Combinatorial control of cell differentiation by cAMP and DIF-1 during development of Dictyostelium discoideum

At least three distinct types of cell arise from a population of similar amoebae during Dictyostelium development: prespore, prestalk A and prestalk B cells. We report evidence suggesting that this cellular diversification can be brought about by the combinatorial action of two diffusible signals, cAMP and DIF-1. Cells at different stages of normal development were transferred to shaken suspension, challenged with various combinations of signal molecules and the expression of cell-type-specific mRNA markers measured 1-2 h later. pDd63, pDd56 and D19 mRNAs were used for prestalk A, prestalk B and prespore cells respectively. We find the following results. (1) Cells first become responsive to DIF-1 for prestalk A differentiation and to cAMP for prespore differentiation at the end of aggregation, about 2 h before these cell types normally appear. (2) At the first finger stage of development, when the rate of accumulation of the markers is maximal, the expression of each is favoured by a unique combination of effectors: prespore differentiation is stimulated by cAMP and inhibited by DIF-1; prestalk A differentiation is stimulated by both cAMP and DIF-1 and prestalk B differentiation is stimulated by DIF-1 and inhibited by cAMP. (3) Half-maximal effects are produced by 10-70 nM DIF-1, which is in the physiological range. (4) Ammonia and adenosine, which can affect cell differentiation in other circumstances, have no significant pathway-specific effect in our conditions. These results suggest that cell differentiation could be brought about in normal development by the localized action of cAMP and DIF-1.     

DIF-1 induces its own breakdown in Dictyostelium.

DIF-1 is a novel chlorinated alkyl phenone which induces differentiation of prestalk cells in Dictyostelium discoideum. It is broken down and inactivated by a cytoplasmic enzyme, DIF-1 3(5)-dechlorinase (hereafter referred to as DIF-1 dechlorinase), which is found only in prestalk cells. We show that DIF-1 dechlorinase levels are induced at least 50-fold when cells are treated with DIF-1. This response is rapid--enzyme activity doubles within 15 min and is fully induced within an hour--and occurs early in development, before other prestalk markers can be induced by DIF-1. Maximum inducibility is seen towards the end of aggregation, when DIF-1 dechlorinase is barely detectable in uninduced cells. The dose-dependence reveals a threshold concentration of DIF-1 (15 nM) below which almost no response is seen. Cyclic AMP, which is the chemoattractant during aggregation and plays a key role in later development, suppresses the induction of DIF-1 dechlorinase by DIF-1. We conclude that induction of DIF-1 dechlorinase is one of the first steps on the developmental pathway which leads to prestalk cell differentiation, and suggest that the resulting negative feedback on DIF-1 levels is an important part of the mechanism by which cells decide whether to become prestalk or prespore cells.     

Differentiation inducing factor-1 (DIF-1) induces gene and protein expression of the Dictyostelium nuclear calmodulin-binding protein nucleomorphin

The nucleomorphin gene numA1 from Dictyostelium codes for a multi-domain, calmodulin binding protein that regulates nuclear number. To gain insight into the regulation of numA, we assessed the effects of the stalk cell differentiation inducing factor-1 (DIF-1), an extracellular signalling molecule, on the expression of numA1 RNA and protein. For comparison, the extracellular signalling molecules cAMP (mediates chemotaxis, prestalk and prespore differentiation) and ammonia (NH₃/NH₄⁺; antagonizes DIF) were also studied. Starvation, which is a signal for multicellular development, results in a greater than 80% decrease in numA1 mRNA expression within 4 h. Treatment with ammonium chloride led to a greater than 90% inhibition of numA1 RNA expression within 2 h. In contrast, the addition of DIF-1 completely blocked the decrease in numA1 gene expression caused by starvation. Treatment of vegetative cells with cAMP led to decreases in numA1 RNA expression that were equivalent to those seen with starvation. Western blotting after various morphogen treatments showed that the maintenance of vegetative levels of numA1 RNA by DIF-1 in starved cells was reflected in significantly increased numA1 protein levels. Treatment with cAMP and/or ammonia led to decreased protein expression and each of these morphogens suppressed the stimulatory effects of DIF-1. Protein expression levels of CBP4a, a calcium-dependent binding partner of numA1, were regulated in the same manner as numA1 suggesting this potential co-regulation may be related to their functional relationship. NumA1 is the first calmodulin binding protein shown to be regulated by developmental morphogens in Dictyostelium being upregulated by DIF-1 and down-regulated by cAMP and ammonia.     

Arrestins function in cAR1 GPCR-mediated signaling and cAR1 internalization in the development of Dictyostelium discoideum

Oscillation of chemical signals is a common biological phenomenon, but its regulation is poorly understood. At the aggregation stage of Dictyostelium discoideum development, the chemoattractant cAMP is synthesized and released at 6-min intervals, directing cell migration. Although the G protein–coupled cAMP receptor cAR1 and ERK2 are both implicated in regulating the oscillation, the signaling circuit remains unknown. Here we report that D. discoideum arrestins regulate the frequency of cAMP oscillation and may link cAR1 signaling to oscillatory ERK2 activity. Cells lacking arrestins (adcB⁻C⁻) display cAMP oscillations during the aggregation stage that are twice as frequent as for wild- type cells. The adcB⁻C⁻ cells also have a shorter period of transient ERK2 activity and precociously reactivate ERK2 in response to cAMP stimulation. We show that arrestin domain–containing protein C (AdcC) associates with ERK2 and that activation of cAR1 promotes the transient membrane recruitment of AdcC and interaction with cAR1, indicating that arrestins function in cAR1-controlled periodic ERK2 activation and oscillatory cAMP signaling in the aggregation stage of D. discoideum development. In addition, ligand-induced cAR1 internalization is compromised in adcB⁻C⁻ cells, suggesting that arrestins are involved in elimination of high-affinity cAR1 receptors from cell surface after the aggregation stage of multicellular development.     

Cell differentiation and patterning in Dictyostelium.

In Dictyostelium development, prestalk cells first differentiate at scattered positions in the aggregate and then sort out, probably by chemotaxis to cAMP. They may regulate their proportions by selective depletion of the stalk cell inducer, DIF-1. Once sorted, prestalk cells form a DIF-1 sink, which can produce gradients of DIF-1 and its metabolites in the slug. Global movements of cells in the slug may be regulated by cAMP signals, as in aggregation. Terminal differentiation of stalk and spore cells requires activation of cAMP-dependent protein kinase, possibly brought about by ammonia depletion. Finally, a technique for insertional mutagenesis promises the ready isolation of developmental genes.     

CulB,a putative ubiquitin ligase subunit,regulates prestalk cell differentiation and morphogenesis in Dictyostelium spp

Dictyostelium amoebae accomplish a starvation-induced developmental process by aggregating into a mound and forming a single fruiting body with terminally differentiated spores and stalk cells. culB was identified as the gene disrupted in a developmental mutant with an aberrant prestalk cell differentiation phenotype. The culB gene product appears to be a homolog of the cullin family of proteins that are known to be involved in ubiquitin-mediated protein degradation. The culB mutants form supernumerary prestalk tips atop each developing mound that result in the formation of multiple small fruiting bodies. The prestalk-specific gene ecmA is expressed precociously in culB mutants, suggesting that prestalk cell differentiation occurs earlier than normal. In addition, when culB mutant cells are mixed with wild-type cells, they display a cell-autonomous propensity to form stalk cells. Thus, CulB appears to ensure that the proper number of prestalk cells differentiate at the appropriate time in development. Activation of cyclic AMP-dependent protein kinase (PKA) by disruption of the regulatory subunit gene (pkaR) or by overexpression of the catalytic subunit gene (pkaC) enhances the prestalk/stalk cell differentiation phenotype of the culB mutant. For example, culB⁻ pkaR⁻ cells form stalk cells without obvious multicellular morphogenesis and are more sensitive to the prestalk O (pstO) cell inducer DIF-1. The sensitized condition of PKA activation reveals that CulB may govern prestalk cell differentiation in Dictyostelium, in part by controlling the sensitivity of cells to DIF-1, possibly by regulating the levels of one or more proteins that are rate limiting for prestalk differentiation.     

A possible role for DIF-2 in the formation of stalk cells during Dictyostelium development.

The differentiation inducing factor (DIF) is essential for stalk cell formation in monolayers of Dictyostelium discoideum and is necessary for the expression of several prestalk cell-specific genes. DIF activity has been fractionated into a major species, designated DIF-1, and several minor species, including DIF-2. Although DIF-1 is an excellent inducer of stalk cell formation from vegetative cells, it is a poor inducer of stalk cell formation from prestalk cells. In contrast, DIF-2 is more active for the conversion of prestalk cells into stalk cells, than for the conversion of vegetative cells to stalk cells. The same results were obtained regardless of whether chemically synthesized or naturally occurring components were utilized. In addition, stalk cell formation was three- to fourfold higher when vegetative cells were incubated with DIF-1 for a suboptimal period and then subsequently incubated with DIF-2, than when cells were incubated with DIF-2 first and then subsequently with DIF-1. These results indicate a distinct role for DIF-2 during stalk cell formation and suggest the possibility that DIF-1 and DIF-2 act sequentially.     

A role for cAMP in angiotensin II mediated inhibition of cell growth in AT1A receptor-transfected CHO-K1 cells

G-protein coupled Angiotensin II receptors (AT_1A), mediate cellular responses through multiple signal transduction pathways. In AT_1A receptor-transfected CHO-K1 cells (T3CHO/AT_1A), angiotensin II (AII) stimulated a dose-dependent (EC₅₀=3.3 nM) increase in cAMP accumulation, which was inhibited by the selective AT₁, nonpeptide receptor antagonist EXP3174. Activation of protein kinase C, or increasing intracellular Ca²⁺ with ATP, the calcium ionophore A23187 or ionomycin failed to stimulate cAMP accumulation. Thus, AII-induced cAMP accumulation was not secondary to activation of a protein kinase C- or Ca²⁺/calmodulin-dependent pathway. Since cAMP has an established role in cellular growth responses, we investigated the effect of the AII-mediated increase in cAMP on cell number and [³H]thymidine incorporation in T3CHOA/AT_1A cells. AII (1 M) significantly inhibited cell number (51% at 96 h) and [³H]thymidine incorporation (68% at 24 h) compared to vehicle controls. These effects were blocked by EXP3174, confirming that these responses were mediated through the AT₁ receptor. Forskolin (10 M) and the cAMP analog dibutyryl-cAMP (1 mM) also inhibited [³H]thymidine incorporation by 55 and 25% respectively. We extended our investigation on the effect of AII-stimulated increases in cAMP, to determine the role for established growth related signaling events, i.e., mitogen-activated protein kinase activity and tyrosine phosphorylation of cellular proteins. AII-stimulated mitogen-activated protein kinase activity and phosphorylation of the 42 and 44 kD forms. These events were unaffected by forskolin stimulated increases in cAMP, thus the AII-stimulated mitogen-activated protein kinase activity was independent of cAMP in these cells. AII also stimulated tyrosine phosphorylation of a number of cellular proteins in T3CHO/AT_1A cells, in particular a 127 kD protein. The phosphorylation of the 127 kD protein was transient, reaching a maximum at 1 min, and returning to basal levels within 10 min. The dephosphorylation of this protein was blocked by a selective inhibitor of cAMP dependent protein kinase A, H89-dihydrochloride and preexposure to forskolin prevented the AII-induced transient tyrosine phosphorylation of the 127 kD protein. These data suggest that cAMP, and therefore protein kinase A can contribute to AII-mediated growth inhibition by stimulating the dephosphorylation of substrates that are tyrosine phosphorylated in response to AII.     

DIF-1 induces the basal disc of the Dictyostelium fruiting body

The polyketide DIF-1 induces Dictyostelium amoebae to form stalk cells in culture. To better define its role in normal development, we examined the phenotype of a mutant blocking the first step of DIF-1 synthesis, which lacks both DIF-1 and its biosynthetic intermediate, dM-DIF-1 (des-methyl-DIF-1). Slugs of this polyketide synthase mutant (stlB⁻) are long and thin and rapidly break up, leaving an immotile prespore mass. They have ∼ 30% fewer prestalk cells than their wild-type parent and lack a subset of anterior-like cells, which later form the outer basal disc. This structure is missing from the fruiting body, which perhaps in consequence initiates culmination along the substratum. The lower cup is rudimentary at best and the spore mass, lacking support, slips down the stalk. The dmtA⁻ methyltransferase mutant, blocked in the last step of DIF-1 synthesis, resembles the stlB⁻ mutant but has delayed tip formation and fewer prestalk-O cells. This difference may be due to accumulation of dM-DIF-1 in the dmtA⁻ mutant, since dM-DIF-1 inhibits prestalk-O differentiation. Thus, DIF-1 is required for slug migration and specifies the anterior-like cells forming the basal disc and much of the lower cup; significantly the DIF-1 biosynthetic pathway may supply a second signal - dM-DIF-1.     

The role of DIF-1 signaling in Dictyostelium development

We have constructed a mutant blocked in the biosynthesis of DIF-1, a chlorinated signal molecule proposed to induce differentiation of both major prestalk cell types formed during Dictyostelium development. Surprisingly, the mutant still forms slugs retaining one prestalk cell type, the pstA cells, and can form mature stalk cells. However, the other major prestalk cell type, the pstO cells, is missing. Normal pstO cell differentiation and their patterning in the slug are restored by development on a uniform concentration of DIF-1. We conclude that pstO and pstA cells are in fact induced by separate signals and that DIF-1 is the pstO inducer. Positional information, in the form of DIF-1 gradients, is evidently not required for pstO cell induction.     

Evidence that DIF-1 and hyper-osmotic stress activate a Dictyostelium STAT by inhibiting a specific protein tyrosine phosphatase

STATc becomes tyrosine phosphorylated and accumulates in the nucleus when Dictyostelium cells are exposed to the prestalk cell inducer Differentiation inducing factor 1 (DIF-1), or are subjected to hyper-osmotic stress. We show that the protein tyrosine phosphatase PTP3 interacts directly with STATc and that STATc is refractory to activation in PTP3 overexpressing cells. Conversely, overexpression of a dominant inhibitor of PTP3 leads to constitutive tyrosine phosphorylation and ectopic nuclear localisation of STATc. Treatment of cells with DIF-1 or exposure to hyper-osmotic stress induces a decrease in biochemically assayable PTP3 activity and both agents also induce serine-threonine phosphorylation of PTP3. These observations suggest a novel mode of STAT activation, whereby serine-threonine phosphorylation of a cognate protein tyrosine phosphatase results in the inhibition of its activity, shifting the phosphorylation-dephosphorylation equilibrium in favour of phosphorylation.     

Identification of Regulatory Phosphorylation Sites in a Cell Volume– and Ste20 Kinase–dependent ClC Anion Channel

Changes in phosphorylation regulate the activity of various ClC anion transport proteins. However, the physiological context under which such regulation occurs and the signaling cascades that mediate phosphorylation are poorly understood. We have exploited the genetic model organism Caenorhabditis elegans to characterize ClC regulatory mechanisms and signaling networks. CLH-3b is a ClC anion channel that is expressed in the worm oocyte and excretory cell. Channel activation occurs in response to oocyte meiotic maturation and swelling via serine/threonine dephosphorylation mediated by the type I phosphatases GLC-7α and GLC-7β. A Ste20 kinase, germinal center kinase (GCK)-3, binds to the cytoplasmic C terminus of CLH-3b and inhibits channel activity in a phosphorylation-dependent manner. Analysis of hyperpolarization-induced activation kinetics suggests that phosphorylation may inhibit the ClC fast gating mechanism. GCK-3 is an ortholog of mammalian SPAK and OSR1, kinases that bind to, phosphorylate, and regulate the cell volume–dependent activity of mammalian cation-Cl⁻ cotransporters. Using mass spectrometry and patch clamp electrophysiology, we demonstrate here that CLH-3b is a target of regulatory phosphorylation. Concomitant phosphorylation of S742 and S747, which are located 70 and 75 amino acids downstream from the GCK-3 binding site, are required for kinase-mediated channel inhibition. In contrast, swelling-induced channel activation occurs with dephosphorylation of S747 alone. Replacement of both S742 and S747 with glutamate gives rise to kinase- and swelling-insensitive channels that exhibit activity and biophysical properties similar to those of wild-type CLH-3b inhibited by GCK-3. Our studies provide novel insights into ClC regulation and mechanisms of cell volume signaling, and provide the foundation for studies aimed at defining how conformational changes in the cytoplasmic C terminus alter ClC gating and function in response to intracellular signaling events.     

Ciliary transport regulates PDGF-AA/αα signaling via elevated mammalian target of rapamycin signaling and diminished PP2A activity

Primary cilia are built and maintained by intraflagellar transport (IFT), whereby the two IFT complexes, IFTA and IFTB, carry cargo via kinesin and dynein motors for anterograde and retrograde transport, respectively. Many signaling pathways, including platelet- derived growth factor (PDGF)-AA/αα, are linked to primary cilia. Active PDGF-AA/αα signaling results in phosphorylation of Akt at two residues: P-Akt^T308 and P-Akt^S473, and previous work showed decreased P-Akt^S473 in response to PDGF-AA upon anterograde transport disruption. In this study, we investigated PDGF-AA/αα signaling via P-Akt^T308 and P-Akt^S473 in distinct ciliary transport mutants. We found increased Akt phosphorylation in the absence of PDGF-AA stimulation, which we show is due to impaired dephosphorylation resulting from diminished PP2A activity toward P-Akt^T308. Anterograde transport mutants display low platelet-derived growth factor receptor (PDGFR)α levels, whereas retrograde mutants exhibit normal PDGFRα levels. Despite this, neither shows an increase in P-Akt^S473 or P-Akt^T308 upon PDGF-AA stimulation. Because mammalian target of rapamycin complex 1 (mTORC1) signaling is increased in ciliary transport mutant cells and mTOR signaling inhibits PDGFRα levels, we demonstrate that inhibition of mTORC1 rescues PDGFRα levels as well as PDGF-AA–dependent phosphorylation of Akt^S473 and Akt^T308 in ciliary transport mutant MEFs. Taken together, our data indicate that the regulation of mTORC1 signaling and PP2A activity by ciliary transport plays key roles in PDGF-AA/αα signaling.     

The effects of presumptive morphogens on prestalk and prespore cell gene expression in monolayers of Dictyostelium discoideum

Abstract. The expression of three prestalk cell-specific genes ( ecm A, ecm B and pDd26) was examined during in vitro differentiation in cell monolayers, in an attempt to explain the spatial heterogeneity of the prestalk region of migrating Dictyostelium pseudoplasmodia. Under these conditions ecm A, ecm B and pDd26 mRNAs were expressed sequentially in response to the addition of differentiation inducing factor-1 (DIF)-1, a temporal sequence similar to that observed during normal development. ecm A and ecm B mRNAs reached a maximum level 2–4 h after DIF-1 supplementation and then declined, whereas pDd26 mRNA levels increased more slowly but remained high 24 h after DIF addition. The increases in expression in response to increasing concentrations of either DIF-1 or DIF-2 were identical for the three genes, suggesting that neither alteration in DIF concentration nor species was an important determinant of spatial heterogeneity. Ammonia had the same inhibitory effect on the expression of all three prestalk cell-specific genes and stimulated the expression of the prespore cell-specific gene, D19. These results indicate that ammonia is also not responsible for the spatial heterogeneity of the prestalk cell region. In contrast, cyclic AMP had a differential effect on the expression of the prestalk cell specific genes: ecm A expression was variably stimulated, pDd26 expression was inhibited and ecm B expression was sometimes stimulated and sometimes inhibited. These results are difficult to explain in terms of a gradient of cyclic AMP in the prestalk region. We postulate that temporal responses are more important than spatial responses to cyclic AMP in regulating stalk cell differentiation.