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.     

cAMP production by adenylyl cyclase G induces prespore differentiation in Dictyostelium slugs

Encystation and sporulation are crucial developmental transitions for solitary and social amoebae, respectively. Whereas little is known of encystation, sporulation requires both extra- and intracellular cAMP. After aggregation of social amoebae, extracellular cAMP binding to surface receptors and intracellular cAMP binding to cAMP-dependent protein kinase (PKA) act together to induce prespore differentiation. Later, a second episode of PKA activation triggers spore maturation. Adenylyl cyclase B (ACB) produces cAMP for maturation, but the cAMP source for prespore induction is unknown. We show that adenylyl cyclase G (ACG) protein is upregulated in prespore tissue after aggregation. acg null mutants show reduced prespore differentiation, which becomes very severe when ACB is also deleted. ACB is normally expressed in prestalk cells, but is upregulated in the prespore region of acg null structures. These data show that ACG induces prespore differentiation in wild-type cells, with ACB capable of partially taking over this function in its absence.     

The regulatory subunit of cAMP-dependent protein kinase from Dictyostelium discoideum: cellular localization and developmental regulation analyzed by immunoblotting

The level of the regulatory subunit of the cAMP-dependent protein kinase from Dictyostelium discoideum was analyzed in subcellular fractions of cells at various stages of development by Western blotting. The protein was found only in the cytosolic fraction. A small amount of regulatory subunit was present in vegetative cells, and its level increased sharply during the first hours of aggregation; a further increase also occurred during culmination. Analysis of mature spores and of the stalky mutant HL 65 revealed that the protein is present only in prespore cells.     

Culmination in Dictyostelium is regulated by the cAMP-dependent protein kinase.

We placed a specific inhibitor of cyclic AMP-dependent protein kinase (PKA) under the control of a prestalk-specific promoter. Cells containing this construct form normally patterned slugs, but under environmental conditions that normally trigger immediate culmination, the slugs undergo prolonged migration. Slugs that eventually enter culmination do so normally but arrest as elongated, hairlike structures that contain neither stalk nor spore cells. Mutant cells do not migrate to the stalk entrance when codeveloped with wild-type cells and show greatly reduced inducibility by DIF, the stalk cell morphogen. These results suggest that the activity of PKA is necessary for the altered pattern of movement of prestalk cells at culmination and their differentiation into stalk cells. We propose a model whereby a protein repressor, under the control of PKA, inhibits precocious induction of stalk cell differentiation by DIF and so regulates the choice between slug migration and culmination.     

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.     

Induction of post-aggregative differentiation in Dictyostelium discoideum by cAMP: Evidence of involvement of the cell surface cAMP receptor

Exogenous cAMP is known to induce post-aggregative differentiation in Dictyostelium discoideum under conditions that normal development is blocked. We have analysed the cyclic nucleotide specificity, the effect of modulation of the cAMP signal and the dose-response relationship of the induction of two independent markers of post-aggregative differentiation, i.e., a prespore cell-specific antigen detected by a monoclonal antibody, and the activity of glycogen phosphorylase. Our results confirm that high concentrations of cAMP (10(-6)-10(-3)M) are required for the induction of these markers. The cells are shown not to adapt to the cAMP signal. The cyclic nucleotide specificity of induction agrees with the specificity of the cell surface cAMP receptor, but is very dissimilar to the specificity of the intracellular cAMP-dependent protein kinase. It is thus unlikely that cAMP leaks into the cell and activates the cAMP-dependent protein kinase directly. Instead, the induction of post-aggregative differentiation by cAMP seems to be mediated by cell surface cAMP receptors.     

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.     

Different synthetic profiles and developmental fates of prespore versus prestalk proteins of Dictyostelium

Abstract. Depending upon environmental conditions, developing cells of the cellular slime mold Dictyostelium discoideum may enter a slug stage in which the cell mass migrates in response to gradients of light and temperature. This developmental stage has often been used to study the divergent differentiation of the cells that will subsequently form spores and stalk in the mature fruiting body. However, still debated is the extent to which the differentiation evident in slug cells is a precondition for development of the mature cells in fruits. Using two-dimensional gel electrophoresis of polypeptides, we have examined the proteins made by prespore and prestalk cells of migrating slugs and by maturing spore and stalk cells. The data indicate that many of the cell-type specific polypeptides in prespore cells of slugs persist as cell-type specific polypeptides of mature spores. Prestalk slug cells, in contrast, do not contain significant amounts of stalk-specific proteins; these proteins appear only during culmination. The precursor cell types also differ in the times and rates of synthesis of cell-specific proteins: prestalk proteins appear much earlier in development than do the prespore, but never reach the levels of expression that the prespore proteins do later in culmination. These findings may explain the well established ability of prespore cells to regulate their cell type more rapidly than do prestalk cells. There are also implications for our general understanding of what is a 'prestalk' gene product.     

Hypertonic signal promotes stability of Dictyostelium spores via a PKA-independent pathway

Differentiation of Dictyostelium spores initiates with rapid encapsulation of prespore cells under the control of cAMP-dependent protein kinase (PKA), followed by further maturation processes involving cytoskeletal reorganization. Constitutive activation of PKA induces precocious formation of viable spores in development and confers the ability to encapsulate under specific submerged conditions. In this study, we show that the stability of these spores depends upon conditions of high osmotic strength during spore differentiation, indicating that a hypertonic signal is required in addition to PKA to induce maturation to stable spores. The formation of stable spores under hypertonic conditions requires high cell density, suggesting the involvement of additional cellular signaling.     

Re-expression of 117 antigen, a cell surface glycoprotein of aggregating cells, during terminal differentiation of Dictyostelium discoideum prespore cells

117 antigen is a glycoprotein expressed on the surface of D. discoideum cells at aggregation. It then disappears and is later re-expressed on the surface of a subpopulation of cells at culmination, the terminal differentiation stage (Sadeghi et al. 1987). A cDNA clone was used to show that the appearance of cell surface 117 antigen accurately reflects the expression of the 117 gene as measured by mRNA levels. It was also shown that during multicellular development there is a reciprocal relationship between the levels of 117 mRNA and the mRNA which codes for prespore surface glycoprotein, PsA. Dual parameter flow cytometry was used to demonstrate that the 117 antigen is found on the surface of maturing prespore cells after the PsA glycoprotein disappears, but that it is not found on mature spores. Using three monoclonal antibodies which identify respectively 117 antigen, PsA, and MUD3 antigen (a spore coat glycoprotein--probably Sp96), two new stages of final spore maturation were defined. These results indicate that there is a recapitulation of at least one aggregative cell surface glycoprotein in the prespore subpopulation of cells as they rise up the stalk during final spore development. This raises the possibility that culmination, which involves complex three dimensional morphogenetic movements not unlike those observed during animal embryogenesis, involves components of the two-dimensional pattern seen during aggregation.     

Developmental decisions in Dictyostelium discoideum.

A few hours after the onset of starvation, amoebae of Dictyostelium discoideum start to form multicellular aggregates by chemotaxis to centers that emit periodic cyclic AMP signals. There are two major developmental decisions: first, the aggregates either construct fruiting bodies directly, in a process known as culmination, or they migrate for a period as "slugs." Second, the amoebae differentiate into either prestalk or prespore cells. These are at first randomly distributed within aggregates and then sort out from each other to form polarized structures with the prestalk cells at the apex, before eventually maturing into the stalk cells and spores of fruiting bodies. Developmental gene expression seems to be driven primarily by cyclic AMP signaling between cells, and this review summarizes what is known of the cyclic AMP-based signaling mechanism and of the signal transduction pathways leading from cell surface cyclic AMP receptors to gene expression. Current understanding of the factors controlling the two major developmental choices is emphasized. The weak base ammonia appears to play a key role in preventing culmination by inhibiting activation of cyclic AMP-dependent protein kinase, whereas the prestalk cell-inducing factor DIF-1 is central to the choice of cell differentiation pathway. The mode of action of DIF-1 and of ammonia in the developmental choices is discussed.     

Role of cyclic AMP and ammonia in induction and maintenance of post-aggregative differentiation in a suspension culture of Dictyostelium discoideum

The effects of ammonia and cAMP on prespore and prestalk differentiation of Dictyostelium discoideum were investigated by monitoring eight developmentally regulated proteins as differentiation markers under the shake culture conditions in glucose/albumin medium. In the medium containing cAMP, cells form small agglomerates and undergo prespore differentiation [19]. Under the conditions where agglomeration was prevented, ammonia induced four marker proteins out of eight tested in the presence of cAMP, which included not only a prespore specific enzyme but also cell-type non-specific proteins. No inhibitory effect of ammonia was observed in presumptive cell differentiation. These results suggest that ammonia is an inducer of differentiation at the protein level as well as the mRNA level as found previously [24]. The effects of cAMP were examined with special attention to the difference between induction of differentiation and maintenance of differentiated state in this specific medium. The induction of differentiation from early aggregative cells was cAMP-dependent with all the marker proteins tested. This agrees with the observations so far obtained in other culture systems. However, when already differentiated cell masses (slugs) were dissociated and shaken in this specific medium, only two enzymes required cAMP to maintain the activity while five out of eight kinds of the proteins continued to be expressed as in undisturbed slugs even without cAMP. This suggests that for the maintenance of the differentiated state after slug disaggregation cAMP may not be required with respect to the majority of proteins, if cells are provided with some favorable conditions such as glucose/albumin medium.     

From drought sensing to developmental control: evolution of cyclic AMP signaling in social amoebas

Amoebas and other protists commonly encyst when faced with environmental stress. Although little is known of the signaling pathways that mediate encystation, the analogous process of spore formation in dictyostelid social amoebas is better understood. In Dictyostelium discoideum, secreted cyclic AMP (cAMP) mediates the aggregation of starving amoebas and induces the differentiation of prespore cells. Intracellular cAMP acting on cAMP-dependent protein kinase (PKA) triggers the maturation of spores and prevents their germination under the prevalent conditions of high osmolality in the spore head. The osmolyte-activated adenylate cyclase, ACG, produces cAMP for prespore differentiation and inhibition of spore germination. To retrace the origin of ACG function, we investigated ACG gene conservation and function in species that span the dictyostelid phylogeny. ACG genes, osmolyte-activated ACG activity, and osmoregulation of spore germination were detected in species that represent the 4 major groups of Dictyostelia. Unlike the derived species D. discoideum, many basal Dictyostelia have retained the ancestral mechanism of encystation from solitary amoebas. In these species and in solitary amoebas, encystation is independently triggered by starvation or by high osmolality. Osmolyte-induced encystation was accompanied by an increase in cAMP and prevented by inhibition of PKA, indicating that ACG and PKA activation mediate this response. We propose that high osmolality signals drought in soil amoebas and that developmental cAMP signaling in the Dictyostelia has evolved from this stress response.     

The function of PP2A/B56 in non-metazoan multicellular development

DB56, the Dictyostelium B56 homolog, displayed high sequence homology to other eukaryotic B56 subunits of the PP2A and a specific association with the PP2A catalytic subunit. Cells lacking DB56, psrA(-), displayed higher PP2A phosphatase activity compared with the wild type, approximately 10 hr of delayed expression of ecmA and ecmB prestalk markers, and inefficient culmination. The prespore marker cotB declined as wild-type cells culminate, but no such decline was observed from psrA(-) cells. Interestingly, psrA(-) cells exhibited higher GSK3 kinase activity. Furthermore, the expression of the dominant negative GSK3 mutant (K84/85M) in psrA(-) cells improved both prestalk and prespore expression patterns similarly to wild-type cells. However, culmination was not restored in psrA(-) cells expressing dominant negative GSK3, which suggests that PP2A/DB56 has an extra target during terminal differentiation. This report shows that PP2A/DB56 controls not only metazoan development, but also non-metazoan cell fate decision processes.     

Three steps in prespore differentiationin Dictyostelium discoideum with different requirements of cellular interaction

Abstract. Extracellular cAMP and a secreted factors have been known to be involved in prespore differentiation of Dictyostelium discoideum . Here we show that cAMP, a secreted factor(s) and some other interactions are required for prespore differentiation and that they work in completely different periods; a secreted factor(s) and other interactions are required only in the stages earlier than the cAMP-dependent stage. According to the results the process of prespore differentiation can be dissected into three sequential stages, stage I, II and III. The processes in stage I and II depend on high cell density. The requirement for high cell density in stage II could be replaced with a secreted factor(s) in conditioned medium, whereas it could not in stage I. The factor(s) in conditioned medium does not appear to be cAMP, ammonia, or methionine. In contrast to these two stages, the process in stage III, the last stage, proceeds even at low cell density if cAMP is supplied, where other interactions would be negligible. Therefore cells that have proceeded to the end of stage II are considered to have acquired a competence to differentiate to prespore cells without further cellular interactions other than cAMP.
cAMP pulses are not essential for the processes of any stage of prespore differentiation, since they proceed in the presence of caffeine, an inhibitor of cAMP pulse production, or in a mutant strain (Frigid A) which is deficient in cAMP relay systems.     

Functional modulation of brain sodium channels by cAMP-dependent phosphorylation.

Voltage-gated Na+ channels, which are responsible for the generation of action potentials in brain, are phosphorylated by cAMP-dependent protein kinase in vitro and in intact neurons. Phosphorylation by cAMP-dependent protein kinase reduces peak Na+ currents 40%--50% in membrane patches excised from rat brain neurons or from CHO cells expressing type IIA Na+ channels. Inhibition of basal cAMP-dependent protein kinase activity by transfection with a plasmid encoding a dominant negative mutant regulatory subunit increases Na+ channel number and activity, indicating that even the basal level of kinase activity is sufficient to reduce Na+ channel activity significantly. Na+ currents in membrane patches from kinase-deficient cells were reduced up to 80% by phosphorylation by cAMP-dependent protein kinase. These effects could be blocked by a specific peptide inhibitor of cAMP-dependent protein kinase and reversed by phosphoprotein phosphatases. Convergent modulation of brain Na+ channels by neurotransmitters acting through the cAMP and protein kinase C signaling pathways may result in associative regulation of electrical activity by different synaptic inputs.     

The timing of cell-type-specific differentiation in Dictyostelium discoideum

We have used two-dimensional gel electrophoresis to identify over 30 proteins which are specific to one or other of the two cell types of Dictyostelium discoideum, either at the slug stage or in mature fruiting bodies. Our results support the idea that there is a continuous developmental program that begins in prespore cells at the hemispherical mound stage (10-12 hr) and results in spore differentiation (24 hr). Prestalk differentiation, on the other hand, appeared largely unrelated to stalk differentiation, which was first detectable at the onset of culmination (18 hr). We have also used this approach to study the differentiation of stalk-only mutants and have found that the cells can switch from spore to stalk differentiation as late as 2 hr before the end of the wild-type developmental program.     

A prespore gene, Dd31, expressed during culmination of Dictyostelium discoideum

During culmination of Dictyostelium fruiting bodies, prespore and prestalk cells undergo terminal differentiation to form spores and a cellular stalk. A genomic fragment was isolated by random cloning that hybridizes to a 1.4-kb mRNA present during culmination. Cell type separations at culmination showed that the mRNA is present in prespore cells and spores, but not in prestalk or stalk cells. After genomic mapping, an additional 3 kb of DNA surrounding the original 1-kb fragment was cloned. The gene was sequenced and named Dd31 after the size of the predicted protein product in kilodaltons. Accumulation of Dd31 mRNA occurs immediately prior to sporulation. Addition of 20 mM 8-Br-cAMP to cells dissociated from Mexican hat stage culminants induced sporulation and the accumulation of Dd31 mRNA, while 20 mM cAMP did not. Dd31 mRNA does not accumulate in the homeotic mutant stalky in which prespore cells are converted to stalk cells rather than spores. Characterization of Dd31 extends the known temporal dependent sequence of molecular differentiations to sporulation.