Pharmacological Evidence That Dictyostelium Differentiation-Inducing Factor 1 Promotes Glucose Uptake Partly via an Increase in Intracellular cAMP Content in Mouse 3T3-L1 Cells.

Differentiation-inducing factor 1 (DIF-1) isolated from the cellular slime mold Dictyostelium discoideum can inhibit mammalian calmodulin-dependent cAMP/cGMP phosphodiesterase (PDE1) in vitro. DIF-1 also promotes glucose uptake, at least in part, via a mitochondria- and AMPK-dependent pathway in mouse 3T3-L1 fibroblast cells, but the mechanism underlying this effect has not been fully elucidated. In this study, we investigated the effects of DIF-1 on intracellular cAMP and cGMP levels, as well as the effects that DIF-1 and several compounds that increase cAMP and cGMP levels have on glucose uptake in confluent 3T3-L1 cells. DIF-1 at 20 µM (a concentration that promotes glucose uptake) increased the level of intracellular cAMP by about 20% but did not affect the level of intracellular cGMP. Neither the PDE1 inhibitor 8-methoxymethyl-3-isobutyl-1-methylxanthine at 10-200 µM nor the broad-range PDE inhibitor 3-isobutyl-1-methylxanthine at 40-400 µM had any marked effects on glucose uptake. The membrane-permeable cAMP analog 8-bromo-cAMP at 200-1000 µM significantly promoted glucose uptake (by 20-25%), whereas the membrane-permeable cGMP analog 8-bromo-cGMP at 3-100 µM did not affect glucose uptake. The adenylate cyclase activator forskolin at 1-10 µM promoted glucose uptake by 20-30%. Thus, DIF-1 may promote glucose uptake by 3T3-L1 cells, at least in part, via an increase in intracellular cAMP level.

Derivatives of Differentiation-Inducing Factor 1 Differentially Control Chemotaxis and Stalk Cell Differentiation in Dictyostelium discoideum.

Differentiation-inducing factors 1 and 2 (DIF-1 and DIF-2) are small lipophilic signal molecules that induce stalk cell differentiation but differentially modulate chemotaxis toward cAMP in the cellular slime mold Dictyostelium discoideum; DIF-1 suppresses chemotactic cell movement in shallow cAMP gradients, whereas DIF-2 promotes it. The receptor(s) for DIF-1 and DIF-2 have not yet been identified. We examined the effects of nine derivatives of DIF-1 on chemotactic cell movement toward cAMP and compared their chemotaxis-modulating activity and stalk cell differentiation-inducing activity in wild-type and mutant strains. The DIF derivatives differentially affected chemotaxis and stalk cell differentiation; for example, TM-DIF-1 suppressed chemotaxis and showed poor stalk-inducing activity, DIF-1(3M) suppressed chemotaxis and showed strong stalk-inducing activity, and TH-DIF-1 promoted chemotaxis. These results suggest that DIF-1 and DIF-2 have at least three receptors: one for stalk cell induction and two for chemotaxis modulation. In addition, our results show that the DIF derivatives can be used to analyze the DIF-signaling pathways in D. discoideum.

Deletion of gmfA induces keratocyte-like migration in Dictyostelium.

Glia maturation factor (GMF) has been established as an inactivating factor of the actin-related protein 2/3 (Arp2/3) complex, which regulates actin assembly. Regulation of actin assembly and reorganization is crucial for various cellular events, such as cell migration, cell division, and development. Here, to examine the roles of ADF-H domain-containing protein (also known as glia maturation factor; GmfA), the product of a single GMF homologous gene in Dictyostelium, gmfA-null cells were generated. They had moderate defects in cell growth and cytokinesis. Interestingly, they showed a keratocyte-like fan shape with a broader pseudopod, where Arp3 accumulated at higher levels than in wild-type cells. They migrated with higher persistence, but their velocities were comparable to those of wild-type cells. The polar pseudopods during cell division were also broader than those in wild-type cells. However, GmfA did not localize at the pseudopods in migrating cells or the polar pseudopods in dividing cells. Adhesions of mutant cells to the substratum were much stronger than that of wild-type cells. Although the mutant cells showed chemotaxis comparable to that of wild-type cells, they formed disconnected streams during the aggregation stage; however, they finally formed normal fruiting bodies. These results suggest that GmfA plays a crucial role in cell migration.

Mg, K-containing microparticle: A possible active principle of a culture extract produced by a microbial consortium.

A synthetic microbial consortium called Effective Microorganisms (EM) consists mainly of photosynthetic bacteria, lactic acid bacteria and yeast. Various effects of EM∙XGOLD, a health drink produced by EM, on life cycle of Dictyostelium discoideum were described previously. Here, we report our attempt to identify the active principle, termed EMF, that brought about the observed effects. Throughout the purification processes, the presence of the active principle was monitored by promoted fruiting body formation. By liquid-liquid separation the activity was recovered in aqueous phase, which, after concentration, was further subjected to reverse-phase column chromatography. No activity was detected in any eluant, while almost all the activity was recovered in residual insoluble material. The application of conventional organic chemistry procedures to the residual fraction did not lead to any informative results. Acid treatment of the insoluble material produced air bubbles, suggesting it to be composed of some inorganic carbonate. Viewed under scanning electronmicroscope, the residue revealed spherical particles of µm size range. Energy Dispersive X-ray (EDX) Spectroscopy pointed to the existence, on the surface of the particles, of magnesium and, to a certain extent, of potassium. In separate experiments, acid treatment and alkali neutralization of EM∙XGOLD completely wiped out the stimulatory activity of fruiting body formation. These lines of evidence indicate these Mg, K-containing microparticles to be an active principle of EM culture extract. How these particles exert their effect is currently under intensive investigation.

Peptide nucleic acid as a template for Taq DNA polymerase.

Peptide nucleic acid (PNA), an artificial DNA analog, comprises a purine or pyrimidine base and a pseudo-peptide backbone instead of deoxyribose-phosphate. PNA has been found to have stronger adhesion and higher stability in binding to its complementary DNA than deoxyribose-phosphate. Thus, it could serve as an agent for gene modulation, demonstrating potential in antisense therapy, molecular diagnostics, and nanotechnology. However, the applications of PNA remain limited because its biological activities are not fully known. Here, I demonstrate that a thermostable DNA polymerase, Thermus aquaticus (Taq) polymerase, exhibits transcriptase activity when a PNA oligomer is used as a template and that genetic information of the oligomer can be amplified by PCR using DNA primers. Furthermore, the insertion of a glutamine peptide stretch in the middle part of the PNA template did not interfere with transcription; it was transcribed into a guanosine or adenosine stretch. Intriguingly, this amino acid-to-DNA transcription did not occur when glycine residues were inserted. A synthetic PNA oligomer can, therefore, function as a template for a DNA polymerase, and polyglutamine peptides can be transcribed into guanosine or adenosine. These findings provide a cornerstone to reveal all amino acid genetic codes and transcription activity in the future.

Polar pattern formation induced by contact following locomotion in a multicellular system.

Biophysical mechanisms underlying collective cell migration of eukaryotic cells have been studied extensively in recent years. One mechanism that induces cells to correlate their motions is contact inhibition of locomotion, by which cells migrating away from the contact site. Here, we report that tail-following behavior at the contact site, termed contact following locomotion (CFL), can induce a non-trivial collective behavior in migrating cells. We show the emergence of a traveling band showing polar order in a mutant Dictyostelium cell that lacks chemotactic activity. We find that CFL is the cell-cell interaction underlying this phenomenon, enabling a theoretical description of how this traveling band forms. We further show that the polar order phase consists of subpopulations that exhibit characteristic transversal motions with respect to the direction of band propagation. These findings describe a novel mechanism of collective cell migration involving cell-cell interactions capable of inducing traveling band with polar order.

The cells of animals and many other living things are able to migrate together in groups. This collective cell migration plays crucial roles in many processes in animals such as forming organs and limbs, and healing wounds. A soil-dwelling amoeba called Dictyostelium discoideum ­ or just Dicty for short ­ is commonly used as a model to study how groups of cells migrate collectively. Individual Dicty cells may live alone but sometimes many cells come together to form a larger mobile structure called a "slug". Chemical signals coordinate how the cells collectively migrate to form the multicellular slug. Mutant Dicty cells that lack these chemical signal processes can still move together as a band that travels across a surface. This movement resembles a type of collective motion that has previously been observed in physics experiments using self-propelled particles. However, it remains unclear how this collective behavior works. Hayakawa et al. have now combined genetics, cell biology and computational approaches to study how groups of the mutant Dicty cells migrate together. The experiments showed that the traveling band is dynamically maintained by cells joining or leaving, and that this turnover is caused by simple interactions between the cells known as "contact following locomotion". Contact following locomotion has been also reported in mammalian cells so the findings of Hayakawa et al. may aid research into how animals develop and how errors in cell migration may lead to diseases. Further studies are required to find out whether other cells showing contact following locomotion also travel in a band.

The Life Cycle of Dictyostelium discoideum Is Accelerated via MAP Kinase Cascade by a Culture Extract Produced by a Synthetic Microbial Consortium.

A cellular slime mold, Dictyostelium discoideum, is an amoeboid organism that has a unique life cycle consisting of distinctly separated vegetative and developmental phases. Thus, this organism presents a rare opportunity in which to examine the effects of bioactive substances on separate cellular activities. In this research, we investigated the effect of a culture extract, termed EMXG, produced by a synthetic microbial consortium. EMXG promoted proliferative response of amoeba cells. It further accelerated the developmental phase, leading to the preferred fruiting body formation from fewer cells. Furthermore, EMXG modulated biological rhythm of this organism, that is, interval of oscillation of cAMP level observed in suspension starvation was significantly shortened. Concomitantly, the level of ERKB, a MAP kinase, was found to oscillate in a similar fashion to that of cAMP. Additionally, ErkB-deficient mutant amoeboid cells did not respond to proliferative stimulation by EMXG. These lines of evidence point to a likelihood that MAP kinase cascade is involved and further that ErkB could be the molecular target of EMXG.

Some chemotactic mutants can be progress through development in chimeric populations.

Cell migration in response to morphogen gradients affects morphogenesis. Chemotaxis towards adenosine 3', 5'-monophosphate (cAMP) is essential for the early stage of morphogenesis in the slime mold Dictyostelium discoideum. Here, we show that D. discoideum completes morphogenesis without cAMP-chemotaxis-dependent cell migration. The extracellular cAMP gradient is believed to cause cells to form a slug-shaped multicellular structure and fruiting body. The cAMP receptor, cAR1, was not expressed at the cell surface during these stages, correlating with reduced chemotactic activity. Gß-null cells expressing temperature sensitive Gß are unable to generate extracellular cAMP (Jin et al., 1998) and thus unable to aggregate and exhibit proper morphogenesis under restrictive temperature. However, when mixed with wild type cells ts-Gß expressing gß-null cells normally aggregated and exhibited normal morphogenesis under restrictive temperature. Furthermore, cells migrated after aggregation in a mixture containing wild-type cells. KI-5 cells, which do not show aggregation or morphogenesis, spontaneously migrated to a transplanted wild-type tip and underwent normal morphogenesis and cell differentiation; this was not observed in cells lacking tgrB1and tgrC1 cells adhesion molecules. Thus, cAMP gradient-dependent cell migration may not be required for multicellular pattern formation in late Dictyostelium development.

Chemoattractant receptors activate, recruit and capture G proteins for wide range chemotaxis.

The wide range sensing of extracellular signals is a common feature of various sensory cells. Eukaryotic chemotactic cells driven by GPCRs and their cognate G proteins are one example. This system endows the cells directional motility towards their destination over long distances. There are several mechanisms to achieve the long dynamic range, including negative regulation of the receptors upon ligand interaction and spatial regulation of G proteins, as we found recently. However, these mechanisms are insufficient to explain the 105-fold range of chemotaxis seen in Dictyostelium. Here, we reveal that the receptor-mediated activation, recruitment, and capturing of G proteins mediate chemotactic signaling at the lower, middle and higher concentration ranges, respectively. These multiple mechanisms of G protein dynamics can successfully cover distinct ranges of ligand concentrations, resulting in seamless and broad chemotaxis. Furthermore, single-molecule imaging analysis showed that the activated Gα subunit forms an unconventional complex with the agonist-bound receptor. This complex formation of GPCR-Gα increased the membrane-binding time of individual Gα molecules and therefore resulted in the local accumulation of Gα. Our findings provide an additional chemotactic dynamic range mechanism in which multiple G protein dynamics positively contribute to the production of gradient information.

Evidence that differentiation-inducing factor-1 controls chemotaxis and cell differentiation, at least in part, via mitochondria in D. discoideum.

Differentiation-inducing factor-1 [1-(3,5-dichloro-2,6-dihydroxy-4-methoxyphenyl)hexan-1-one (DIF-1)] is an important regulator of cell differentiation and chemotaxis in the development of the cellular slime mold Dictyostelium discoideum However, the entire signaling pathways downstream of DIF-1 remain to be elucidated. To characterize DIF-1 and its potential receptor(s), we synthesized two fluorescent derivatives of DIF-1, boron-dipyrromethene (BODIPY)-conjugated DIF-1 (DIF-1-BODIPY) and nitrobenzoxadiazole (NBD)-conjugated DIF-1 (DIF-1-NBD), and investigated their biological activities and cellular localization. DIF-1-BODIPY (5 µM) and DIF-1 (2 nM) induced stalk cell differentiation in the DIF-deficient strain HM44 in the presence of cyclic adenosine monosphosphate (cAMP), whereas DIF-1-NBD (5 µM) hardly induced stalk cell differentiation under the same conditions. Microscopic analyses revealed that the biologically active derivative, DIF-1-BODIPY, was incorporated by stalk cells at late stages of differentiation and was localized to mitochondria. The mitochondrial uncouplers carbonyl cyanide m-chlorophenylhydrazone (CCCP), at 25-50 nM, and dinitrophenol (DNP), at 2.5-5 µM, induced partial stalk cell differentiation in HM44 in the presence of cAMP. DIF-1-BODIPY (1-2 µM) and DIF-1 (10 nM), as well as CCCP and DNP, suppressed chemotaxis in the wild-type strain Ax2 in shallow cAMP gradients. These results suggest that DIF-1-BODIPY and DIF-1 induce stalk cell differentiation and modulate chemotaxis, at least in part, by disturbing mitochondrial activity.

Two HAP2-GCS1 homologs responsible for gamete interactions in the cellular slime mold with multiple mating types: Implication for common mechanisms of sexual reproduction shared by plants and protozoa and for male-female differentiation.

Fertilization is a central event in sexual reproduction, and understanding its molecular mechanisms has both basic and applicative biological importance. Recent studies have uncovered the molecules that mediate this process in a variety of organisms, making it intriguing to consider conservation and evolution of the mechanisms of sexual reproduction across phyla. The social amoeba Dictyostelium discoideum undergoes sexual maturation and forms gametes under dark and humid conditions. It exhibits three mating types, type-I, -II, and -III, for the heterothallic mating system. Based on proteome analyses of the gamete membranes, we detected expression of two homologs of the plant fertilization protein HAP2-GCS1. When their coding genes were disrupted in type-I and type-II strains, sexual potency was completely lost, whereas disruption in the type-III strain did not affect mating behavior, suggesting that the latter acts as female in complex organisms. Our results demonstrate the highly conserved function of HAP2-GCS1 in gamete interactions and suggest the presence of additional allo-recognition mechanisms in D. discoideum gametes.

Differentiation-inducing factor 2 modulates chemotaxis via the histidine kinase DhkC-dependent pathway in Dictyostelium discoideum.

Differentiation-inducing factor 1(DIF-1) and DIF-2 are signaling molecules that control chemotaxis in Dictyostelium discoideum. Whereas DIF-1 suppresses chemotaxis in shallow cAMP gradients, DIF-2 enhances chemotaxis under the same conditions via a phosphodiesterase, response regulator A (RegA), which is a part of the DhkC-RdeA-RegA two-component signaling system. In this study, to investigate the mechanism of the chemotaxis regulation by DIF-2, we examined the effects of DIF-2 (and DIF-1) on chemotaxis in rdeA(-) and dhkC(-) mutant strains. In the parental wild-type strains, chemotactic cell movement was suppressed with DIF-1 and enhanced with DIF-2 in shallow cAMP gradients. In contrast, in both rdeA(-) and dhkC(-) strains, chemotaxis was suppressed with DIF-1 but unaffected by DIF-2. The results suggest that DIF-2 modulates chemotaxis via the DhkC-RdeA-RegA signaling system.

Glutathione S-transferase 4 is a putative DIF-binding protein that regulates the size of fruiting bodies in Dictyostelium discoideum.

In the development of the cellular slime mold Dictyostelium discoideum, two chlorinated compounds, the differentiation-inducing factors DIF-1 and DIF-2, play important roles in the regulation of both cell differentiation and chemotactic cell movement. However, the receptors of DIFs and the components of DIF signaling systems have not previously been elucidated. To identify the receptors for DIF-1 and DIF-2, we here performed DIF-conjugated affinity gel chromatography and liquid chromatography-tandem mass spectrometry and identified the glutathione S-transferase GST4 as a major DIF-binding protein. Knockout and overexpression mutants of gst4 (gst4- and gst4OE, respectively) formed fruiting bodies, but the fruiting bodies of gst4- cells were smaller than those of wild-type Ax2 cells, and those of gst4OE cells were larger than those of Ax2 cells. Both chemotaxis regulation and in vitro stalk cell formation by DIFs in the gst4 mutants were similar to those of Ax2 cells. These results suggest that GST4 is a DIF-binding protein that regulates the sizes of cell aggregates and fruiting bodies in D. discoideum.

Root of Dictyostelia based on 213 universal proteins.

Dictyostelia are common soil microbes that can aggregate when starved to form multicellular fruiting bodies, a characteristic that has also led to their long history of study and widespread use as model systems. Ribosomal RNA phylogeny of Dictyostelia identified four major divisions (Groups 1-4), none of which correspond to traditional genera. Group 1 was also tentatively identified as sister lineage to the other three Groups, although not consistently or with strong support. We tested the dictyostelid root using universal protein-coding genes identified by exhaustive comparison of six completely sequenced dictyostelid genomes, which include representatives of all four major molecular Groups. A set of 213 genes are low-copy number in all genomes, present in at least one amoebozoan outgroup taxon (Acanthamoeba castellanii or Physarum polycephalum), and phylogenetically congruent. Phylogenetic analysis of a concatenation of the deduced protein sequences produces a single topology dividing Dictyostelia into two major divisions: Groups 1+2 and Groups 3+4. All clades in the tree are fully supported by maximum likelihood and Bayesian inference, and all alternative roots are unambiguously rejected by the approximately unbiased (AU) test. The 1+2, 3+4 root is also fully supported even after deleting clusters with strong individual support for this root, or concatenating all clusters with low support for alternative roots. The 213 putatively ancestral amoebozoan proteins encode a wide variety of functions including 21 KOG categories out of a total of 25. These comprehensive analyses and consistent results indicate that it is time for full taxonomic revision of Dictyostelia, which will also enable more effective exploitation of its unique potential as an evolutionary model system.

Comparative genome and transcriptome analyses of the social amoeba Acytostelium subglobosum that accomplishes multicellular development without germ-soma differentiation.

BACKGROUND: Social amoebae are lower eukaryotes that inhabit the soil. They are characterized by the construction of a starvation-induced multicellular fruiting body with a spore ball and supportive stalk. In most species, the stalk is filled with motile stalk cells, as represented by the model organism Dictyostelium discoideum, whose developmental mechanisms have been well characterized. However, in the genus Acytostelium, the stalk is acellular and all aggregated cells become spores. Phylogenetic analyses have shown that it is not an ancestral genus but has lost the ability to undergo cell differentiation. RESULTS: We performed genome and transcriptome analyses of Acytostelium subglobosum and compared our findings to other available dictyostelid genome data. Although A. subglobosum adopts a qualitatively different developmental program from other dictyostelids, its gene repertoire was largely conserved. Yet, families of polyketide synthase and extracellular matrix proteins have not expanded and a serine protease and ABC transporter B family gene, tagA, and a few other developmental genes are missing in the A. subglobosum lineage. Temporal gene expression patterns are astonishingly dissimilar from those of D. discoideum, and only a limited fraction of the ortholog pairs shared the same expression patterns, so that some signaling cascades for development seem to be disabled in A. subglobosum. CONCLUSIONS: The absence of the ability to undergo cell differentiation in Acytostelium is accompanied by a small change in coding potential and extensive alterations in gene expression patterns.

Cross-species functional complementation of cellulose synthase during the development of cellular slime molds.

Cellulose is a major and important component of the extracellular matrix during the development of Dictyostelium discoideum. Upon starvation, solitary amoebae of D. discoideum gather and form fruiting bodies in which cells differentiate into stalk cells and spores. The stalk tubes and walls of spores and stalk cells are made of cellulose. In the genus Acytostelium, however, all cells are destined to become spores and the stalks comprise only a cellulose tube, suggesting species-specific regulation of cellulose synthesis. In this study, we cloned a putative cellulose synthase gene (cesA) of Acytostelium subglobosum and performed comparative analyses with the D. discoideum cellulose synthase gene (dcsA). Although the deduced amino acid sequences were highly conserved between cesA and dcsA, the numbers of transmembrane spans preceding the catalytic domain were dissimilar; 2 and 3, respectively. Since ectopic expression of cesA in dcsA(-) null cells failed to restore the developmental defects of the mutant, we constructed a series of chimerical genes for complementation analyses and found that the catalytic domain of cesA was functional in D. discoideum cells if the preceding transmembrane region was swapped with dcsA. The non-functional products that contained the cesA-derived transmembrane region were localized to lysosomes. These results indicate that the transmembrane region of cellulose synthase is essential for proper accumulation of cellulose during the development of D. discoideum and that its differential localization in A. subglobosum may be related to the characteristic morphogenesis in this species.

A RabGAP regulates life-cycle duration via trimeric G-protein cascades in Dictyostelium discoideum.

BACKGROUND: The life-cycle of cellular slime molds comprises chronobiologically regulated processes. During the growth phase, the amoeboid cells proliferate at a definite rate. Upon starvation, they synthesize cAMP as both first and second messengers in signalling pathways and form aggregates, migrating slugs, and fruiting bodies, consisting of spores and stalk cells, within 24 h. In Dictyostelium discoideum, because most growth-specific events cease during development, proliferative and heterochronic mutations are not considered to be interrelated and no genetic factor governing the entire life-cycle duration has ever been identified. METHODOLOGY/PRINCIPAL FINDINGS: Using yeast 2-hybrid library screening, we isolated a Dictyostelium discoideum RabGAP, Dd Rbg-3, as a candidate molecule by which the Dictyostelium Gα2 subunit directs its effects. Rab GTPase-activating protein, RabGAP, acts as a negative regulator of Rab small GTPases, which orchestrate the intracellular membrane trafficking involved in cell proliferation. Deletion mutants of Dd rbg-3 exhibited an increased growth rate and a shortened developmental period, while an overexpression mutant demonstrated the opposite effects. We also show that Dd Rbg-3 interacts with 2 Gα subunits in an activity-dependent manner in vitro. Furthermore, both human and Caenorhabditis elegans rbg-3 homologs complemented the Dd rbg-3-deletion phenotype in D. discoideum, indicating that similar pathways may be generally conserved in multicellular organisms. CONCLUSIONS/SIGNIFICANCE: Our findings suggest that Dd Rbg-3 acts as a key element regulating the duration of D. discoideum life-span potentially via trimeric G-protein cascades.

Biological soliton in multicellular movement.

Solitons have been observed in various physical phenomena. Here, we show that the distinct characteristics of solitons are present in the mass cell movement of non-chemotactic mutants of the cellular slime mould Dictyostelium discoideum. During starvation, D. discoideum forms multicellular structures that differentiate into spore or stalk cells and, eventually, a fruiting body. Non-chemotactic mutant cells do not form multicellular structures; however, they do undergo mass cell movement in the form of a pulsatile soliton-like structure (SLS). We also found that SLS induction is mediated by adhesive cell-cell interactions. These observations provide novel insights into the mechanisms of biological solitons in multicellular movement.

Temporal and non-permanent division of labor during sorocarp formation in the social amoeba Acytostelium subglobosum.

Somatic cell differentiation is crucial for the development of multicellular organisms. While the development of a fruiting body in Dictyostelium discoideum represents a simple model of this process with separation of stalk cells from the spore lineage, that of Acytostelium subglobosum is not accompanied by cell type separation. This species produces acellular stalks and, seemingly, all aggregated amoebae become spores; however, it possesses homologs for the stalk-cell marker genes of D. discoideum. In this study, we analyzed the spatio-temporal expression of A. subglobosum orthologs for D. discoideum stalk- or spore-lineage markers to clarify the developmental process of A. subglobosum. We first found that the prespore vesicles, which contained spore coat proteins, started to accumulate in the tip region and were observed in the entire sorogen throughout later development, confirming that all A. subglobosum cells became spores. The expression of a stalk-lineage gene ortholog, As-ecmA, started at the mound stage and was prominent in the protruding sorogen. Although two spore-lineage gene orthologs, As-cotD1 and -cotD2, were likewise detected shortly after cell aggregation and increased in intensity until tip formation, their expression diminished in the protruding sorogen. Double-fluorescence staining of these prestalk and prespore marker genes revealed that the expression of these marker genes was mutually exclusive and that expression switching occurred in the early tip. Our results indicate that A. subglobosum cells become committed to the spore lineage first, and then, while keeping this commitment intact, participate in stalk formation. Instead of the permanent division of labor observed in D. discoideum, A. subglobosum produces fruiting bodies by all cells contributing to the formation of the stalk as well as forming spores.

Arachidonic acid enhances caffeine-induced cell death via caspase-independent cell death.

Caffeine is a globally consumed psychostimulant but can be fatal to cells at overdose exposures. Although caspase-dependent apoptosis plays a role in caffeine-induced cell death, the responsible intracellular signalling cascade remains incompletely understood. The cellular slime mould, Dictyostelium discoideum, does not possess caspase-dependent apoptotic machinery. Here, we observed that ablation of D. discoideumplaA, which encodes a phospholipase A2 (PLA2) homolog, leads to a decreased rate of cell death under high caffeine concentrations and to enhanced cell death with the addition of arachidonic acid. Moreover, the inhibition of PLA2 activity lead to a recovery of the survival rate in caspase-inhibited Hela cervical carcinoma cells under high caffeine concentrations, indicating that caffeine-induced cell death is enhanced via PLA2-dependent signalling. Our results indicate that arachidonic acid may be a general second messenger that negatively regulates caffeine tolerance via a caspase-independent cell death cascade, which leads to multiple effects in eukaryotic cells.