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.     

A Dictyostelium SH2 adaptor protein required for correct DIF-1 signaling and pattern formation

Dictyostelium is the only non-metazoan with functionally analyzed SH2 domains and studying them can give insights into their evolution and wider potential. LrrB has a novel domain configuration with leucine-rich repeat, 14-3-3 and SH2 protein–protein interaction modules. It is required for the correct expression of several specific genes in early development and here we characterize its role in later, multicellular development. During development in the light, slug formation in LrrB null (lrrB-) mutants is delayed relative to the parental strain, and the slugs are highly defective in phototaxis and thermotaxis. In the dark the mutant arrests development as an elongated mound, in a hitherto unreported process we term dark stalling. The developmental and phototaxis defects are cell autonomous and marker analysis shows that the pstO prestalk sub-region of the slug is aberrant in the lrrB- mutant. Expression profiling, by parallel micro-array and deep RNA sequence analyses, reveals many other alterations in prestalk-specific gene expression in lrrB- slugs, including reduced expression of the ecmB gene and elevated expression of ampA. During culmination ampA is ectopically expressed in the stalk, there is no expression of ampA and ecmB in the lower cup and the mutant fruiting bodies lack a basal disc. The basal disc cup derives from the pstB cells and this population is greatly reduced in the lrrB- mutant. This anatomical feature is a hallmark of mutants aberrant in signaling by DIF-1, the polyketide that induces prestalk and stalk cell differentiation. In a DIF-1 induction assay the lrrB- mutant is profoundly defective in ecmB activation but only marginally defective in ecmA induction. Thus the mutation partially uncouples these two inductive events. In early development LrrB interacts physically and functionally with CldA, another SH2 domain containing protein. However, the CldA null mutant does not phenocopy the lrrB- in its aberrant multicellular development or phototaxis defect, implying that the early and late functions of LrrB are affected in different ways. These observations, coupled with its domain structure, suggest that LrrB is an SH2 adaptor protein active in diverse developmental signaling pathways.     

Dictyopyrones, novel alpha-pyronoids isolated from Dictyostelium spp., promote stalk cell differentiation in Dictyostelium discoideum

Dictyopyrones A and B (DpnA and B), whose function(s) is not known, were isolated from fruiting bodies of Dictyostelium discoideum. In the present study, to assess their function(s), we examined the effects of Dpns on in vitro cell differentiation in D. discoideum monolayer cultures with cAMP. Dpns at 1-20 microM promoted stalk cell formation to some extent in the wild-type strain V12M2. Although Dpns by themselves could hardly induce stalk cell formation in a differentiation-inducing factor (DIF)-deficient strain HM44, both of them dose-dependently promoted DIF-1-dependent stalk cell formation in the strain. In the sporogenous strain HM18, Dpns at 1-20 microM suppressed spore formation and promoted stalk cell formation in a dose-dependent manner. Analogs of Dpns were less effective in affecting cell differentiation in both HM44 and HM18 cells, indicating that the activity of Dpns should be chemical structure specific. It was also shown that DpnA at 2-20 microM dose-dependently suppressed spore formation induced with 8-bromo cAMP and promoted stalk cell formation in V12M2 cells. Interestingly, it was shown by the use of RT-PCR that DpnA at 10 microM slightly promoted both prespore- and prestalk-specific gene expressions in an early phase of V12M2 and HM18 in vitro differentiation. The present results suggest that Dpns may have functions (1) to promote both prespore and prestalk cell differentiation in an early stage of development and (2) to suppress spore formation and promote stalk cell formation in a later stage of development in D. discoideum.     

The novel ankyrin-repeat containing kinase ARCK-1 acts as a suppressor of the Spalten signaling pathway during Dictyostelium development

Spalten (Spn), a member of the PP2C family of Ser/Thr protein phosphatases, is required for Dictyostelium cell-type differentiation and morphogenesis. We have identified a new protein kinase, ARCK-1, through a second site suppressor screen for mutants that allow spn null cells to proceed further through development. ARCK-1 has a C-terminal kinase domain most closely related to Ser/Thr protein kinases and an N-terminal putative regulatory domain with ankyrin repeats, a 14-3-3 binding domain, and a C1 domain, which is required for binding to RasB^GTP in a two-hybrid assay. Disruption of the gene encoding ARCK-1 results in weak, late developmental defects. However, overexpression of ARCK-1 phenocopies the spn null phenotype, consistent with Spn and ARCK-1 being on the same developmental pathway. Our previous analyses of Spn and the present analysis of ARCK-1 suggest a model in which Spn and ARCK-1 differentially control the phosphorylation state of a protein that regulates cell-type differentiation. Dephosphorylation of the substrate by Spn is required for cell-type differentiation. Control of ARCK-1 and Spn activities by upstream signals is proposed to be part of the developmental regulatory program mediating cell-fate decisions in Dictyostelium.     

Functional analysis of a novel gene, DD3-3, from Dictyostelium discoideum

Mutations in MYOC gene encoding myocilin are responsible for primary open-angle glaucoma (POAG). In order to search for protein(s) that can interact with myocilin, we screened a human skeletal muscle cDNA library using yeast two-hybrid system and identified flotillin-1, a structural protein of lipid raft that is detergent-resistant and a liquid ordered microdomain, as a protein interacting with myocilin. The interaction was confirmed by in vitro glutathione S-transferase pulldown and in vivo co-immunoprecipitation studies. In yeast two-hybrid assay, the C-terminus of myocilin, an olfactomedin-like domain in which most mutations related to POAG are scattered, was found to be necessary and sufficient for the interaction. However, myocilins with mutations such as G364V, K423E, and Y437H on the domain failed to interact with flotillin-1. Although the physiological significance of the interaction has yet to be elucidated, our results showed that the alteration of the interaction by mutations in MYOC might be a key factor of the pathogenesis of POAG.     

Dictyostelium cell death: early emergence and demise of highly polarized paddle cells

Cell death in the stalk of Dictyostelium discoideum, a prototypic vacuolar cell death, can be studied in vitro using cells differentiating as a monolayer. To identify early events, we examined potentially dying cells at a time when the classical signs of Dictyostelium cell death, such as heavy vacuolization and membrane lesions, were not yet apparent. We observed that most cells proceeded through a stereotyped series of differentiation stages, including the emergence of "paddle" cells showing high motility and strikingly marked subcellular compartmentalization with actin segregation. Paddle cell emergence and subsequent demise with paddle-to-round cell transition may be critical to the cell death process, as they were contemporary with irreversibility assessed through time-lapse videos and clonogenicity tests. Paddle cell demise was not related to formation of the cellulose shell because cells where the cellulose-synthase gene had been inactivated underwent death indistinguishable from that of parental cells. A major subcellular alteration at the paddle-to-round cell transition was the disappearance of F-actin. The Dictyostelium vacuolar cell death pathway thus does not require cellulose synthesis and includes early actin rearrangements (F-actin segregation, then depolymerization), contemporary with irreversibility, corresponding to the emergence and demise of highly polarized paddle cells.     

SadA,a novel adhesion receptor in Dictyostelium

Little is known about cell-substrate adhesion and how motile and adhesive forces work together in moving cells. The ability to rapidly screen a large number of insertional mutants prompted us to perform a genetic screen in Dictyostelium to isolate adhesion-deficient mutants. The resulting substrate adhesion-deficient (sad) mutants grew in plastic dishes without attaching to the substrate. The cells were often larger than their wild-type parents and displayed a rough surface with many apparent blebs. One of these mutants, sadA-, completely lacked substrate adhesion in growth medium. The sadA- mutant also showed slightly impaired cytokinesis, an aberrant F-actin organization, and a phagocytosis defect. Deletion of the sadA gene by homologous recombination recreated the original mutant phenotype. Expression of sadA-GFP in sadA-null cells restored the wild-type phenotype. In sadA-GFP-rescued mutant cells, sadA-GFP localized to the cell surface, appropriate for an adhesion molecule. SadA contains nine putative transmembrane domains and three conserved EGF-like repeats in a predicted extracellular domain. The EGF repeats are similar to corresponding regions in proteins known to be involved in adhesion, such as tenascins and integrins. Our data combined suggest that sadA is the first substrate adhesion receptor to be identified in Dictyostelium.     

An immediate-early gene, srsA: its involvement in the starvation response that initiates differentiation of Dictyostelium cells

When nutrients are depleted, Dictyostelium cells undergo cell cycle arrest and initiate a differentiation program for survival. We have found a novel gene, srsA, which is rapidly expressed in the first 5 min following the removal of nutrients and is turned off within an hour. This gene encodes a small protein with no significant similarity to previously characterized proteins. Disruption of srsA results in delayed expression of the early genes acaA and carA that encode adenylyl cyclase and the cAMP receptor necessary for chemotactic aggregation, respectively. Streaming is delayed several hours and the aggregates are larger than normal in the mutant strains. These phenotypes are cell-autonomous. Overexpression of srsA also results in delayed aggregation. Some of the slugs of the srsA(OE) strains showed stalked migration reminiscent of the slugs of the related species Dictyostelium mucoroides. The terminal structures formed by srsA(OE) cells were grossly abnormal and contained very few viable spores. When cells overexpressing srsA were developed together with an excess of wild-type cells, the fruiting bodies were still abnormal, indicating that the mutant cells have a dominant effect on late development. These findings suggest that srsA may be involved in both the starvation response and late differentiation.     

Ethylene induces zygote formation through an enhanced expression of zyg1 in Dictyostelium mucoroides

We have previously demonstrated that a potent plant hormone, ethylene induces sexual development including zygote formation in Dictyostelium cells, and that a novel gene (zyg1) is also involved in zygote formation. Based on these findings, the present work was mainly designed to reveal (1) the precise relationship between the ethylene amount and zygote formation, and (2) the relation of in situ ethylene synthesis to zyg1 expression, using transformants that over- or under-produce ACC-oxidase (Dd-aco) involved in ethylene biosynthesis. ACO(OE) cells overexpressing Dd-aco gene overproduced ethylene and exhibited the augmented zygote formation. In contrast, ACO-RNAi cells, in which the expression of Dd-aco was suppressed by the RNAi method, showed a reduced level of ethylene production, thus resulting in inhibition of zygote formation. Importantly, the expression of zyg1 was affected by the amount of ethylene produced: Zyg1 expression was augmented in ACO(OE) cells, but was significantly suppressed in ACO-RNAi cells. In another experiment, we found that 1-methylcyclopropene (1-MCP), which is known to inhibit the function of ethylene by binding specifically to ethylene receptors, greatly suppresses zygote formation. These results indicate that ethylene is capable of inducing zygote formation through the expression of zyg1.     

Tortoise, a novel mitochondrial protein, is required for directional responses of Dictyostelium in chemotactic gradients

We have identified a novel gene, Tortoise (TorA), that is required for the efficient chemotaxis of Dictyostelium discoideum cells. Cells lacking TorA sense chemoattractant gradients as indicated by the presence of periodic waves of cell shape changes and the localized translocation of cytosolic PH domains to the membrane. However, they are unable to migrate directionally up spatial gradients of cAMP. Cells lacking Mek1 display a similar phenotype. Overexpression of Mek1 in torA- partially restores chemotaxis, whereas overexpression of TorA in mek1- does not rescue the chemotactic phenotype. Regardless of the genetic background, TorA overexpressing cells stop growing when separated from a substrate. Surprisingly, TorA-green fluorescent protein (GFP) is clustered near one end of mitochondria. Deletion analysis of the TorA protein reveals distinct regions for chemotactic function, mitochondrial localization, and the formation of clusters. TorA is associated with a round structure within the mitochondrion that shows enhanced staining with the mitochondrial dye Mitotracker. Cells overexpressing TorA contain many more of these structures than do wild-type cells. These TorA-containing structures resist extraction with Triton X-100, which dissolves the mitochondria. The characterization of TorA demonstrates an unexpected link between mitochondrial function, the chemotactic response, and the capacity to grow in suspension.     

Anti-leukemic activities of Dictyostelium secondary metabolites: a novel aromatic metabolite, 4-methyl-5-n-pentylbenzene-1,3-diol, isolated from Dictyostelium mucoroides suppresses cell growth in human leukemia K562 and HL-60 cells

It has previously been shown that DIF-1, a differentiation-inducing factor of the cellular slime mold Dictyostelium discoideum, possesses antitumor activities in mammalian tumor cells and that neuronal differentiation of PC12 cells can be induced with furanodictines (FDs), aminosugar analogs found in D. discoideum, or dictyoglucosamines (DGs), N-acetyl glucosamine derivatives (DG-A from D. purpureum and DG-B from D. discoideum). Thus, cellular slime molds are attractive natural resources that may provide valuable lead compounds to be utilized in the field of pharmacology and medicine. In this study, we have isolated a novel aromatic compound, 4-methyl-5-n-pentylbenzene-1,3-diol (MPBD), from fruiting bodies of the cellular slime mold D. mucoroides and assessed the in vitro antiproliferative activities of MPBD, FDs, and DGs in human leukemia K562 and HL-60 cells. MPBD at 20-80 microM dose-dependently suppressed cell growth in both K562 and HL-60 cells. While FDs at 10-80 microM did not affect cell growth, DGs at 10-40 microM dose-dependently suppressed cell growth in the cells. Although we failed to find the roles of FDs and DGs in the original organisms, MPBD at 5-20 microM was found to promote stalk cell formation in D. discoideum. The present results indicate that MPBD, DGs or their derivatives may have therapeutic potential in the treatment of cancer and confirm our expectations regarding cellular slime molds as drug resources.     

Tetrahydropteridines possess antioxidant roles to guard against glucose-induced oxidative stress in Dictyostelium discoideum

Glucose effects on the vegetative growth of Dictyostelium discoideum Ax2 were studied by examining oxidative stress and tetrahydropteridine synthesis in cells cultured with different concentrations (0.5X, 7.7 g L^-1; 1X, 15.4 g L^-1; 2X, 30.8 g L^-1) of glucose. The growth rate was optimal in 1X cells (cells grown in 1X glucose) but was impaired drastically in 2X cells, below the level of 0.5X cells. There were glucose-dependent increases in reactive oxygen species (ROS) levels and mitochondrial dysfunction in parallel with the mRNA copy numbers of the enzymes catalyzing tetrahydropteridine synthesis and regeneration. On the other hand, both the specific activities of the enzymes and tetrahydropteridine levels in 2X cells were lower than those in 1X cells, but were higher than those in 0.5X cells. Given the antioxidant function of tetrahydropteridines and both the beneficial and harmful effects of ROS, the results suggest glucose-induced oxidative stress in Dictyostelium, a process that might originate from aerobic glycolysis, as well as a protective role of tetrahydropteridines against this stress. [BMB Reports 2013; 46(2): 86-91]     

Involvement of the TRAP-1 homologue, Dd-TRAP1, in spore differentiation during Dictyostelium development

Tumor necrosis factor receptor-associated protein 1 (TRAP1) is a member of the molecular chaperone HSP90 (90-kDa heat shock protein) family. We have previously demonstrated that Dictyostelium discoideum TRAP1 (Dd-TRAP1) synthesized at the vegetative growth phase is retained during the whole course of D. discoideum development, and that at the multicellular slug stage, it is located in prespore-specific vacuoles (PSVs) of prespore cells as well as in the cell membrane and mitochondria. Thereupon, we examined the function of Dd-TRAP1 in prepore and spore differentiation, using Dd-TRAP1-knockdown cells (TRAP1-RNAi cells) produced by the RNA interference method. As was expected, Dd-TRAP1 contained in the PSV was found to be exocytosed during sporulation to constitute the outer-most layer of the spore cell wall. In the TRAP1-RNAi cells, PSV formation and therefore prespore differentiation were significantly impaired, particularly under a heat stress condition. Although the TRAP1-RNAi cells formed apparently normal-shaped spores with a cellulosic wall, the spores were less resistant to heat and detergent treatments, as compared with those of parental MB35 cells derived from Ax-2 cells. These findings strongly suggest that Dd-TRAP1 may be closely involved in late development including spore differentiation, as well as in early development as realized by its induction of prestarvation response.     

Phospholipase D controls Dictyostelium development by regulating G protein signaling

Dictyostelium discoideum cells normally exist as individual amoebae, but will enter a period of multicellular development upon starvation. The initial stages of development involve the aggregation of individual cells, using cAMP as a chemoattractant. Chemotaxis is initiated when cAMP binds to its receptor, cAR1, and activates the associated G protein, Gα2βγ. However, chemotaxis will not occur unless there is a high density of starving cells present, as measured by high levels of the secreted quorum sensing molecule, CMF. We previously demonstrated that cells lacking PldB bypass the need for CMF and can aggregate at low cell density, whereas cells overexpressing pldB do not aggregate even at high cell density. Here, we found that PldB controlled both cAMP chemotaxis and cell sorting. PldB was also required by CMF to regulate G protein signaling. Specifically, CMF used PldB, to regulate the dissociation of Gα2 from Gβγ. Using fluorescence resonance energy transfer (FRET), we found that along with cAMP, CMF increased the dissociation of the G protein. In fact, CMF augmented the dissociation induced by cAMP. This augmentation was lost in cells lacking PldB. PldB appears to mediate the CMF signal through the production of phosphatidic acid, as exogenously added phosphatidic acid phenocopies overexpression of pldB. These results suggest that phospholipase D activity is required for CMF to alter the kinetics of cAMP-induced G protein signaling.     

Prespore-to-stalk conversion involves the production of a pathway-specific glycoprotein, wst25, in the cellular slime mould Dictyostelium discoideum

We have previously identified a stalk-specific wheat germ agglutinin (WGA)-binding protein, wst34, in the cellular slime mould Dictyostelium discoideum [Biochem. Cell Biol. 68 (1990) 699]. Here, we found another stalk-specific WGA-binding protein, wst25, which was detected with two antisera that recognize wst34. Using the two marker proteins, we then analyzed and compared the pathways of prestalk-to-stalk maturation and prespore-to-stalk conversion in vitro and in vivo. Prestalk cells isolated from normally formed slugs can be converted to stalk cells (designated StI) in vitro with 8-bromo-cAMP (Br-cAMP), whereas prespore cells isolated from slugs can be converted to fully vacuolated stalk cells (designated StII) in vitro with Br-cAMP and DIF-1. During the process of prespore-to-stalk conversion, prespore-specific mRNAs, D19 and 2H3, disappeared rapidly, while prestalk-specific mRNAs, ecmA and ecmB, appeared at 2h of incubation and increased thereafter. Most importantly, however, the StII cells thus formed were biochemically different from the StI cells originated from prestalk cells; that is, StI cells expressed wst34 but not wst25, while StII cells expressed wst25 but not wst34. When prespore cells isolated from slugs were allowed to develop on a substratum, they differentiated into spores and stalk cells and formed fruiting bodies, and the stalk cells formed from prespore cells in vivo expressed wst25 but not wst34. The present results indicate that there are two types of stalk cells, StI (prestalk-origin) and StII (prespore-origin), and that wst34 and wst25 are the specific markers for StI and StII, respectively.     

Naringenin is a novel inhibitor of Dictyostelium cell proliferation and cell migration

Naringenin is a flavanone compound that alters critical cellular processes such as cell multiplication, glucose uptake, and mitochondrial activity. In this study, we used the social amoeba, Dictyostelium discoideum, as a model system for examining the cellular processes and signaling pathways affected by naringenin. We found that naringenin inhibited Dictyostelium cell division in a dose-dependent manner (IC(50) approximately 20 microM). Assays of Dictyostelium chemotaxis and multicellular development revealed that naringenin possesses a previously unrecognized ability to suppress amoeboid cell motility. We also found that naringenin, which is known to inhibit phosphatidylinositol 3-kinase activity, had no apparent effect on phosphatidylinositol 3,4,5-trisphosphate synthesis in live Dictyostelium cells; suggesting that this compound suppresses cell growth and migration via alternative signaling pathways. In another context, the discoveries described here highlight the value of using the Dictyostelium model system for identifying and characterizing the mechanisms by which naringenin, and related compounds, exert their effects on eukaryotic cells.     

Phenotypic plasticity of Escherichia coli at initial stage of symbiosis with Dictyostelium discoideum

We observed the change in the physiological state of Escherichia coli cells at the initial stage for establishing a new symbiotic relationship with Dictyostelium discoideum cells. For the physiological state, we monitored green fluorescence intensity due to a green fluorescent protein (GFP) gene integrated into the chromosome by flow cytometry (FCM). On co-cultivation of the two species, a new population of E. coli cells with increased GFP concentration appeared, and when the formation of mucoidal colonies housing the coexisting two species began, most E. coli cells were from the new population. Further experiments suggest that the physiological change is induced by interaction with D. discoideum cells and is reversible, although the processes of the changes in both directions seem to proceed gradually. The observed phenotypic plasticity, together with natural selection under a co-cultivation environment, may be important for leading to the evolution of a new symbiotic system.