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.     

Production and turnover of cAMP signals by prestalk and prespore cells in Dictyostelium discoideum cell aggregates

Dictyostelium discoideum prestalk cells and prespore cells from migrating slugs and culminating cell aggregates were isolated by Percoll density centrifugation. Several activities relevant to the generation, detection, and turnover of extracellular cyclic AMP (cAMP) signals were determined. It was found that: the two cell types have the same basal adenylate cyclase activity; prespore cells and prestalk cells are able to relay the extracellular cAMP signal equally well; intact prestalk cells show a threefold higher cAMP phosphodiesterase activity on the cell surface than prespore cells, whereas their cytosolic activity is the same; intact prestalk cells bind three to four times more cAMP than prespore cells; no large differences in cAMP metabolism and detection were observed between cells derived from migrating slugs and culminating aggregates. The results are discussed in relation to the possible morphogenetic role of extracellular cAMP in Dictyostelium cell aggregates. On the basis of the properties of the isolated cells we assume that a gradient of extracellular cAMP exists in Dictyostelium aggregates. This gradient appears to be involved in the formation and stabilization of the prestalk-prespore cell pattern.     

Pharmacological characterization of cyclic AMP receptors mediating gene regulation in Dictyostelium discoideum.

Extracellular molecules regulate gene expression in eucaryotes. Exogenous cyclic AMP (cAMP) affects the expression of a large number of developmentally regulated genes in Dictyostelium discoideum. Here, we determine the specificity of the receptor(s) which mediates gene expression by using analogs of cAMP. The order of potency with which these analogs affect the expression of specific genes is consistent with the specificity of their binding to a cell surface receptor and is distinct from their affinity for intracellular cAMP-dependent protein kinase. Dose-response curves with cAMP and adenosine 3',5'-monophosphorothioate, a nonhydrolyzable analog, revealed that the requirement for high concentrations of exogenous cAMP for regulating gene expression is due to the rapid degradation of cAMP by phosphodiesterase. The addition of low concentrations of cAMP (100 nM) or analogs in pulses also regulates gene expression. Both the genes that are positively regulated by exogenous cAMP and the discoidin gene, which is negatively regulated, respond to cAMP analogs to the same degree. Genes expressed in prespore or prestalk cells are also similarly regulated. These data suggest that the effects are mediated through the same receptor. The specificity of this receptor is indistinguishable from that of the well-characterized cell surface cAMP receptor.     

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.     

The prespore vesicles of Dictyostelium discoideum. Purification, characterization, and developmental regulation

The coordinate fusion of the prespore vesicles (PSVs) with the plasma membrane at the terminal stage of spore differentiation in Dictyostelium discoideum is an important example of developmentally regulated protein secretion. However, little is known about the composition of the vesicles, the molecular signals regulating secretion, or the mechanics of the membrane fusion. Taking a biochemical approach, we purified PSVs from different developmental stages. These preparations are highly enriched for their specific cargo of spore coat proteins while devoid of markers for other cellular compartments. Electron microscopic observations show that the PSV preparations are homogenous, with the soluble spore coat protein PsB/SP85 distributed throughout the lumen and the acid mucopolysaccharide localized in the central core. During development the PSVs increase in size and density concomitant with an increase in their protein cargo. The PSVs contain approximately 80 proteins, and we have identified a PSV-specific GTP-binding protein that may be involved in regulating vesicle fusion. The PSVs are not clathrin-coated and do not contain the SpiA spore coat protein. The PSV preparations are ideal for a global proteome analysis to identify proteins involved in signal reception, vesicle movement, docking, and fusion in this developmentally regulated organelle.     

Unexpected localisation of cells expressing a prespore marker of Dictyostelium discoideum

Abstract. We show that the anterior, prestalk region of the Dictyostelium slug contains cells which express, or have expressed, a prespore-specific marker. We term these cells "prespore-like cells" (PLC). In newly formed slugs there is a sharp prespore/prestalk boundary, with very few PLC, but after several days of migration the clear demarcation between prespore and prestalk zones breaks down because the number of PLC increases dramatically. This is consistent with previous observations showing there to be rapid interchange of cells between the prestalk and prespore regions. This is not, however, their only source, as a scattering of PLC appear when separate prestalk and prespore regions first become apparent at the time of tip formation. Also, at culmination, there is respecification of "prespore" cells at the pre-stalk/prespore boundary to form part of the mature stalk. The existence of these cells, and of PLC, may explain why we find prespore-specific mRNAs in mature stalk cells.     

Changes of plasma membrane proteins during prespore differentiation in Dictyostelium discoideum

By the use of a shake culture system, we have previously shown (Oyama, M., Okamoto, K., & Takeuchi, I. (1982) J. Cell Sci. 56, 223-232) that both cAMP and cAMP-dependent cell contact are required for prespore differentiation in Dictyostelium discoideum. The present study was undertaken to examine changes of the plasma membrane proteins during prespore differentiation in the shake culture system. Rabbit antibodies prepared against the plasma membrane fraction of the differentiated cells inhibited the reaggregation of the differentiated cells but not that of aggregation-competent cells. This result indicates that new contact sites are formed in the differentiated cells. By the combined use of the antibody-conjugated immuno-adsorbent with sodium dodecyl sulfate-polyacrylamide gel electrophoresis, changes of membrane proteins were analyzed with the cells incubated under various conditions. Three proteins were found to be present specifically in the differentiated cells only in the presence of cAMP, one of which (105K protein) appeared when cells became adhesive, but before prespore specific proteins were detected. Two others (80K and 58K proteins) appeared during prespore differentiation after cells formed agglomerates.     

cAMP regulation of cell differentiation in Dictyostelium discoideum and the role of the cAMP receptor

DNA polymerases and DNA ligases have been studied during development of the amphibian, axolotl. Three forms of DNA polymerase, I, II, and III, with sedimentation coefficients in sucrose of 9, 6, and 3.1 S, respectively, have been found in the axolotl egg. The activity of these three DNA polymerases is unchanged during early embryonic development. The activity of DNA polymerase III then increases significantly, beginning at the tailbud stage, while the activity of DNA polymerase II increases at the larval stage. DNA polymerase I does not show significant variations during this time. On the basis of their catalytic properties, it appears that DNA polymerases I and II are α-type DNA polymerases whereas DNA polymerase III is a β-type enzyme. Two different DNA ligases are found in the axolotl, one showing a sedimentation coefficient in sucrose of 8.2 S (heavy form) and the other, 6 S (light form). The 6 S enzyme is the major DNA ligase activity found in the egg before and after fertilization. Its activity then decreases during embryonic development. It can be observed again, as the only DNA ligase activity, in some adult tissues. The 8.2 S enzyme appears during the first division cycle of the fertilized egg, is present at all stages of embryonic development, and is absent from the adult tissues tested. Properties of the two DNA ligases at different stages of embryonic development have also been compared.     

Characterization of a cAMP-stimulated cAMP phosphodiesterase in Dictyostelium discoideum

A cyclic nucleotide phosphodiesterase, PdeE, that harbors two cyclic nucleotide binding motifs and a binuclear Zn(2+)-binding domain was characterized in Dictyostelium. In other eukaryotes, the Dictyostelium domain shows greatest homology to the 73-kDa subunit of the pre-mRNA cleavage and polyadenylation specificity factor. The Dictyostelium PdeE gene is expressed at its highest levels during aggregation, and its disruption causes the loss of a cAMP-phosphodiesterase activity. The pdeE null mutants show a normal cAMP-induced cGMP response and a 1.5-fold increase of cAMP-induced cAMP relay. Overexpression of a PdeE-yellow fluorescent protein (YFP) fusion construct causes inhibition of aggregation and loss of the cAMP relay response, but the cells can aggregate in synergy with wild-type cells. The PdeE-YFP fusion protein was partially purified by immunoprecipitation and biochemically characterized. PdeE and its Dictyostelium ortholog, PdeD, are both maximally active at pH 7.0. Both enzymes require bivalent cations for activity. The common cofactors Zn(2+) and Mg(2+) activated PdeE and PdeD maximally at 10 mm, whereas Mn(2+) activated the enzymes to 4-fold higher levels, with half-maximal activation between 10 and 100 microm. PdeE is an allosteric enzyme, which is approximately 4-fold activated by cAMP, with half-maximal activation occurring at about 10 microm and an apparent K(m) of approximately 1 mm. cGMP is degraded at a 6-fold lower rate than cAMP. Neither cGMP nor 8-Br-cAMP are efficient activators of PdeE activity.     

Spore coat proteins of Dictyostelium discoideum are packaged in prespore vesicles

Previous studies have shown that Dictyostelium discoideum spore coat proteins are found in prespore cells, which are localized to the posterior region of migrating slugs, and in the coats of mature spores. Prespore vesicles, identified by morphology and by staining with anti-D. mucoroides spore serum, are also localized in the posterior region of migrating slugs. Using antisera specific to the spore coat proteins, we show that the spore coat proteins are packaged in prespore vesicles. They are present in the vesicles as a complex which can be dissociated by denaturation. The anti-D. mucoroides spore serum reacts with at least five proteins in whole spore extracts including the spore coat proteins SP96 and SP70.     

Analysis of a novel diacylglycerol kinase from Dictyostelium discoideum: DGKA

Diacylglycerol kinases (DGKs) catalyze the ATP-dependent phosphorylation of diacylglycerols to generate phosphatidic acid and have been investigated in prokaryotic and eukaryotic organisms. Recently, a protein that is significantly similar to human DGK-theta, DGKA, was identified in Dictyostelium discoideum. It has been shown to possess DGK activity when assayed using a medium-chain diacylglycerol, 1,2-dioctanoyl-sn-glycerol (DiC8). A complete understanding of DGK catalytic and regulatory mechanisms, as well as physiological roles, requires an understanding of its biochemical and kinetic properties. This report presents an analysis of these properties for DGKA. The enzyme catalyzes the phosphorylation of DiC8, and another medium-chain DAG, DiC6 (1,2-dihexanoyl-sn-glycerol), in a Michaelis-Menten manner. Interestingly, the kinetics of DGKA using physiologically relevant long-chain DAGs was dependent on substrate surface concentration and the detergent that was used. DGKA displayed Michaelis-Menten kinetics with respect to bulk substrate concentration (1,2-dioleoyl-sn-glycerol) in octyl glucoside mixed micelles when the surface substrate concentration was at or below 3.5 mol %. At higher surface concentrations, however, there was a sigmoidal relationship between the initial velocity and bulk substrate concentration. In contrast, DGKA displayed sigmoidal kinetics with respect to bulk substrate concentrations at all surface concentrations in Triton X-100 mixed micelles. Finally, we show the catalytic activity of DGKA was significantly enhanced by phosphatidylserine (PS) and phosphatidic acid (PA).     

DC6, a novel type of Dictyostelium discoideum gene regulated by secreted factors but not by cAMP

We have isolated a gene, DC6, which is induced in the early aggregative stages of development in Dictyostelium discoideum. The increase in DC6 expression is dependent on high cell density, indicating that cellular interactions are required for DC6 induction. In low-cell-density cultures, the induction of DC6 occurs if supplied with conditioned medium of developing cells, suggesting that secreted factors are involved in DC6 induction. The expression of DC6 is not affected (1) in the presence of caffeine or adenosine, which block the production or the action of cAMP pulses, (2) in the presence of high concentrations of cAMP, or (3) in mutant strains (Synag7 and FrigidA), which are defective in transduction pathways of cAMP pulse signals. These results indicate that the induction of DC6 does not require extracellular cAMP pulse signals, which are known to regulate the expression of many genes in the early development. Independence of cAMP signals and dependence on other unknown cellular interactions are prominent characteristics of DC6.     

Structure, expression, and inactivation by gene disruption of the Dictyostelium discoideum prespore gene EB4

We present the nucleotide sequence of the cell type specific prespore gene EB4 which encodes a protein containing a hydrophobic leader sequence and two distinct domains of amino acid repeats. By RNase protection experiments we have determined the genomic organization of the gene which differs substantially from the previously published data. An antibody directed against one of the repeat structures (hexamer repeat) specifically reacts with a developmentally regulated antigen of 58 kd. Gene disruption transformants were obtained by transformation with a genomic DNA fragment. The transformants express a 3' truncated mRNA and do not react with the anti-hexamer antibody. So far, we could not detect any phenotypic aberrations in the transformed cell line.     

Continuous requirement of cAMP for pre-spore differentiation in Dictyostelium discoideum

Abstract Using a shaking culture system, we have previously shown that both cell contact and cAMP are required for pre-spore differentiation in Dictyostelium discoideum [2]. In the present study, cAMP was removed from the medium by the use of a hydrolysing enzyme after cells had formed agglomerates. This treatment left the agglomerates unchanged, but caused a rapid decrease in the activity of UDP galactose transferase, a pre-spore-specific enzyme. This result indicates that cAMP is required even after agglomerate formation to maintain pre-spore differentiation.     

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.     

Stalk cell formation in monolayers from isolated prestalk and prespore cells of Dictyostelium discoideum: evidence for two populations of prestalk cells

Cells from the pseudoplasmodial stage of Dictyostelium discoideum differentiation were dispersed and separated on Percoll gradients into prestalk and prespore cells. The requirements for stalk cell formation in low-density monolayers from the two cell types were determined. The isolated prespore cells required both the Differentiation Inducing Factor (DIF) and cyclic AMP for stalk cell formation. In contrast, only part of the isolated prestalk cell population required both cyclic AMP and DIF, the remainder requiring DIF alone, suggesting the possibility that there were two populations of prestalk cells, one independent of cyclic AMP and one dependent on cyclic AMP for stalk cell formation. The finding that part of the prestalk cell population required only a brief incubation in the presence of DIF to induce stalk cell formation, whilst the remainder required a considerably longer incubation in the presence of both DIF and cyclic AMP was consistent with this idea. In addition, stalk cell formation from cyclic-AMP-dependent prestalk cells was relatively more sensitive to caffeine inhibition than stalk cell formation from cyclic-AMP-independent prestalk cells. The latter cells were enriched in the most anterior portion of the migrating pseudoplasmodium, indicating that there is spatial segregation of the two prestalk cell populations. The conversion of prespore cells to stalk cells took longer and was more sensitive to caffeine when compared to stalk cell formation from cyclic-AMP-dependent prestalk cells.