Cyclic AMP inhibits dedifferentiation in the cellular slime mold Dictyostelium discoideum

When aggregating amoebas of the cellular slime mold Dictyostelium discoideum are disaggregated and morphogenesis is reinitiated, the amoebas will reaggregate in less than $\text{1}\text{10}\text{th}$ the original time. When aggregating amoebas are disaggregated and resuspended either in full nutrient medium or in buffered salts solution containing dextrose, they retain this developmentally acquired capacity to rapidly reaggregate for approximately 1 hr and then lose it completely in a synchronous and discrete step which we have referred to as the “erasure event.” In this report, it is demonstrated that micromolar concentrations of cAMP completely block this transition from the developmental to vegetative state, and that other cyclic nucleotides also inhibit it, but they do so at 20-fold higher concentrations. Neither the hydrolysis products of cAMP nor the vegetative chemoattractant folic acid inhibit dedifferentiation at concentrations as high as 10^?3M, demonstrating a specificity for cyclic nucleotides and cAMP in particular. The addition of cAMP at any time during the lag period preceding the erasure event inhibits it and addition immediately after the erasure event reverses it. Since cAMP may inhibit the transition from the developmental to vegetative state intracellularly or extracellularly, we have also examined the intracellular concentration of cAMP and the levels of cAMP binding sites on the cell surface during the erasure process. Evidence is presented that the majority of cAMP binding sites on the cell surface are not necessary for the inhibition of erasure by cAMP. The results of these latter studies are discussed in terms of alternative models for the involvement of cAMP in the transition from the developing to vegetative state.     

Cyclic AMP gradient in migrating pseudoplasmodia of the cellular slime mold Dictyostelium discoideum.

Several lines of experimental evidence suggest that an anterior-posterior gradient of cyclic AMP exists in migrating pseudoplasmodia of the cellular slime mold Dictyostelium discoideum, and that this gradient may be responsible for control of the proportions of stalk and spore cells that form during culmination. In experiments reported here, the amounts of cyclic AMP present in the anterior and posterior portions of pseudoplasmodia were measured using a protein binding assay, and the levels obtained normalized to protein and DNA. For a variety of pseudoplasmodia migration conditions examined, the anterior portion was found to contain cyclic AMP concentrations 40 to 70% higher than the posterior portions.     

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.     

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.     

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.     

RNA metabolsim during vegetative growth and morphogenesis of the cellular slime mold Dictyostelium discoideum

During vegetative growth of the cellular slime mold Dictyostelium discoideum, RNA is rapidly labeled by radioactive precursor and both the 25 S and the 17 S ribosomal RNA species appear in the cytoplasm 6–7 min after the onset of labeling. Thirty minutes after further incorporation of radioactive RNA precursors has been blocked, less than 10% of the label in RNA is associated with the nuclear fraction. After aggregation of the slime mold amoebae, RNA appears in the cytoplasm at a reduced rate, the small ribosomal subunit appearing in the cytoplasmic fraction more slowly than the larger ribosomal subunit. Some labeled RNA remains in the nuclei of developing cells long after the incorporation of ³H-uridine is blocked.     

'Ghosts' formation and their isolation from the cellular slime mold Dictyostelium discoideum.

Conditions for isolating ghosts from the cellular slime mold Dictyostelium discoideum are described. The cells were washed with a 20 mM KCl, 2.5 mM MgCl₂ solution and homogenized vigorously in 15 mM lactate buffer, pH 4.8, using a tight-fitting Dounce homogenizer. The resultant spherical ghosts were purified by the dextran-polyethylene glycol aqueous two-phase system described by Brunette &; Till [1], The proportion of ghosts which are finally purified by 3rd partition in the aqueous two-phase system is 5.6% of those present in the homogenate. As shown by phase-contrast and scanning electron microscopy, the plasma membrane fractions are almost completely uncontaminated by other identifiable subcellular components. On the basis of enzyme assays the ghosts isolated showed a 9- to 11-fold enrichment of alkaline phosphatase relative to the homogenate. They are free of succinic dehydrogenase, glucose 6-phosphatase but do contain some acid phosphatase and N-acetylglucosaminidase activity. Further purification using a sucrose-density gradient removes the residual lysosomal enzyme activities.     

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.     

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.