Periodic activation of ERK2 and partial involvement of G protein in ERK2 activation by cAMP in Dictyostelium cells.

Intercellular signaling mediated by cAMP plays a pivotal role in coordinating cell movement into aggregates at the early stage of Dictyostelium development when the extracellular level of cAMP periodically changes at 6- to 7-min intervals. We have shown that MAP kinase ERK2 is activated via the cAMP receptor CAR1 in phase with this periodic change. This was revealed by assessing the level of ERK2 activation with immunoblots using two kinds of antibodies, commercially available anti-phospho-p44/p42 MAP-kinase antibody and anti-Dictyostelium ERK2 antibody. In this chapter, we describe the methods we have used to assess the level of activated ERK2 and partial involvement of G protein in cAMP-induced ERK2 activation.

Novel patterns of the gene expression regulation in the prestalk region along the antero-posterior axis during multicellular development of Dictyostelium.

Simultaneous hybridization with differentially labeled fluorescent probes for in situ hybridization analysis revealed several novel expression patterns of prestalk genes during multicellular development of Dictyostelium. Seven prestalk genes and one prespore gene (pspA) were analyzed in this study. The patterns identified here indicate that prestalk cells are more heterogeneous than previously thought. Heterogeneity was observed in peripheral prestalk tissues such as the pstAO domain of a slug and the prestalk region surrounding a stalk tube of a culminant. Heterogeneity was also observed in the core pstAB cells of the slug and immature stalk cells within the stalk tube. The upper- and lower-cups of a late culminant were also composed of several subdomains.

Evidence that the Dictyostelium STAT protein Dd-STATa plays a role in the differentiation of inner basal disc cells and identification of a promoter element essential for expression in these cells.

Dd-STATa, a Dictyostelium homolog of the metazoan STAT (signal transducers and activators of transcription) proteins, is necessary in the slug for correct entry into culmination. Dd-STATa-null mutant fails to culminate and its phenotype correlates with the loss of a funnel-shaped core region, the pstAB core region, which expresses both the ecmA and ecmB genes. To understand how the differentiation of pstAB core cells is regulated, we identified an EST that is expressed in the core cells of normal slugs but down-regulated in the Dd-STATa-null mutant. This EST, SSK348, encodes a close homolog of the Dictyostelium acetyl-CoA synthetase (ACS). A promoter fragment of the cognate gene, aslA (acetyl-CoA synthetase-like A), was fused to a lacZ reporter and the expression pattern determined. As expected from the behavior of the endogenous aslA gene, the aslA::lacZ fusion gene is not expressed in Dd-STATa-null slugs. In parental cells, the aslA promoter is first activated in the funnel-shaped core cells located at the slug anterior, the "pstAB core." During culmination, the pstAB core cells move down, through the prespore cells, to form the inner part of the basal disc. As the spore mass climbs the stalk, the aslA gene comes to be expressed in cells of the upper and lower cups, structures that cradle the spore head. Deletion and point mutation analyses of the promoter identified an AT-rich sequence that is necessary for expression in the pstAB core. This acts in combination with repressor regions that prevent ectopic aslA expression in the pre-stalk regions of slugs and the stalks of culminants. Thus, this study confirms that Dd-STATa is necessary for the differentiation of pstAB core cells, by showing that it is needed for the activation of the aslA gene. It also identifies aslA promoter elements that are likely to be regulated, directly or indirectly, by Dd-STATa.

Identification of new modes of Dd-STATa regulation of gene expression in Dictyostelium by in situ hybridisation.

Signal Transducers and Activators of Transcription (STATs) are transcription factors which lie at the end of cytokine and growth signal transduction pathways. Dictyostelium Dd-STATa is a functional homologue of metazoan STATs. It is activated by cAMP and, at the slug stage, it translocates into the nuclei of the tip cells, which are a subset of the anterior, prestalk A (pstA) cells. Here we searched for novel Dd-STATa regulated genes by in situ hybridisation. A set of 54 cDNA clones whose gene expression patterns are known to be prestalk-specific (Maeda et al., 2003), were chosen as probes and we compared their expression patterns in parental and Dd-STATa-null strains. We identified 13 genes which are candidates for direct induction by Dd-STATa. In the parental strain, most of these genes are expressed in the cone shaped mass of pstAB cells which is located within the prestalk region. These cDNAs show little or no expression in the Dd-STATa-null strain. This contrasts markedly with the paradigmatic ecmB gene which is expressed in pstAB cells in parental cells, but which is expressed throughout the prestalk zone in the Dd-STATa-null strain. We also identified several genes which are normally expressed in pstA cells, or throughout the prestalk region, but whose expression is markedly down-regulated in the null mutant. Again, this contrasts with markers derived from the paradigmatic, ecmA gene which are expressed normally in the Dd-STATa-null strain. The identification of these novel genes provides valuable tools to investigate the role of Dd-STATa.

Control of cell type proportioning in Dictyostelium discoideum by differentiation-inducing factor as determined by in situ hybridization.

We have determined the proportions of the prespore and prestalk regions in Dictyostelium discoideum slugs by in situ hybridization with a large number of prespore- and prestalk-specific genes. Microarrays were used to discover genes expressed in a cell type-specific manner. Fifty-four prespore-specific genes were verified by in situ hybridization, including 18 that had been previously shown to be cell type specific. The 36 new genes more than doubles the number of available prespore markers. At the slug stage, the prespore genes hybridized to cells uniformly in the posterior 80% of wild-type slugs but hybridized to the posterior 90% of slugs lacking the secreted alkylphenone differentiation-inducing factor 1 (DIF-1). There was a compensatory twofold decrease in prestalk cells in DIF-less slugs. Removal of prespore cells resulted in cell type conversion in both wild-type and DIF-less anterior fragments. Thus, DIF-1 appears to act in concert with other processes to establish cell type proportions.

Novel development rescuing factors (DRFs) secreted by the developing Dictyostelium cells, that are involved in the restoration of a mutant lacking MAP-kinase ERK2.

We found novel development rescuing factors (DRFs) secreted from developing Dictyostelium cells, by using a mutant (erkB-) which is missing MAP-kinase ERK2 as a test strain for bioassay. The mutant erkB- fails to undergo multicellular morphogenesis due to impaired cAMP signaling. However, such developmental defect can be restored by the presence of low-molecular weight DRFs that are secreted from developing wild-type cells. We previously showed that DIF-1 (Differentiation-Inducing Factor 1 for stalk cells) possesses this activity, indicating a newly discovered role of DIF-1. Surprisingly, however, the mutant dmtA-, which is incapable of DIF-1 synthesis still exerts a strong inducing activity of the multicellular morphogenesis of erkB-. After analysis of HPLC fractions of conditioned media prepared from both wild type Ax2 and dmtA- strains revealed that both strains secrete at least two novel DRF activities with DIF-like mobility. However, these activities were not derived from other DIFs such as DIF-2 and DIF-3. Identification of these DRFs found in this study would provide insight into the mechanism by which the development of the erkB- mutant is restored and how these factors act in the normal development of Dictyostelium.

Extracellular matrix family proteins that are potential targets of Dd-STATa in Dictyostelium discoideum.

Dd-STATa is a functional Dictyostelium homologue of metazoan STAT (signal transducers and activators of transcription) proteins, which is activated by cAMP and is thereby translocated into the nuclei of anterior tip cells of the prestalk region of the slug. By using in situ hybridization analyses, we found that the SLF308 cDNA clone, which contains the ecmF gene that encodes a putative extracellular matrix protein and is expressed in the anterior tip cells, was greatly down-regulated in the Dd-STATa-null mutant. Disruption of the ecmF gene, however, resulted in almost no phenotypic change. The absence of any obvious mutant phenotype in the ecmF-null mutant could be due to a redundancy of similar genes. In fact, a search of the Dictyostelium whole genome database demonstrates the existence of an additional 16 homologues, all of which contain a cellulose-binding module. Among these homologues, four genes show Dd-STATa-dependent expression, while the others are Dd-STATa-independent. We discuss the potential role of Dd-STATa in morphogenesis via its effect on the interaction between cellulose and these extracellular matrix family proteins.

Periodic signaling controlled by an oscillatory circuit that includes protein kinases ERK2 and PKA.

Self-regulating systems often use robust oscillatory circuits. One such system controls the chemotactic signaling mechanism of Dictyostelium, where pulses of adenosine 3',5'-monophosphate (cAMP) are generated with a periodicity of 7 minutes. We have observed spontaneous oscillations in activation of the mitogen-activated protein (MAP) kinase ERK2 that occur in phase with peaks of cAMP, and we show that ERK2 modulates cAMP levels through the phosphodiesterase RegA. Computer modeling and simulations of the underlying circuit faithfully account for the ability of the cells to spontaneously generate periodic pulses during specific stages of development. Similar oscillatory processes may occur in cells of many different species.

Defect in peroxisomal multifunctional enzyme MFE1 affects cAMP relay in Dictyostelium.

We have previously reported that cells of Dictyostelium discoideum lacking the fatty acid oxidation enzyme MFE1 accumulate excess cyclopropane fatty acids from ingested bacteria. Cells in which mfeA(-) is disrupted fail to develop when grown in association with bacteria but form normal fruiting bodies when grown in axenic media. Bacterially grown mfeA(-) cells express the genes for the cyclic AMP (cAMP) receptor (carA) and adenylyl cyclase (acaA) but fail to respond to a cAMP pulse by synthesis of additional cAMP which normally relays the signal. Moreover, they do not accumulate the adhesion protein, gp80, which is encoded by the cAMP-induced gene, csaA. As a consequence, they do not acquire developmentally regulated EDTA-resistant cell-cell adhesion. When mutant cells are mixed with wild-type cells and allowed to develop together, they co-aggregate and differentiate into both spores and stalk cells. Thus, most of the developmental consequences of excess cyclopropane fatty acids appear to result from impaired cAMP relay.

Analyses of cDNAs from growth and slug stages of Dictyostelium discoideum.

Dictyostelium is a favored model for studying problems in cell and developmental biology. To comprehend the genetic potential and networks that direct growth and multicellular development, we are performing a large-scale analysis of Dictyostelium cDNAs. Here, we newly determine 7720 nucleotide sequences of cDNAs from the multicellular, slug stage (S) and 10 439 from the unicellular, vegetative stage (V). The combined 26 954 redundant ESTs were computer assembled using the PHRAP program to yield 5381 independent sequences. These 5381 predicted genes represent about half of the estimated coding potential of the organism. One-third of them were classified into 12 functional categories. Although the overall classification patterns of the V and S libraries were very similar, stage-specific genes exist in every category. The majority of V-specific genes function in some aspect of protein translation, while such genes are in a minority in the S-specific and common populations. Instead, genes for signal transduction and multicellular organization are enriched in the population of S-specific genes. Genes encoding the enzymes of basic metabolism are mainly found in the common gene population. These results therefore suggest major differences between growing and developing Dictyostelium cells in the nature of the genes transcribed.

A neutral ceramidase homologue from Dictyostelium discoideum exhibits an acidic pH optimum.

The nucleotide sequence reported for the Dictyostelium discoideum ceramidase is available on the DNA Data Bank of Japan (DDBJ). Ceramidases (CDases) are currently classified into three categories (acid, neutral and alkaline) based on their optimal pHs and primary structures. Here, we report the first exception to this rule. We cloned the CDase cDNA, consisting of 2142 nucleotides encoding 714 amino-acid residues, from the slime mould, Dictyostelium discoideum. The putative amino-acid sequence indicates 32-42% identity with various neutral CDases, but does not show any similarity to the acid and alkaline CDases, indicating the enzyme should be classified as a neutral CDase. However, overexpression of the cDNA in D. discoideum resulted in increased CDase activity at an acidic, but not a neutral pH range. Knockout of the gene in slime mould eliminated CDase activity at acidic pH. The recombinant enzyme expressed in the slime mould was purified and then characterized. Consequently, the purified CDase was found to exhibit the maximal activity at approx. pH 3.0. The singular pH dependency of slime mould CDase is not derived from the specific post-translational modification in the slime mould, because the enzyme showed an acidic pH optimum even when expressed in Chinese hamster ovary cells, whereas rat neutral-CDase exhibited a neutral pH optimum when expressed in slime mould.

A novel gene trap method using terminator-REMI and 3' rapid amplification of cDNA ends (RACE) in Dictyostelium.

We describe a novel restriction enzyme-mediated integration (REMI) method for gene trapping in Dictyostelium based on the use of a terminator-deficient vector. The vector has a blasticidin deaminase (bsr) gene as a selectable marker but lacks a terminator containing a poly(A) addition signal (AATAAA). Thus, the vector was expected to integrate into the coding region of a gene to create a fusion transcript flanked by the 3' proximal region of the trapped gene. The trapped gene can be identified by simply amplifying the fusion transcript by 3' rapid amplification of cDNA ends (3'-RACE). In the analysis of 35 integration events into known genes, the vectors were found to be integrated 20 times in close proximity to the 3' ends of the genes and in the direction of transcription. This strictly localized insertion seemed to be mediated by negative selection via the surveillance system referred to nonsense-mediated mRNA decay. In contrast, in 15 events the vector integrated in the opposite direction to transcription and at random positions throughout the coding sequence. Analysis of the trapped 3' sequences showed that the transcription of the fusion gene terminated prematurely without the apparent use of an endogenous terminator; nevertheless the transcript did exhibit a poly(A) tail. Based on these results, we designated the method terminator-REMI. Using this method, we have generated a library of tagged Dictyostelium clones from which we have thus far isolated 242 developmental mutants.

Changing patterns of gene expression in dictyostelium prestalk cell subtypes recognized by in situ hybridization with genes from microarray analyses.

We used microarrays carrying most of the genes that are developmentally regulated in Dictyostelium to discover those that are preferentially expressed in prestalk cells. Prestalk cells are localized at the front of slugs and play crucial roles in morphogenesis and slug migration. Using whole-mount in situ hybridization, we were able to verify 104 prestalk genes. Three of these were found to be expressed only in cells at the very front of slugs, the PstA cell type. Another 10 genes were found to be expressed in the small number of cells that form a central core at the anterior, the PstAB cell type. The rest of the prestalk-specific genes are expressed in PstO cells, which are found immediately posterior to PstA cells but anterior to 80% of the slug that consists of prespore cells. Half of these are also expressed in PstA cells. At later stages of development, the patterns of expression of a considerable number of these prestalk genes changes significantly, allowing us to further subdivide them. Some are expressed at much higher levels during culmination, while others are repressed. These results demonstrate the extremely dynamic nature of cell-type-specific expression in Dictyostelium and further define the changing physiology of the cell types. One of the signals that affect gene expression in PstO cells is the hexaphenone DIF-1. We found that expression of about half of the PstO-specific genes were affected in a mutant that is unable to synthesize DIF-1, while the rest appeared to be DIF independent. These results indicate that differentiation of some aspects of PstO cells can occur in the absence of DIF-1.

MFE1, a member of the peroxisomal hydroxyacyl coenzyme A dehydrogenase family, affects fatty acid metabolism necessary for morphogenesis in Dictyostelium spp.

Beta-oxidation of long-chain fatty acids and branched-chain fatty acids is carried out in mammalian peroxisomes by a multifunctional enzyme (MFE) or D-bifunctional protein, with separate domains for hydroxyacyl coenzyme A (CoA) dehydrogenase, enoyl-CoA hydratase, and steroid carrier protein SCP2. We have found that Dictyostelium has a gene, mfeA, encoding MFE1 with homology to the hydroxyacyl-CoA dehydrogenase and SCP2 domains. A separate gene, mfeB, encodes MFE2 with homology to the enoyl-CoA hydratase domain. When grown on a diet of bacteria, Dictyostelium cells in which mfeA is disrupted accumulate excess cyclopropane fatty acids and are unable to develop beyond early aggregation. Axenically grown mutant cells, however, developed into normal fruiting bodies composed of spores and stalk cells. Comparative analysis of whole-cell lipid compositions revealed that bacterially grown mutant cells accumulated cyclopropane fatty acids that remained throughout the developmental stages. Such a persistent accumulation was not detected in wild-type cells or axenically grown mutant cells. Bacterial phosphatidylethanolamine that contains abundant cyclopropane fatty acids inhibited the development of even axenically grown mutant cells, while dipalmitoyl phosphatidylethanolamine did not. These results suggest that MFE1 protects the cells from the increase of the harmful xenobiotic fatty acids incorporated from their diets and optimizes cellular lipid composition for proper development. Hence, we propose that this enzyme plays an irreplaceable role in the survival strategy of Dictyostelium cells to form spores for their efficient dispersal in nature.

A STAT-regulated, stress-induced signalling pathway in Dictyostelium.

The Dictyostelium stalk cell inducer differentiation-inducing factor (DIF) directs tyrosine phosphorylation and nuclear accumulation of the STAT (signal transducer and activator of transcription) protein Dd-STATc. We show that hyperosmotic stress, heat shock and oxidative stress also activate Dd-STATc. Hyperosmotic stress is known to elevate intracellular cGMP and cAMP levels, and the membrane-permeant analogue 8-bromo-cGMP rapidly activates Dd-STATc, whereas 8-bromo-cAMP is a much less effective inducer. Surprisingly, however, Dd-STATc remains stress activatable in null mutants for components of the known cGMP-mediated and cAMP-mediated stress-response pathways and in a double mutant affecting both pathways. Also, Dd-STATc null cells are not abnormally sensitive to hyperosmotic stress. Microarray analysis identified two genes, gapA and rtoA, that are induced by hyperosmotic stress. Osmotic stress induction of gapA and rtoA is entirely dependent on Dd-STATc. Neither gene is inducible by DIF but both are rapidly inducible with 8-bromo-cGMP. Again, 8-bromo-cAMP is a much less potent inducer than 8-bromo-cGMP. These data show that Dd-STATc functions as a transcriptional activator in a stress-response pathway and the pharmacological evidence, at least, is consistent with cGMP acting as a second messenger.

DIF-1, an anti-tumor substance found in Dictyostelium discoideum, inhibits progesterone-induced oocyte maturation in Xenopus laevis.

Differentiation-inducing factor-1 (DIF-1; 1-(3,5-dichloro-2,6-dihydroxy-4-methoxyphenyl)hexan-1-one) is a putative morphogen that induces stalk-cell formation in the cellular slime mold Dictyostelium discoideum. DIF-1 has previously been shown to suppress cell growth in mammalian cells. In this study, we examined the effects of DIF-1 on the progesterone-induced germinal vesicle breakdown in Xenopus laevis, which is thought to be mediated by a decrease in intracellular cAMP and the subsequent activation of mitogen-activated protein kinase (MAPK) and maturation-promoting factor, a complex of cdc2 and cyclin B, which regulates germinal vesicle breakdown. DIF-1 at 10-40 microM inhibited progesterone-induced germinal vesicle breakdown in de-folliculated oocytes in a dose-dependent manner. Progesterone-induced cdc2 activation, MAPK activation, and c-Mos accumulation were inhibited by DIF-1. Furthermore, DIF-1 was found to inhibit the progesterone-induced cAMP decrease in the oocytes. These results indicate that DIF-1 inhibits progesterone-induced germinal vesicle breakdown possibly by blocking the progesterone-induced decrease in [cAMP](i) and the subsequent events in Xenopus oocytes.

Identification and characterization of novel calcium-binding proteins of Dictyostelium and their spatial expression patterns during development.

Five putative Ca2(+)-binding proteins, CBP5, 6, 7, 8 and 9, all having EF-hand motifs, were found by searching the Dictyostelium cDNA database (http://www.csm.biol.tsukuba.ac.jp/cDNAproject.html). 45Ca2(+)-overlay experiments revealed that four of these (excluding CBP9) are real Ca2(+)-binding proteins. Northern blot analysis revealed that the genes encoding CBP5, 6, 7 and 8 are all developmentally regulated. In situ hybridization analyses revealed that spatial expression of these genes was regulated in several different ways. CBP1, 2, 3, 5, 6 and 7 are expressed in prespore cells in the slug stage. Transcripts of the genes for CBP1 and 5 are enriched in prestalk subtype PstO cells. In contrast, CBP4 is expressed predominantly in PstO cells. CBP8 is evenly expressed at a very low level throughout the whole slug. Such distinct spatial expression patterns suggest that the CBP might be involved in morphogenesis and might have their own roles either in prespore or in prestalk cell differentiation of Dictyostelium.

A transcriptional profile of multicellular development in Dictyostelium discoideum.

A distinct feature of development in the simple eukaryote Dictyostelium discoideum is an aggregative transition from a unicellular to a multicellular phase. Using genome-wide transcriptional analysis we show that this transition is accompanied by a dramatic change in the expression of more than 25% of the genes in the genome. We also show that the transcription patterns of these genes are not sensitive to the strain or the nutritional history, indicating that Dictyostelium development is a robust physiological process that is accompanied by stereotypical transcriptional events. Analysis of the two differentiated cell types, spores and stalk cells, and their precursors revealed a large number of differentially expressed genes as well as unexpected patterns of gene expression, which shed new light on the timing and possible mechanisms of cell-type divergence. Our findings provide new perspectives on the complexity of the developmental program and the fraction of the genome that is regulated during development.

Protein Phosphorylation during the Process of Prestalk-to-Stalk Conversion in Dictyostelium discoideum: (Dictyostelium discoideum/8-bromo cyclic AMP/stalk differentiation/protein kinase A/protein kinase inhibitor).

We have previously shown that 8-bromo cAMP (Br-cAMP) efficiently induces prestalk-to-stalk conversion in in vitro cultures of the cellular slime mold Dictyostelium discoideum, and that protein kinase A (PKA) plays an important role(s) in the conversion process (Kubohara et al. Exp. Cell Res. vol. 207, pp. 107-114, 1993). In the present study, we analyzed protein phosphorylation during the Br-cAMP-induced process, and identified two phosphoproteins, p60 and p40, that were specifically induced (phosphorylated) by Br-cAMP. The protein kinase inhibitors, staurosporine and K252a, which blocked the Br-cAMP-induced stalk cell formation, also inhibited the phosphorylations of these proteins. These results suggest that p60 and p40 may be the target proteins of PKA and thus be involved in the prestalk-to-stalk conversion.

Efficient Induction of Sporulation of Dictyostelium Prespore Cells by 8-Bromocyclic AMP under Both Submerged- and Shaken-Culture Conditions and Involvement of Protein Kinase(s) in Its Action.

It is important to establish an experimental system in which sporulation of Dictyostelium can be induced at high cell densities to obtain sufficient amounts of materials for analysis of the molecular events leading to sporulation. 8-Bromo cAMP (Br-cAMP) was found to be effective for inducing sporulation by prespore cells of Dictyostelium discoideum NC4 at high cell densities under both submerged- and shaken-culture conditions. Ultrastructural studies revealed that the morphological changes associated with this sporulation proceeded normally in vitro. The effect of Br-cAMP was inhibited by two protein kinase inhibitors, K252a and staurosporine. Protein-phosphorylation experiments showed that Br-cAMP induced increased phosphorylations of a 96 kDa spore coat protein (SP96) and a protein with a mobility corresponding to a molecular weight of 50 kDa (p50-4). The protein kinase inhibitor K252a blocked the phosphorylations of both proteins. These proteins may be targets of particular protein kinase(s) that is activated by Br-cAMP. These findings indicate that the present experimental system should be useful for elucidating the molecular events involved in normal sporulation and the mechanism by which Br-cAMP induces sporulation in vitro.