indicate that accumulation of some substance(s) such as lipoprotein on the cell surface is responsible for the increase in adhesiveness during the interphase. EDTA and periodic acid had no noticeable effect on the adhesiveness of the interphase amoebae. EDTA, however, decreased the adhesiveness in co-operation with trypsin or lipase. The cells disaggregated from the anterior part of the migrating slug showed higher adhesiveness than those from the posterior part. The adhesiveness of either cells was higher than that of the interphase amoebae.     

Preferential synthesis of actin during early development of the slime mold Dictyostelium discoideum

The changes in protein synthesis during differentiation of the cellular slime mold Dictyostelium were studied by SDS-polyacrylamide gel electrophoresis. Total cell protein was analyzed following a 2-hr pulse-label. It was found that during the preaggregation stage, comprising the first third of the developmental cycle, a single major band accounts for more than 20% of the total labeled protein on the gel. This species was produced in at least 5–10-fold lower amounts, relative to total cell protein synthesis, in vegetative cells and in later developing stages. Actin was purified from vegetative cells and was found to correspond to the major band in several respects. The discovery of a single protein being synthesized in such quantity at a specific developmental stage provides a powerful tool for the isolation of a specific messenger RNA molecule and for an intensive study of all the factors involved in regulating protein synthesis in a eukaryotic organism.     

The calcium content of the cellular slime mold, Dictyostelium discoideum, during development and differentiation

A high calcium concentration is known to induce stalk differentiation of the cellular slime mold D. discoideum. Therefore, the change in the calcium content of this organism during differentiation was studied and found to vary during development, more calcium being found in the anterior prestalk cells of the pseudoplasmodium (slug) than in the posterior prespore cells. It is concluded from the results that calcium is of importance in the cell differentiation of this organism and particularly in stalk formation.     

Cytokinin oxidase activity in the cellular slime mold, Dictyostelium discoideum

The cytokinin N6-(delta 2-isopentenyl)adenine (i6Ade) is produced during the development of the cellular slime mold, Dictyostelium discoideum, and functions in this organism as the immediate precursor of the spore germination inhibitor, discadenine. The metabolism of i6Ade in axenic cultures of D. discoideum Ax-3 amoebae has been investigated in the present study. An enzyme activity that specifically catalyzes the degradation of i6Ade has been detected in Ax-3 amoebae. This enzyme is similar to the cytokinin oxidases present in higher plant systems and cleaves the N6-side chain of i6Ade to form adenine. Discadenine synthase activity was also detected in axenically cultured Ax-3 amoebae. The cytokinin oxidase activity detected in Dictyostelium decreased during aggregation and development of Ax-3 amoebae and in starving Ax-3 amoebae maintained under either fast-shake (230 rpm) or slow-shake (70 rpm) conditions. In the latter case, the fall in enzyme activity was accelerated by treatment with cyclic AMP. In contrast to these results, discadenine synthase activity in Ax-3 amoebae rose sharply during the culmination phase of development, exhibited little change in starving Ax-3 amoebae maintained under fast-shake conditions, and fell under slow-shake conditions unless the amoebae were treated with cyclic AMP. Possible functions of the Dictyostelium cytokinin oxidase and the significance of the i6Ade metabolism observed in vegetative Dictyostelium amoebae are discussed.     

Cyclic AMP levels and turnover during development of the cellular slime mold Dictyostelium discoideum.

Changes in intracellular and extracellular cAMP levels are reported for the cellular slime mold Dictyostelium discoideum during its development on filter supports. Examined were axenically and bacterially grown strain A3 and bacterially grown NC-4. In each case a major peak in cAMP occurred during aggregation. In addition, axenically grown A3 showed minor rises in cAMP at 16 hr and during culmination; in contrast, NC-4 showed no increase at 16 hr but gave a very large increase at culmination. Both cell-associated phosphodiesterase and the extracellular phosphodiesterase present in the top filter were measured throughout development. Both showed activity peaks during aggregation with much lower plateau values thereafter. At aggregation about 80% of the activity per filter was contributed by the cell-associated phosphodiesterase. The rate of cAMP turnover during aggregation was estimated by following the hydrolysis of applied [³H]cAMP. A minimum rate of about 7% turnover/sec was obtained. From this turnover rate a minimum value for the stimulated activity of the adenylate cyclase was estimated as 224 pmoles/min-mg. Although this level is already over threefold greater than the highest value obtained in vitro, other experiments indicate that the in vivo adenylate cyclase activity may exceed 700 pmoles/min-mg.     

Nuclear ribonucleoprotein particles in the cellular slime mold Dictyostelium discoideum

The nuclear ribonucleoprotein (RNP) particles containing rapidly labeled RNA were isolated from interphase cells of the cellular slime mold Dictyostelium discoideum and characterized. The size of the isolated RNP particles was small (10S to 50S) in comparison with that of nuclear RNP particles found in higher eukaryotes. These small RNP particles do not seem to be artifacts due to degradation during the preparation of nuclear extracts. The rapidly labeled RNA of the nuclear RNP particles was heterogeneous in size and a considerable amount contained polyadenylic acid sequences. Synthesis of RNA in the nuclear RNP particles was resistant to a relatively high concentration of actinomycin D. The protein component of the RNP particle consists of at least four proteins with molecular weights of 80,000, 66,000, 60,000, and 42,000. Thus it is suggested that almost all of the nuclear RNP particles containing rapidly labeled RNA in interphase cells are RNP complexes consisting of Heterogeneous nuclear RNA and several protein species.     

Characterization of pyrimidine metabolism in the cellular slime mold, Dictyostelium discoideum

The arginine-independent, de novo biosynthetic pathway of pyrimidines in Dictyostelium discoideum is initiated by a class II carbamoyl-phosphate synthetase (EC 6.3.5.5) specific for pyrimidine biosynthesis which utilized L-glutamine as its N donor and was partially inhibited by both UTP and CTP. The second step in the de novo pathway was provided by an unregulated aspartate transcarbamoylase (EC 2.1.3.2) which primarily appeared as a multimeric enzyme of 105 kilodaltons. The next enzyme, dihydroorotase (EC 3.5.2.3), was approximately 90-100 kilodaltons. Although the early enzymatic activities of the pyrimidine pathway appeared to reside in independent protein complexes, various unstable molecular species were observed. These structural variants may represent proteolytic fragments of a multienzyme complex. In addition to de novo synthesis, the amoeba demonstrated the capacity for salvage utilization of uracil, uridine, and cytidine. Upon starvation on a solid substratum, axenically grown amoebas began a concerted developmental program accompanied by a restructuring of nucleotide metabolism. The absolute levels of the ribonucleotide pools droppedby 98% within 30 h; however, both the adenylate energy charge and the GTP/ATP ratios were maintained for 50 h after the initiation of development. The maintenance of these metabolic energy parameters required the tight cell-cell contact necessary for development, and the capacity for pyrimidine metabolism was maintained throughout developmental morphogenesis.