Distribution of cAMP-dependent protein kinase during development in Dictyostelium discoideum

During the developmental cycle of Dictyostelium discoideum cyclic AMP functions as both a chemotactic signal for aggregation and a regulatory molecule during later events of differentiation. Morphological and biochemical data suggest that cAMP may direct cells during morphogenesis and differentiation. We utilized microtechniques to determine the stage- and cell-specific levels of the cAMP-dependent protein kinase, the probable intracellular cAMP receptor. Kinase activity was low and non-cAMP-dependent in amoebae and early aggregates but increased and became cAMP-dependent in aggregates after the formation of tight cell contacts. Maximum kinase activity and cAMP dependency occurred during the slug and culmination stages. The only differential distribution of the kinase within a single stage occurred during culmination when the activity in the stalks was approximately one-fourth of that in the prespore mass. Preliminary evidence indicates that this difference is not due to an inhibitor. In all other stages tested cAMP-dependent protein kinase activity was equal in prespore and prestalk cells.     

The cyclic adenosine 3':5'-monophosphate receptor of Dictyostelium discoideum. Binding characteristics of aggregation-competent cells and variation of binding levels during the life cycle.

Both cyclic guanosine 3':5'-monophosphate and dithiothreitol stimulate binding of cyclic adenosine 3':5'-monophosphate (cAMP) to aggregation-competent amoebae. Both compounds appear to function solely by preventing the hydrolysis of cAMP by the cell-bound phosphodiesterase. The dissociation constant for binding of cAMP is 36 nM. Both cAMP binding and membrane-bound phosphodiesterase activities increase dramatically as cells develop aggregation competence, reach a maximum at about 11 hours, and remain at high levels for up to 48 hours if cells are maintained in shaken suspension. When amoebae are allowed to aggregate and develop naturally, binding of cAMP increases during aggregation, decreases during tip formation, and disappears during culmination. Phosphodiesterase activity parallels binding activity except that the decreased level after tip formation is retained throughout culmination. Two N-6-modified cAMP derivatives compete with cAMP for binding sites. One derivative is fluorescent (1,N-6-etheno-cAMP); the other is photolyzable [N-6(ethyl-2-diazomalonyl)cAMP]. This result opens the possibilities of using fluorescence quenching for assay of in vitro binding and of affinity labeling of binding sites. Competition by the derivatives is only partial, indicating possible heterogeneity of binding sites. Both compounds inhibit hydrolysis of cAMP by the membrane-bound phosphodiesterase.     

Propagating chemoattractant waves coordinate periodic cell movement in Dictyostelium slugs.

Migration and behaviour of Dictyostelium slugs results from coordinated movement of its constituent cells. It has been proposed that cell movement is controlled by propagating waves of cAMP as during aggregation and in the mound. We report the existence of optical density waves in slugs; they are initiated in the tip and propagate backwards. The waves reflect periodic cell movement and are mediated by cAMP, as injection of cAMP or cAMP phosphodiesterase disrupts wave propagation and results in effects on cell movement and, therefore, slug migration. Inhibiting the function of the cAMP receptor cAR1 blocks wave propagation, showing that the signal is mediated by cAR1. Wave initiation is strictly dependent on the tip; in decapitated slugs no new waves are initiated and slug movement stops until a new tip regenerates. Isolated tips continue to migrate while producing waves. We conclude from these observations that the tip acts as a pacemaker for cAMP waves that coordinate cell movement in slugs.     

Guanylate Cyclase Activity in Dictyostelium discoideum and Its Increase during Cell Development

Following consumption of the food supply, cells of the cellular slime mould Dictyostelium discoideum aggregate and form a multicellular organism. The mechanism for cell aggregation is chemotaxis. The chemotactic signal in D. discoideum is released periodically from aggregation centers and propagated from cell to cell. cAMP mediates cell aggregation by acting as chemotactic attractant and as propagator of the signal. cAMP signals are measured by cell-surface receptors. Recent evidence indicates a role for cGMP during cAMP-mediated cell aggregation in D. discoideum .
During cell differentiation to aggregation competence, cAMP binding sites appear at the cell surface, and the activity of the enzymes adenylate cyclase and phosphodiesterase increases several-fold. In the present work we investigate the synthesis of cGMP in D. discoideum . Conditions for the assay of guanylate cyclase in cell homogenates are described. Guanylate cyclase activity was followed during cell differentiation to aggregation competence and found to increase fourfold. These results indicate that cGMP is involved in cell differentiation of D. discoideum . In contrast to adenylate cyclase, which is activated by cAMP, guanylate cyclase was under our conditions activated neither by cAMP, nor by folic acid.     

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.     

Purification and properties of a cyclic-AMP phosphodiesterase that is active in only one cell type during the multicellular development of Dictyostelium discoideum

Cyclic-AMP phosphodiesterase (PDE) accumulates during the aggregation stage of Dictyostelium where it functions in maintaining extracellular levels of cyclic AMP (cAMP). The activity decreases during the subsequent multicellular slug stage and then accumulates again during sorocarp construction, but the enzyme is active only in the developing stalk. Because of the possible significance of this localized activity in only one of the two cell types, we have purified the enzyme from the multicellular stage in order to understand its mode of regulation in vivo. We find that the enzyme which is localized in the prestalk cells is similar in many respects to the extracellular PDE which is active at the aggregation stage. The enzyme from both stages is inhibited by a low molecular weight protein. The mechanism of this inhibition is through a shift in the apparent Km for cAMP from micromolar to millimolar levels. The inhibited form of the enzyme can be activated by preincubation with MgSO4 and dithiothreitol (DTT). This activation treatment releases the inhibitor from the enzyme, thus restoring the low Km form, changes the molecular weight of the culmination stage enzyme from 95 000-100 000 to 68 000 by releasing the Mr 35 000-40 000 inhibitor protein, and causes irreversible loss of inhibitor activity. Although the inhibitor could be obtained in high yield from the aggregation stage by simply heating the extracellular fluid, it could not be detected from culmination stage extracts when prepared by this method. However, inclusion of calcium in the extraction buffer resulted in release of inhibitor from both heated and nonheated samples. The results indicate that the stalk cell specific PDE is regulated similarly to the aggregation stage PDE and opens the possibility of differential regulation of PDE in the two cell types.     

Localization of cyclic nucleotide phosphodiesterase in the multicellular stages of Dictyostelium discoideum

Cyclic 3',5'-adenosine monophosphate (cAMP) is secreted as the chemotactic signal by aggregating amoebae of the cellular slime mold Dictyostelium discoideum. We have used ultramicrotechniques in the biochemical analysis of cyclic nucleotide phosphodiesterase (PD) distribution in individual aggregates at various stages of development. With handmade constriction pipettes in microliter volumes, sections of lyophilized individuals weighing 20-100 ng could be assayed in a reaction coupled to 5'-nucleotidase. Phosphodiesterase activity was measured at pH 7.5 with 12 microM cAMP, cAMP-PD activity in aggregates ranged from 20-40 mmol/h/kg. In the pseudoplasmodium it had dropped to 5-10 mmol/h/kg and a difference in activity between the anterior prestalk cells and posterior prespore cells began to appear. The utmost posterior sections showed elevated phosphodiesterase from this stage onward. During culmination, activity rose to 40-60 mmol/h/kg associated with the developing stalk, while it declined in the spore mass. The papilla remained constant at 5-10 mmol/h/kg. The pattern of localization in the stalk was the same when cGMP was used as substrate. Extracellular phosphodiesterase inhibitor produced at the aggregation stage was found to reduce the localized activity in the culmination stage by 50-80%, with the most marked inhibition occurring in the center of the papilla. We found no evidence of endogenous heat-stable phosphodiesterase inhibitor within the culminating sorocarp.     

Function of ammonium transporter A in the initiation of culmination of development in Dictyostelium discoideum

The histidine kinase DhkC controls a phosphorelay involved in regulating the slug versus culmination choice during the multicellular developmental program of Dictyostelium discoideum. When the relay is active, slug migration is favored due to the activation of a cyclic AMP (cAMP) phosphodiesterase and the resultant lowering of the intracellular and extracellular levels of cAMP. Ammonia signaling represents one input into the DhkC phosphorelay, and previous studies indicated that the ammonium transporter C inhibits the relay in response to low ammonia levels. Evidence is presented that another member of the family of ammonium transporters, AmtA, also regulates the slug/culmination choice. Under standard conditions of development, the wild-type strain requires a transitional period of 2 to 3 h to go from fingers to culminants, with some slugs forming and migrating briefly prior to culmination. In contrast, amtA null cells, like cells that lack DhkC, possessed a transitional period of only 1 to 2 h and rarely formed slugs. Disruption of amtA in an amtC null strain overcame the slugger phenotype of that strain and restored its ability to culminate. Strains lacking AmtA were insensitive to the ability of ammonia to promote and prolong slug migration. These findings lead to the proposal that AmtA functions in ammonia sensing as an activator of the DhkC phosphorelay in response to perceived high ammonia levels.     

Involvement of cyclic AMP cell surface receptors and G-proteins in signal transduction during slug migration of Dictyostelium discoideum.

The presence of G-proteins, interacting with cAMP surface receptors, was investigated in vegetative cells, aggregation-competent cells, and migrating slugs of Dictyostelium discoideum. Our results indicate that G-proteins are present in all stages. In vegetative cells there is a limited number of cAMP receptors but no effect of GTP tau S on cAMP binding could be detected; in addition, no effect of cAMP on GTP tau S binding or GTPase activity was observed. In both aggregation-competent cells and slugs GTP tau S inhibits cAMP binding, while cAMP stimulates GTP tau S binding and high-affinity GTPase. Since the presence of G-proteins coupled to cAMP receptors could be demonstrated in slugs, the involvement of the effector enzymes adenylate cyclase and phospholipase C was investigated. The results show that adenylate cyclase activity is stimulated by GTP tau S in both stages and that in cells from migrating slugs the Ins(1,4,5)P3 production is increased upon stimulation with cAMP. The possible involvement of G-proteins in signal transduction during the slug stage of D. discoideum is discussed.     

Cyclic nucleotides and cyclic nucleotide phosphodiesterase during development of Polysphondylium violaceum

Polysphondylium violaceum is shown to produce and excrete cyclic nucleotides and to produce a cell-associated cyclic nucleotide phosphodiesterase(s). The amount of adenosine 3′,5′-cyclic monophosphate (cAMP) excreted by the amebae reaches a maximum during development when aggregation centers are just forming and then falls off rapidly. Measurements of total cAMP show that the amount synthesized increases more than 15-fold throughout development with the majority of the increase coming during the culmination stages. Guanosine 3′,5′-cyclic monophosphate (cGMP) is either not excreted or is excreted at levels below our limits of detection. An increase in the total cGMP synthesized occurs at mid-aggregation when two or three sharp peaks of synthesis are observed. However, development of P. violaceum is not affected by the addition of high concentrations of either cAMP or cGMP (or their dibutyryl derivatives) to the medium despite the fact that the cells produce these nucleotides. Cell-associated cyclic nucleotide phosphodiesterase activity, which hydrolyses both cAMP and cGMP, is greatest at the onset of starvation with a second increase in activity during aggregation.     

Quantitative studies on cell differentiation during morphogenesis of the cellular slime mold Dictyostelium discoideum

Taking advantage of the fact that differentiation of the prespore cell of Dictyostelium discoideum is characterized by synthesis of a prespore specific antigen, the process of its differentiation during the course of morphogenesis was quantitatively studied by determining the proportion of prespore cells and their cellular contents of the antigen, using the method of microfluorometry in combination with immunocytochemistry with antispore serum. The cells synthesizing the antigen became first detectable in the early aggregation center which was about to form a papilla. As the papilla elongated, the number of prespore cells rapidly increased up to the stationary level (70–80% of total cells) before completion of slug formation. During the process antigenic contents of prespore cells were gradually increased and leveled off in the early migration stage. When culmination was induced, antigenic contents were markedly increased to the maximum, which was followed by a sudden decrease immediately before spore formation. On the other hand, the proportions of prespore to total cells were kept constant at the stationary level all through the migration and culmination stages, in spite of a persistent decrease during culmination in the total number of cells due to continuous differentiation of the prestalk into the mature stalk cells. These results were discussed in relation to possible mechanisms of differentiation in this organism.     

cAMP relay during early culmination of Dictyostelium minutum

Abstract. It has recently been found that the culmination process of Dictyostelium minutum is accompanied by the appearance of oscillatory cell movement, cell-surface cAMP receptors, and cAMP phosphodiesterase. In the present study, it is demonstrated that the cAMP analog 2'deoxyc-AMP induces a transient accumulation and secretion of cAMP in culminating structures. At least 50-fold-less cAMP is accumulated during relay of D. minutum than during relay of Dictyostelium discoideum aggregative cells. No cAMP relay could be induced in vegetative or aggregative cells of D. minutum . These combined results yield evidence that oscillatory cAMP secretion and relay are involved in the organization of cell movement during the culmination of D. minutum .     

Cell type specific inhibition of cAMP phosphodiesterase activity during terminal differential in Dictyostelium discoideum

Cyclic AMP phosphodiesterase (PDE) activity reaches a peak during the aggregation stage of development where it functions to regulate extracellular levels of cAMP. During the subsequent differentiation of the two cell types at the culmination stage, the activity reappears but only in stalk cells. We found that extracts from the culmination stage contained PDE which could be activated by preincubation with Mg2+ and dithiothreitol (DTT), a treatment which is known to release an endogenous inhibitor from the aggregation stage enzyme. When the culmination stage extracts were subjected to chromatography on Biogel P300, two peaks of activity were eluted, PDE-I (Mr greater than 260,000) and PDE-II (Mr 100,000). Treatment of the fractions with Mg-DTT did not affect the low-molecular-weight enzyme but caused activation of the high-molecular-weight enzyme and the appearance of a third, intermediate form. Kinetic analysis of the two peaks revealed Km values for cAMP of 2 mM and 10 microM for PDE-I and PDE-II, respectively. We tested the possibility that these forms of the enzyme might be distributed differently in the two cell types by measuring the Km for cAMP and the effect of Mg-DTT treatment on isolated sections of stalk and spore cells. The spore sections contained a high Km form of the enzyme (0.3 mM) which was activated by preincubation with Mg . DTT whereas stalk sections contained a low Km form (3 microM) which was not affected by the activation treatment. We conclude that both cell types contain enzyme protein and that the apparent localization of PDE activity in stalk cells is due to the inhibition of activity in spore cells.     

Correlations between prestalk-prespore tendencies and cAMP-related activities in Dictyostelium discoideum

Amoebae of the cellular slime mould Dictyostelium discoideum (strain Ax2) grown in axenic medium containing 86 mM glucose [G(+) cells] or no glucose [G(−) cells] were examined for the characteristics of aggregation, cAMP secretion rate, cAMP phosphodiesterase activity and cell surface cAMP-binding activity. (When G(+) and G(−) cells are mixed, G(+) cells preferentially differentiate into prespore cells and sort out to the posterior region of a slug.)Under the same conditions, G(−) cells aggregate later than G(+) cells and the formation of stable streams by G(−) cells was particularly delayed. The movements of G(−) cells during aggregation were less organized compared with the ordered behaviour of G(+) cells, yet G(−) cells seemed to be more sensitive to chemotactic signals. Furthermore, the cAMP-binding activity of G(−) cells was considerably higher than that of G(+) cells, a difference that is probably due to the difference in the number of the cAMP receptor sites. Also G(−) cells, compared to G(+) cells, secreted slightly more cAMP and showed higher activity of cell-bound phosphodiesterase activity at the aggregation stage, whereas the extracellular phosphodiesterase activity was lower, although these differences were minor.     

Cell Patterning During Slug Migration and Early Culmination in Dictyostelium discoideum

Abstract. The effects of migration and culmination on patterning of presumptive (prespore and prestalk) cells and mature (spore and stalk) cells of D. discoideum were investigated. The ratio of prespore to total cells, as determined by staining with fluorescein-conjugated antispore globulin, was constant (77%) up until 8 h of slug migration, but then decreased to a level (64%) which thereafter remained unchanged during migration. Cells which lost prespore antigen during migration were located in the posterior (prespore) part next to the agar surface.
Upon induction of culmination, however, the ratio of prespore cells quickly increased to the normal level (77%) within 1–2 h. During the transition between migration and culmination prestalk and prespore cells were considerably intermixed within the cell mass, before the normal prestalk-prespore pattern was reestablished at the preculmination (Mexican hat) stage. Spore: stalk ratios within fruiting bodies were normal irrespective of the lengths of slug migration.     

Apparent Positive Cooperativity at a Surface cAMP Receptor in Dictyostelium

In the large species of the cellular slime mold Dictyostelium , cell aggregation is regulated by extracellular cAMP. During aggregation, cAMP is released in pulses from cells in the aggregation centers and these rhythmic signals are propagated through the population by a signal relay system. In addition to triggering the relay response, the pulsatile signals also regulate the chemotactic movement of the cells and early cell differentiation. These different cellular responses to exogenous cAMP are thought to be mediated via cAMP receptors, which appear on the cell surface shortly after starvation.
Using a sensitive assay, the equilibrium binding properties of these receptors were analyzed at low cAMP concentrations. As reported earlier, Scatchard plots of cAMP binding to preaggregative amoebae of D. discoideum strain NP187 in the concentration range 2–500 nM were curvilinear suggesting either receptor heterogeneity or negative cooperative interactions. However, at cAMP concentrations below approximately 1.5 nM, the affinity of the receptors was found to decline as a function of decreasing receptor occupancy. This apparent positive cooperativity was observed with binding sites on crude plasma membranes as well as on intact cells, and it occurred at both 0°C and 22°C. Moreover, apparent positive cooperativity was a property of the receptors on all strains of D. discoideum examined and on one strain of D. purpureum . Unlike preaggregative cells, receptors on postaggregative cells often lacked this property.
The lowest concentration of cAMP pulses that can appreciably stimulate membrane differentiation in strain NP187 was found to be 0.15–1.5 nM. Since similar concentrations of exogenous cAMP have been reported to trigger minimal chemotactic and relay responses in D. discoideum , the apparent positive cooperative behavior of the cAMP receptor might function to generate a steep cellular response threshold.     

The effects of inhibitors of adenylate cyclase and phosphodiesterase on D. discoideum aggregation.

The action of two adenine analogues on the aggregation of D. discoideum amebae was examined. SQ22536 and SQ20009 are inhibitors of adenylate cyclase and phosphodiesterase, respectively, in higher eukaryotes. Both compounds are shown here to inhibit the differentiation of cells to aggregation-competence. SQ22536-treated cells exhibited normal accumulation of their adenylate cyclase activity as measured in cell lysates but the amebae did not synthesize cAMP. The ability of this drug to compete for cAMP surface-binding sites, and the observation that the effects of the drug could be reversed by imposed pulses of cAMP, suggest that SQ22536 functions as a cAMP antagonist. The effects of SQ20009 on cell differentiation did not appear to be mediated by an inhibition of phosphodiesterase activity or cAMP binding to the cell surface. Amebae were arrested at a very early stage in development and remained unresponsive to external cAMP.