On induction of cell differentiation by cyclic AMP pulses in Dictyostelium discoideum

Repeated pulses of cyclic AMP, applied at intervals of 5 min, efficiently induced differentiation in cells of agip 53, a morphogenetic mutant of Dictyostelium discoideum, strain Ax-2. In contrast, pulses applied at intervals of 2 min did not induce cell differentiation. To analyze this phenomenon the hydrolysis of cyclic AMP between the pulses as well as the effect of the pulses on the intracellular concentrations of cyclic GMP were investigated. Experiments performed in the presence of added cyclic AMP was not the reason of the inefficiency of the pulses applied with a 2-min rhythm. Cyclic AMP pulses applied at intervals of 2 min induced discrete increases of the cyclic GMP concentration. Limited time resolution at the level of cyclic GMP cannot account for the inefficiency of the 2-min pulses.     

On induction of cell differentiation by cyclic AMP pulses in Dictyostelium discoideum

Repeated pulses of cyclic AMP, applied at intervals of 5 min, efficiently induced differentiation in cells of agip 53, a morphogenetic mutant of Dictyostelium discoideum, strain Ax-2. In contrast, pulses applied at intervals of 2 min did not induce cell differentiation. To analyze this phenomenon the hydrolysis of cyclic AMP between the pulses as well as the effect of the pulses on the intracellular concentration of cyclic GMP were investigated. Experiments performed in the presence of added cyclic AMP phosphodiesterase revealed that incomplete hydrolysis of cyclic AMP was not the reason for the inefficiency of the pulses applied with a 2-min rhythm. Cyclic AMP pulses applied at intervals of 2 min induced discrete increases of the cyclic GMP concentration. Limited time resolution at the level of cyclic GMP cannot account for the inefficiency of the 2-min pulses.Based on material presented at the Symposium Intercellular Communication Stuttgart, September 16–17, 1982     

Inhibitors of intracellular cyclic AMP accumulation affect differentiation of sporogenous mutants of Dictyostelium discoideum

Abstract Sporogenous mutants of Dictyostelium discoideum strain V12M2 were used to determine whether the intracellular levels of cyclic AMP or other second messengers regulate differentiation. Increasing external concentrations of cyclic AMP promoted spore formation. Caffeine and progesterone, which lower intracellular cyclic AMP levels by different mechanisms, blocked spore formation and favored stalk cell formation. In contrast, differentiation of both spore and stalk cells occurred normally in the presence of agents that disrupt calcium/calmodulin or protein kinase C-based second messenger systems. The data are in accord with the view that (1) intracellular cyclic AMP is essential for terminal differentiation of both cell types, and (2) higher levels are required for formation of spores than for stalk cells.     

Analysis of gene expression in rapidly developing mutants of Dictyostelium discoideum

Developmentally regulated gene expression has been analyzed in the wild-type D. discoideum strain NC-4 and a series of temporally deranged mutants. The mutants include representatives from each class of rapid development mutation, Fr17(rdeA-) and HT506(rdeC-), and strain HIfm-1, which appears to be defective in the timing of events early in development. We have monitored four prespore-specific genes, three of which show coordinate expression in the wild type. The coordination is maintained in each of the mutant strains though the specific expression pattern varied from strain to strain. Likewise, a series of prestalk-specific genes have been analyzed. They also show coordinated expression in the wild type and in all of the mutants. The timing of expression, however, is different between the prestalk-specific and the prespore-specific with the overall pattern of expression being unique for each strain examined. These results confirm our previous suggestion that the major classes of prestalk- and prespore-specific genes are coordinately regulated and show that a great deal of tolerance is allowed in the timing of specific gene expression as it relates to terminal differentiation. In addition we have analyzed the expression of actin, discoidin I, and I42. These genes, or gene families, are preferentially expressed in either vegetatively growing cells or in cells during the early stages of development. As with the cell-type-specific genes, the pattern of expression of the three early gene classes is unique for each strain examined.     

A cyclic AMP dependent protein kinase in Dictyostelium discoideum

A cyclic AMP-dependent protein kinase was found to appear during the time course of development of $\text{Dictyostelium}\text{discoideum}$. No cyclic AMP dependency was observed at any stage of development in crude 110,000 X G soluble extracts. After partial purification, however, extracts from post-aggregation stages contained enzyme that was activated up to 6-fold by cyclic AMP, whereas protein kinase from earlier stages was not affected by cyclic AMP. Likewise, cyclic AMP binding activity increased from the aggregation to the slug stage of development. Approximately one-half of the total cyclic AMP binding activity co-purified with the cyclic AMP dependent protein kinase. The enzyme from $\text{Dictyostelium}$ showed similarities to mammalian protein kinases with respect to its kinetic properties but differed in its behavior on ion-exchange chromatography.     

Streamers: chemotactic mutants of Dictyostelium discoideum with altered cyclic GMP metabolism

Mutants of Dictyostelium discoideum that developed huge aggregation streams in expanding clones were investigated using optical and biochemical techniques. Representatives of the six complementation groups previously identified (stmA-stmF) were found to be similar to the parental wild-type strain XP55 in both the extent and timing of their ability to initiate and relay chemotactic signals and in the formation of cyclic AMP receptors and phosphodiesterases. The mutants differed from the wild-type in producing an abnormal chemotactic (movement) response visible using both dark-field optics with synchronously aggregating amoebae on solid substrata and light scattering techniques with oxygenated cell suspensions. Mutants of complementation group stmF showed chemotactic movement responses lasting up to 520 s, rather than 100 s as seen in the parental and other strains. Measurements of cyclic GMP formed intracellularly in response to chemotactic pulses of cyclic AMP in stmF mutants showed that abnormally high concentrations of this nucleotide were formed within 10 s and were not rapidly degraded. A causal correlation between defective cyclic GMP metabolism and the altered chemotactic response is suggested, and a model is proposed that accounts for the formation of huge aggregation streams in clones of these mutants.U     

Dictyostelium discoideum cell membranes contain masked chemotactic receptors for cyclic AMP

Aggregating Dictyostelium discoideum cells possess highly specific receptors for the chemoattractant cAMP on their cell surface. Isolated membranes as well as intact cells are shown to contain a large number of latent cAMP receptors. These are reversibly unmasked in the presence of a high salt concentration (0.1–2 M) or in the presence of millimolar concentrations of Ca²⁺.     

Cell differentiation in the absence of intracellular cyclic AMP pulses in Dictyostelium discoideum

Repeated additions of cyclic AMP to a morphogenetic mutant of Dictyostelium discoideum, agip 53, induced cell differentiation to the aggregation competent state as previously reported [Darmon, Brachet, and Pereira da Silva (1975). Proc. Nat. Acad. Sci. USA72, 3163–3166]. Cyclic AMP additions elicited transient increases of the intracellular cyclic GMP concentration but no significant increases of the intracellular cyclic AMP concentration. These results suggest that transient increases of the intracellular cyclic AMP concentration are not necessary for cell differentiation. Agip 53 seems to be unable to relay cyclic AMP signals. A defect in the receptor-mediated activation of adenylate cyclase could be the biochemical basis of the mutant phenotype of agip 53.     

Specific binding proteins for cyclic AMP and cyclic GMP in Dictyostelium discoideum.

In Dictyostelium discoideum both cyclic AMP and cyclic GMP are regulated by chemotactic stimuli. Binding proteins specific for cAMP and cGMP have been found in aggregation competent cells as well as in cells harvested during growth. The activity of binding proteins was, on the average, lower in the growth phase cells. cAMP binding proteins were separated into 3 fractions, whereas the cGMP binding activity appeared in 1 major peak both on DEAE-cellulose and Sephadex G-200. Protein kinase activity was present in most but not all cyclic necleotide binding fractions; evidence for a relationship is however missing.     

Independent regulation of the extracellular cyclic AMP phosphodiesterase-inhibitor system and membrane differentiation by exogenous cyclic AMP in Dictyostelium discoideum.

When amoebae of Dictyostelium discoideum, suspended in buffer, were treated with 100 nM pulses of cAMP, the extracellular cAMP phosphodiesterase (ePD) activity increased dramatically and the synthesis of the phosphodiesterase inhibitor (PDI) was repressed. In addition, the time of appearance on the cell surface of contact sites A, membrane-bound cAMP phosphodiesterase, and cAMP binding sites was accelerated by 3–4 hr and the concentration of intracellular cAMP increased ?20-fold. When the concentration of the cAMP pulse was reduced to 1 nM, the effect of the pulses on membrane differentiation and on the cAMP pool was virtually the same, while the effect on the ePD-PDI system was reduced. When cAMP was added to the suspension continuously, the nucleotide had no effect on membrane differentiation and failed to stimulate the intracellular cAMP pool, however, the ePD-PDI system was regulated normally. When the developmental mutant, HC112, was treated with cAMP pulses, membrane differentiation and the level of the cAMP pool were unaffected, while the ePD-PDI system responded to the exogenous cAMP. In another mutant, HC53, membrane differentiation was stimulated by cAMP pulses and this response was accompanied by a sharp increase in the concentration of the cAMP pool. These results suggest that the ePD-PDI system and membrane differentiation are regulated independently by exogenous cAMP and that regulation of the ePD-PDI system does not require activation of the adenylyl cyclase.     

Cyclic AMP is an inhibitor of stalk cell differentiation in Dictyostelium discoideum

Cyclic AMP and DIF-1 (1-(3,5-dichloro-2,6-dihydroxy-4-methoxyphenyl)-1-hexanone) together induce stalk cell differentiation in vitro in Dictyostelium discoideum strain V12M2. The induction can proceed in two stages: in the first, cyclic AMP brings cells to a DIF-responsive state; in the second, DIF-1 alone can induce stalk cell formation. We report here that during the DIF-1-dependent stage, cyclic AMP is a potent inhibitor of stalk cell differentiation. Addition of cyclic AMP at this stage to V12M2 cells appreciably delays, but does not prevent, stalk cell formation. In contrast, stalk cell differentiation in the more common strain NC4 is completely suppressed by the continued presence of cyclic AMP. This fact explains earlier failures to induce stalk cells in vitro in NC4. We now consistently obtain efficient stalk cell induction in NC4 by removing cyclic AMP in the DIF-1-dependent stage. Cyclic AMP also inhibits the production of a stalk-specific protein (ST310) in both NC4 and a V12M2 derivative. Adenosine, a known antagonist of cyclic AMP action, does not relieve this inhibition by cyclic AMP and does not itself promote stalk cell formation. Finally, stalk cell differentiation of NC4 cells at low density appears to require factors in addition to cyclic AMP and DIF-1, but their nature is not yet known. The inhibition of stalk cell differentiation by cyclic AMP may be important in establishing the prestalk/prespore pattern during normal development, and in preventing the maturation of prestalk into stalk cells until culmination.     

Identification and cyclic AMP-induced modification of the cyclic AMP receptor in Dictyostelium discoideum

We have recently identified a cell surface cAMP-binding protein by specific photoaffinity labeling of intact Dictyostelium discoideum cells with 8-N3-[32P] cAMP. The major photolabeled protein appears as a doublet (Mr = 40,000-43,000) in sodium dodecyl sulfate-polyacrylamide gel electrophoresis autoradiography. In this study, the doublet is shown to have the characteristics of the cAMP receptor responsible for chemotaxis and cAMP signaling. Both specific photoaffinity labeling of the doublet and binding of 8-N3-[32P]cAMP are saturable (KD = 0.3 microM), the levels of both peak at 5 h, and both are inhibited by cAMP and several cAMP analogs in the same order of potency and with K1 values similar to those measured for inhibition of [3H]cAMP binding. When cAMP-binding activity was partially purified (40-fold) and then photoaffinity labeled, the same bands (Mr = 40,000-43,000) were observed. The relative intensities of the upper and lower bands of the doublet alternated at the same frequency as the spontaneous oscillations in cAMP synthesis. When oscillations were suppressed, the lower band of the doublet predominated. Following addition of cAMP, the relative intensity gradually shifted to the upper band. When cAMP was removed, there was a gradual restoration of the lower band form. We propose that the lower band form of the receptor activates chemotaxis and cAMP signaling and that the upper band form does not. This reversible receptor modification may then be the mechanism of adaptation, the process by which the physiological responses cease to be stimulated by persistent cAMP. Several developmentally regulated genes in D. discoideum have been reported to be induced or suppressed by pulses of cAMP (adaptive regulation) and others by continuous cAMP (nonadaptive regulation). These observations may be explained by the receptor modification reported here if the two forms of the receptor, which bind cAMP with the same affinity, independently influence gene expression.     

Biochemical differentiation in a mutant of Dictyostelium discoideum defective in cyclic AMP chemotaxis and in intercellular cohesion

A temperature-sensitive mutant of Dictyostelium discoideum has been isolated based on its lack of chemotaxis toward cyclic AMP at the restrictive temperature, 27 degrees C. The mutant develops normally at the permissive temperature, 22 degrees C, but fails to aggregate or complete development at the restrictive temperature. The temperature-sensitive phenotype can be bypassed by allowing cultures to grown into late log phase or to starve for 60-90 min at 22 degrees C prior to a shift to 27 degrees C. At 27 degrees C, the mutant overproduces cell surface cyclic AMP receptors of both high and low affinity and is capable of spontaneous oscillations in light scattering in cell suspensions. Despite its complete lack of morphological development, the mutant undergoes extensive biochemical differentiation. At the onset of starvation, it shows increased levels of N-acetylglucosaminidase, it express cyclic AMP receptors at the normal time and, although somewhat slowly, suppresses those receptors as if aggregation had been achieved. Metabolic pulse labellings with [35S]methionine revealed that the mutant at 27 degrees C displays the same changes in the patterns of newly synthesized proteins observed during the vegetative-to-aggregation and the aggregation-to-slug stages of normal development. The only clear difference from wild type was the failure of the culmination-stage isozyme of beta-glucosidase to appear. The mutant is defective in establishment of intercellular cohesion mechanisms, correlated with poor agglutination by concanavalin A, at the restrictive temperature. The properties of the mutant place severe constraints on models regarding the role of chemoreception and intercellular cohesion in regulation of gene expression.     

Localization and levels of cyclic AMP during development of Dictyostelium discoideum

Cyclic AMP functions as the chemotactic signal during aggregation of amoebae of the cellular slime mold Dictyostelium discoideum. Evidence suggests that cAMP also acts as a regulatory molecule during Dictyostelium multicellular differentiation. We have used ultramicrotechniques and a sensitive radioimmunoassay to measure the levels of cAMP within the culmination stage individual. We show that there is a peak of cAMP at the culmination stage of development and that in the individual at this stage the molecule is localized in a gradient within the spore mass.     

Extracellular cyclic AMP-phosphodiesterase accelerates differentiation in Dictyostelium discoideum.

Extracellular cyclic AMP-phosphodiesterase accelerates the development of aggregation competence in Dictyostelium discoideum when present during the preaggregation stage. The effect on development appears to depend only on hydrolysis of extracellular cyclic AMP and not on other properties of the phosphodiesterase molecule. Extracellular cyclic AMP-phosphodiesterase, as a promoter of differentiation, acts mainly throughout the first half of interphase. Our evidence supports the proposal that cyclic AMP oscillations control the rate and possibly the initiation of development. Since extracellular cyclic AMP-phosphodiesterase acts from the beginning of interphase cyclic AMP oscillations may also occur from early interphase, at least in the presence of this enzyme. This would imply that the cyclic AMP oscillator is a determinant, but not a product, of the developmental programme.