Postaggregative Development Research Articles

The dual-specificity protein phosphatase MkpB, homologous to mammalian MKP phosphatases, is required for D. discoideum post-aggregative development and cisplatin response

Dual-specificity protein phosphatases participate in signal transduction pathways inactivating mitogen-activated protein kinases (MAP kinases). These signaling pathways are of critical importance in the regulation of numerous biological processes, including cell proliferation, differentiation and development. The social ameba Dictyostelium discoideum harbors 14 genes coding for proteins containing regions very similar to the dual-specificity protein phosphatase domain. One of these genes, mkpB, additionally codes for a region similar to the Rhodanase domain, characteristic of animal MAP kinase-phosphatases, in its N-terminal region. Cells that over-express this gene show increased protein phosphatase activity. mkpB is expressed in D. discoideum ameba at growth but it is greatly induced at 12 h of multicellular development. Although it is expressed in all the cells of developmental structures, mkpB mRNA is enriched in cells with a distribution typical of anterior-like cells. Cells that express a catalytically inactive mutant of MkpB grow and aggregate like wild-type cells but show a greatly impaired post-aggregative development. In addition, the expression of cell-type specific genes is very delayed, indicating that this protein plays an important role in cell differentiation and development. Cells expressing the MkpB catalytically inactive mutant show increased sensitivity to cisplatin, while cells over-expressing wild type MkpB, or MkpA, proteins or mutated in the MAP kinase erkB gene are more resistant to this chemotherapeutic drug, as also shown in human tumor cells.

The Dictyostelium discoideum acaA gene is transcribed from alternative promoters during aggregation and multicellular development.

BackgroundExtracellular cAMP is a key extracellular signaling molecule that regulates aggregation, cell differentiation and morphogenesis during multi-cellular development of the social amoeba Dictyostelium discoideum. This molecule is produced by three different adenylyl cyclases, encoded by the genes acaA, acrA and acgA, expressed at different stages of development and in different structures.Methodology/Principal FindingsThis article describes the characterization of the promoter region of the acaA gene, showing that it is transcribed from three different alternative promoters. The distal promoter, promoter 1, is active during the aggregation process while the more proximal promoters are active in tip-organiser and posterior regions of the structures. A DNA fragment containing the three promoters drove expression to these same regions and similar results were obtained by in situ hybridization. Analyses of mRNA expression by quantitative RT-PCR with specific primers for each of the three transcripts also demonstrated their different temporal patterns of expression.Conclusions/SignificanceThe existence of an aggregation-specific promoter can be associated with the use of cAMP as chemo-attractant molecule, which is specific for some Dictyostelium species. Expression at late developmental stages indicates that adenylyl cyclase A might play a more important role in post-aggregative development than previously considered.

RasC is required for optimal activation of adenylyl cyclase and Akt/PKB during aggregation.

Disruption of Dictyostelium rasC, encoding a Ras subfamily protein, generated cells incapable of aggregation. While rasC expression is enriched in a cell type-specific manner during post-aggregative development, the defect in rasC(-) cells is restricted to aggregation and fully corrected by application of exogenous cAMP pulses. cAMP is not produced in rasC(-) cells stimulated by 2'-deoxy-cAMP, but is produced in response to GTPgammaS in cell lysates, indicating that G-protein-coupled cAMP receptor activation of adenylyl cyclase is regulated by RasC. However, cAMP-induced ERK2 phosphorylation is unaffected in rasC(-) cells, indicating that RasC is not an upstream activator of the mitogen-activated protein kinase required for cAMP relay. rasC(-) cells also exhibit reduced chemotaxis to cAMP during early development and delayed response to periodic cAMP stimuli produced by wild-type cells in chimeric mixtures. Furthermore, cAMP-induced Akt/PKB phosphorylation through a phosphatidylinositide 3-kinase (PI3K)-dependent pathway is dramatically reduced in rasC(-) cells, suggesting that G-protein-coupled serpentine receptor activation of PI3K is regulated by RasC. Cells lacking the RasGEF, AleA, exhibit similar defects as rasC(-) cells, suggesting that AleA may activate RasC.

Multiple Developmental Roles for CRAC, a Cytosolic Regulator of Adenylyl Cyclase

Receptor-mediated activation of adenylyl cyclase (ACA) inDictyosteliumrequires CRAC protein. Upon translocation to the membrane, this pleckstrin homology (PH) domain protein stimulates ACA and thereby mediates developmental aggregation. CRAC may also have roles later in development since CRAC-null cells can respond to chemotactic signals and participate in developmental aggregation when admixed with wild-type cells, but they do not complete development within such chimeras. To test whether the role of CRAC in postaggregative development is related to the activation of ACA, chemotactic aggregation was bypassed in CRAC-null cells by activating the cAMP-dependent protein kinase (PKA). While such strains formed mounds, they did not complete fruiting body morphogenesis or form spores. Expression of CRAC in the prespore cells of these strains rescued sporulation and fruiting body formation. This later function of CRAC does not appear to require its PH domain since the C-terminal portion of the protein (CRAC-ΔPH) can substitute for full-length CRAC in promoting spore cell formation and morphogenesis. No detectable ACA activation was observed in any of the CRAC-null strains rescued by PKA activation and expression of CRAC-ΔPH. Finally, we found that the development of CRAC-null ACA-null double mutants could be rescued by the activation of PKA together with the expression of CRAC-ΔPH. Thus, there appears to be a required function for CRAC in postaggregative development that is independent of its previously described function in the ACA activation pathway.

Two ras genes in Dictyostelium minutum show high sequence homology, but different developmental regulation from Dictyostelium discoideum rasD and rasG genes

The social amoeba Dictyostelium discoideum expresses five ras genes at different stages of development. One of them, DdrasD is expressed during postaggregative development and transcription is induced by extracellular cAMP. A homologue of DdrasD, the DdrasG gene, is expressed exclusively during vegetative growth. We cloned two ras homologues Dmras1 and Dmras2 from the primitive species D. minutum, which show high homology to DdrasD and DdrasG and less homology to the other Ddras genes. In contrast to the DdrasD and DdrasG genes, both the Dmras1 and Dmras2 genes are expressed during the entire course of development. The expression levels are low during growth, increase at the onset of starvation and do not decrease until fruiting bodies have formed. Expression of neither Dmras1 or Dmras2 is regulated by cAMP. So even though the high degree of homology between the ras genes of different species suggests conservation of function, this function is apparently not associated with a specific developmental stage.

Role of cAMP-Dependent Protein Kinase in Controlling Aggregation and Postaggregative Development inDictyostelium

We have examined the role of cAMP-dependent protein kinase (PKA) in controlling aggregation and postaggregative development inDictyostelium.We previously showed that cells in which the gene encoding the PKA catalytic subunit has been disrupted (pkacat−cells) are unable to aggregate [S. K.O. Mann and R.A. Firtel (1991). A developmentally regulated, putative serine/threonine protein kinase is essential for development inDictyostelium. Mech. Dev.35, 89–102]. We show thatpkacat−cells are unable to activate adenylyl cyclase in response to cAMP stimulation due to the inability to express the aggregation-stage, G-protein-stimulated adenylyl cyclase (ACA). Constitutive expression of ACA from an actin promoter results in a high level of Mn2+-stimulated adenylyl cyclase activity and restores chemoattractant- and GTPγS-stimulated adenylyl cyclase activity but not the ability to aggregate. Similarly, expression of the constitutively active, non-G protein-coupled adenylyl cyclase ACG inpkacat−cells also does not restore the ability to aggregate, although ACG can complement cells in which theACAgene has been disrupted. These results indicate thatpkacat−cells lack multiple, essential aggregation-stage functions. As the mound forms, high, continuous levels of extracellular cAMP functioning through the cAMP serpentine receptors activate a transcriptional cascade that leads to cell-type differentiation and morphogenesis. The first step is the induction and activation of the transcription factor GBF and downstream postaggregative genes, followed by the induction of prestalk- and prespore-specific genes. We show thatpkacat−cells induce postaggregative gene expression in response to exogenous cAMP, but the level of induction of some of these genes, includingGBF,is reduced.SP60(a prespore-specific gene) is not induced andecmA(a prestalk-specific gene) is induced to very low levels. Expressing GBF constitutively inpkacat−cells restoresecmAexpression to a moderate level, butSP60is not detectably induced. Overexpression of PKAcat from the Actin 15 (Act15), ecmAprestalk, and thePKAcatpromoters inpkacat−cells results in significant aberrant spatial patterning of prestalk and prespore cells, as determined bylacZ reporter studies. Our studies identify new, essential regulatory roles for PKA in mediating multicellular development.

A prespore-specific gene of Dictyostelium discoideum encodes the small subunit of ribonucleotide reductase

We have isolated the gene, rnrB, that encodes the ribonucleotide reductase small subunit of Dictyostelium discoideum. The deduced amino acid sequence of rnrB exhibits about 60% sequence identity with its homologues in other eukaryotes. As demonstrated by RNA blot analysis the rnrB transcript is detected in growing cells and decreases dramatically at the onset of development. The rnrB transcript reappears after the cells have formed multicellular aggregates. To further examine the pattern of expression, we have fused the rnrB promoter and part of its coding sequence to lacZ. The transgenic strain bearing such a reporter construct expresses the fusion gene with a biphasic profile, which is indistinguishable from that of the endogenous rnrB. The multicellular aggregates of Dictyostelium are differentiated along the anterior-posterior axis. Cells in the anterior give rise to the stalk of the fruiting body while cells in the posterior are precursors of spores. Results from histochemical staining show that β-galactosidase activity is detected exclusively in the posterior two-thirds of the aggregates. These data suggest that rnrB is expressed in prespore cells during postaggregative development and in vegetative cells.

Serpentine cAMP receptors may act through a G protein-independent pathway to induce postaggregative development in dictyostelium

The transcription factor G box-binding factor (GBF) is required for the developmental switch between aggregative and postaggregative gene expression, cell-type differentiation, and morphogenesis. We show that constitutive expression of GBF allows ectopic expression of postaggregative genes, but only in response to exogenous cAMP. GBF activation requires the serpentine cAMP receptors required for aggregation, but not the coupled Gα2 or the Gβ subunit, suggesting a novel signaling pathway. In response to high cAMP, gα2-null cells can bypass the aggregation stage, expressing cell type-specific genes and forming fruiting bodies. Our results demonstrate that the same receptors regulate aggregation and cell-type differentiation, but via distinct pathways depending upon whether the receptor perceives a pulsatile or sustained signal.

Differentiation and patterning in Dictyostelium

Dictyostelium development is orchestrated by diffusible signals. Progress has been made in understanding how cAMP signalling triggers post-aggregative development and in defining the number of cell types that eventually differentiate. Ammonia is an unusual signal that may act by alkalinizing acidic vesicles. A chlorinated signal, differentiation-inducing factor (DIF), may be universal amongst the slime moulds. The first genes have been cloned using restriction enzyme mediated integration (REMI) insertional mutagenesis; one encodes a novel cytosolic protein essential for activation of adenylyl cyclase.

Multiple genes for cell surface cAMP receptors in Dictyostelium discoideum

We have cloned and characterized three genes (CAR1, CAR2, CAR3) encoding potential cell surface, cyclic adenosine 3':5' monophosphate (AMP) receptors from Dictyostelium discoideum. The three proteins are predicted to be substantially similar in amino acid sequence throughout most of their transmembrane (TM) and loop domains but are distinctly different in their carboxyl terminal segments. In addition, all three genes possess an intron which interrupts an equivalent codon of TM3. CAR1 is expressed early in development when the cAMP relay system is being established. As development proceeds multiple size forms of CAR1 RNA are detected which apparently result from differences in their 5'-untranslated regions. Late in development levels of CAR1 RNA decrease. In contrast, CAR2 encodes a single sized RNA which is expressed only during postaggregative development. CAR3 expression is approximately 10% of CAR1 during early development, is maximal during tight aggregate formation but declines thereafter. Only one size class of CAR3 mRNA is detected throughout development. Because RNA for each of the three genes is present in postaggregative cells, it was of interest to determine the cell type distribution of each RNA. Gene-specific probes were hybridized to RNAs isolated from cells of Percoll gradient-enriched prespore and prestalk fractions and relative levels of hybridization compared. CAR1 and CAR3 show approximately the same pattern of accumulation; a 3-4 fold enrichment in prestalk cells. CAR2, however, is highly enriched in prestalk cells, more than 10 fold relative to prespore cells.

Developmental regulation of the folic acid chemosensory system in Dictyostelium discoideum

The developmental regulation of three classes of cell surface folate receptors, of cell surface folate deaminase, and of three folate-stimulated cellular responses (cGMP and cAMP synthesis, chemotaxis) have been monitored. All of these parameters showed a comparable decline during cell aggregation between 4 and 8 hr after the onset of starvation. Many of the parameters showed an increase during the first 3 hr of starvation (A, B S, and B SS receptors, and the cGMP and cAMP responses) while others were constant during this time period (deaminase, chemotaxis). Moreover, some kinetic properties of A and B SS receptors changed substantially during early development. After cell aggregation (at about 8 hr) all parameters except B SS sites were reduced to less than 10% of their maximal values. Cellular responses to folic acid were absent during the postaggregative development. When aggregating cells were removed from the agar and shaken in suspension for an additional 3 hr, sensitivity to folic acid as judged by cGMP and cAMP synthesis reappeared. Additionally, the further disappearance of cell surface B receptors was inhibited, A sites disappeared more rapidly, and the B SS sites regained the kinetic properties of these sites characteristic of unstarved cells. The changes in receptor kinetics are discussed in terms of coupling to/uncoupling from effector proteins.

Detection and developmental regulation of the mRNA for the regulatory subunit of the cAMP-dependent protein kinase of D. discoideum by cell-free translation.

cAMP is an important effector of the development of Dictyostelium discoideum amoebae and could exert its effects on gene expression through the cytosolic cAMP-dependent protein kinase (cAK). Antibodies, specific for the regulatory subunit (R) of the cAK, were used to investigate the developmental regulation of the corresponding mRNA (R-mRNA) by in vitro translation and immunoprecipitation. Under such conditions, a single polypeptide of the same mol. wt. as R (42 kd) is detected, showing that the protein is not synthesized as a large precursor. The level of the R-mRNA, which is low in vegetative cells, increases 10- to 20-fold during the first hours of development. Its expression is stimulated by the treatment of AX3 cells with cAMP either added to a concentration of 1 mM or given as 0.1 microM pulses every 5 min, whereas such treatments have little or no effect in cells of strain AX2. The R-mRNA remains highly expressed (0.01-0.03% of translatable mRNA) throughout post-aggregative development; it is not affected by mechanical disaggregation of the multicellular organism. The parallel developmental time courses of the translatable R-mRNA and the R protein produced in vivo suggest that the expression of this polypeptide is regulated at the level of mRNA synthesis.

Coh A, a mutation affecting the post aggregative related (par) cohesive system of Dictyostelium discoideum

AbstractThree stage‐specific cohesive systems operate in D. discoideum: VEG, elaborated by vegetative cells: AR, by aggregation competent cells; and PAR, by post aggregation stage cells. Previous study of a mutant strain JC‐5 had shown the stability of its PAR system (but not the AR) to be temperature sensitive. However, the phenotypic expression of this mutation termed Coh A is complicated by the presence in that strain of a preexisting mutant gene Rde A, which accelerates developmental events generally and alters the pattern of morphogenesis. Genetic evidence presented here indicates that the two mutations have been separated by parasexual recombination yielding a Coh A, Rde A+ segregant class of which strain JC‐36 is a prototype.At the permissive temperature, JC‐36 follows a morphogenetic sequence like that of the wild type in respect to timing, morphogenetic pattern, and spore appearance. At the restrictive temperature, it forms normal aggregates at the usual time but exhibits two morphogenetic aberrancies during post aggregative development. First, fruit construction is arrested at a stage approximating the 16 hr “Bottle” stage of the wild type, though more squat and blunt tipped, and then the aggregate regresses. Cytodifferentiation into spores and stalk cells is also blocked. Second, a shift of slugs migrating normally at the permissive temperature to the restrictive causes the latter to disintegrate progressively as they leave clumps of cells behind them within the flattened sheath.JC‐36 cells developing at the restrictive temperature also exhibited a decrease in EDTA resistant cohesivity attributable on two grounds to the sensitivity of the PAR system. In addition, the disappearance of the AR system completed in the wild type by the Mexicanhat (18–19 hr) stage is indefinitely arrested at an intermediate level in JC‐36.

Developmentally regulated enzymes and cyclic AMP-binding sites in Dictyostelium discoideum cells blocked during development by alpha-chymotrypsin.

When cells of the slime mould Dictyostelium discoideum are allowed to starve in the presence of alpha-chymotrypsin, they are blocked in development at the stage where tight aggregates form tips. Analysis of developmentally regulated enzymes has shown that alpha-mannosidase, beta-N-acetylglucosaminidase, threonine deaminase, tyrosine aminotransferase, beta-glucosidase and the carbohydrate-binding protein discoidin are unaffected, but enzymes that show an increase in specific activity during post-aggregative development, namely glycogen phosphorylase, UDP-glucose pyrophosphorylase, UDP-galactose 4-epimerase, UDP-galactose polysaccharide transferase and alkaline phosphatase, did not show the characteristic increase when development was blocked by alpha-chymotrypsin. Recovery of cells from the effects of alpha-chymotrypsin was accompanied by the formation of fruiting bodies and a concomitant increase in the specific activity of UDP-glucose pyrophosphorylase. Uptake or efflux of 45Ca2+ was not altered in the presence of alpha-chymotrypsin. Cells allowed to develop in alpha-chymotrypsin, or treated with the enzyme for 15 min, had a markedly reduced ability to bind cyclic AMP with low affinity; high-affinity binding was unaffected. Pronase had a similar effect on cyclic AMP binding, but trypsin, which does not alter developmental processes, has no effect on cyclic AMP binding to D. discoideum cells.

The evolution of excitable behaviour in Dictyostelium

Aggregation of Dictyostelium discoideum amoebae is effected by relayed cyclic AMP (cAMP) signals. The period of this wave propagation drops abruptly at the transition from aggregative to post-aggregative development. In this paper I demonstrate that the light-scattering response induced by a low concentration pulse of cAMP evolves from one lasting 5 min to one lasting 2 min. The definitive establishment of the 2-min response coincides with the beginning of post-aggregative gene expression. Amoebae at the aggregative stage are unable to respond to a second cAMP pulse delivered less than 4 min after the first, whereas at the post-aggregative stage they can respond to 2 pulses separated by 1 min or longer. Addition of cAMP phosphodiesterase to amoebae at the aggregative stage can in part mimic the change in excitable behaviour: the response is shortened and the amoebae can respond more frequently. However, the shortened response is not post-aggregative in shape and the prolonged presence of cyclic nucleotides induces different responses at the aggregative and post-aggregative stage. Both these results suggest that the rate of destruction of the relayed signal is not solely responsible for the change in excitability.