Overexpression of an activated rasG gene during growth blocks the initiation of Dictyostelium development.

Transformants that expressed either the wild-type rasG gene, an activated rasG-G12T gene, or a dominant negative rasG-S17N gene, all under the control of the folate-repressible discoidin (dis1gamma) promoter, were isolated. All three transformants expressed high levels of Ras protein which were reduced by growth in the presence of folate. All three transformants grew slowly, and the reduction in growth rate correlated with the amount of RasG protein produced, suggesting that RasG is important in regulating cell growth. The pVEII-rasG transformant containing the wild-type rasG gene developed normally despite the presence of high levels of RasG throughout development. This result indicates that the down regulation of rasG that normally occurs during aggregation of wild-type strains is not essential for the differentiation process. Dictyostelium transformants expressing the dominant negative rasG-S17N gene also differentiated normally. Dictyostelium transformants that overexpressed the activated rasG-G12T gene did not aggregate. The defect occurred very early in development, since the expression of car1 and pde, genes that are normally induced soon after the initiation of development, was repressed. However, when the transformant cells were pulsed with cyclic AMP, expression of both genes returned to wild-type levels. The transformants exhibited chemotaxis to cyclic AMP, and development was synergized by mixing with wild-type cells. Furthermore, cells that were pulsed with cyclic AMP for 4 h before being induced to differentiate by plating on filters produced small, but otherwise normal, fruiting bodies. These results suggest that the rasG-G12T transformants are defective in cyclic AMP production and that RasG - GTP blocks development by interfering with the initial generation of cyclic AMP pulses.     

Expression of activated Ras during Dictyostelium development alters cell localization and changes cell fate

There is now a body of evidence to indicate that Ras proteins play important roles in development. Dictyostelium expresses several ras genes and each appears to perform a distinct function. Previous data had indicated that the overexpression of an activated form of the major developmentally regulated gene, rasD, caused a major aberration in morphogenesis and cell type determination. We now show that the developmental expression of an activated rasG gene under the control of the rasD promoter causes a similar defect. Our results indicate that the expression of activated rasG in prespore cells results in their transdifferentiation into prestalk cells, whereas activated rasG expression in prestalk causes gross mislocalization of the prestalk cell populations.     

Expression of an activated rasD gene changes cell fate decisions during Dictyostelium development.

It has been previously demonstrated that the expression of an activated rasD gene in wild-type Dictyostelium cells results in formation of aggregates with multitips, instead of the normal single tips, and a block in further development. In an attempt to better understand the role of activated RasD development, we examined cell-type-specific gene expression in a strain stably expressing high levels of RasD[G12T]. We found that the expression of prestalk cell-specific genes ecmA and tagB was markedly enhanced, whereas the expression of the prespore cell-specific gene cotC was reduced to very low levels. When the fate of cells in the multitipped aggregate was monitored with an ecmA/lacZ fusion, it appeared that most of the cells eventually adopted prestalk gene expression characteristics. When mixtures of the [G12T]rasD cells and Ax3 cells were induced to differentiate, chimeric pseudoplasmodia were not formed. Thus, although the [G12T]rasD transformant had a marked propensity to form prestalk cells, it could not supply the prestalk cell population when mixed with wild-type cells. Both stalk and spore cell formation occurred in low cell density monolayers of the [G12T]rasD strain, suggesting that at least part of the inhibition of stalk and spore formation during multicellular development involved inhibitory cell interactions within the cell mass. Models for the possible role of rasD in development are discussed.     

Mediation of cell-substratum adhesion by RasG in Dictyostelium

Previous studies on the functions of the RasG gene in the cellular slime mold, Dictyostelium discoideum, have revealed that it is required for normal motility and cytokinesis. To further understand how the RasG gene regulates various cellular processes, we transformed an activated form of RasG, that is, RasG (G12T), a mutation from glycine to threonine at amino acid position 12 into wild type KAX-3 cells. This produced moderate but constitutive RasG(G12T) protein expression, which causes cells to become significantly more adherent to the substratum than are wild type cells. The RasG(G12T) transformants also grow slowly on bacterial plates, and engulf fewer bacteria on filter surfaces, indicating a defect in phagocytosis when cells are adhered. The expression of the activated RasG also dramatically reduces the number of filopodia on the cell surface. Tyrosine phosphorylation on a 43 kDa protein (most likely actin) of the RasG (G12T) transformants is highly elevated. Taken together, our observations suggest that RasG is crucial for Dictyostelium cell-substratum adhesion during growth and that RasG may play a role in adhesion-mediated phagocytosis. Our results also suggest that RasG is important in filopodial formation and that RasG is involved in the signal pathway that is regulated by tyrosine phosphorylation.     

Dual role of cAMP and involvement of both G-proteins and ras in regulation of ERK2 in Dictyostelium discoideum.

Dictyostelium discoideum expresses two Extracellular signal Regulated Kinases, ERK1 and ERK2, which are involved in growth, multicellular development and regulation of adenylyl cyclase. Binding of extracellular cAMP to cAMP receptor 1, a G-protein coupled cell surface receptor, transiently stimulates phosphorylation, activation and nuclear translocation of ERK2. Activation of ERK2 by cAMP is dependent on heterotrimeric G-proteins, since activation of ERK2 is absent in cells lacking the Galpha4 subunit. The small G-protein rasD also activates ERK2. In cells overexpressing a mutated, constitutively active rasD, ERK2 activity is elevated prior to cAMP stimulation. Intracellular cAMP and cAMP-dependent protein kinase (PKA) are essential for adaptation of the ERK2 response. This report shows that multiple signalling pathways are involved in regulation of ERK2 activity in D.discoideum.     

Rap1 activation in response to cAMP occurs downstream of ras activation during Dictyostelium aggregation

We have used a doubly disrupted rasC(-)/rasG(-) strain of Dictyostelium discoideum, which ectopically expresses the carA gene, to explore the relationship between the activation of RasC and RasG, the two proteins that are necessary for optimum cAMP signaling, and the activation of Rap1, a Ras subfamily protein, that is also activated by cAMP. The ectopic expression of carA restored early developmental gene expression to the rasC(-)/rasG(-) strain, rendering it suitable for an analysis of cAMP signal transduction. Because there was negligible signaling through both the cAMP chemotactic pathway and the adenylyl cyclase activation pathway in the rasC(-)/rasG(-)/[act15]:carA strain, it is clear that RasG and RasC are the only two Ras subfamily proteins that directly control these pathways. The position of Rap1 in the signal transduction cascade was clarified by the finding that Rap1 activation was totally abolished in rasC(-)/rasG(-)/[act15]:carA and rasG(-) cells but only slightly reduced in rasC(-) cells. Rap1 activation, therefore, occurs downstream of the Ras proteins and predominantly, if not exclusively, downstream of RasG. The finding that in vitro guanylyl cyclase activation is also abolished in the rasC(-)/rasG(-)/[act15]:carA strain identifies RasG/RasC as the presumptive monomeric GTPases required for this activation.     

The effect of the disruption of a gene encoding a PI4 kinase on the developmental defect exhibited by Dictyostelium rasC(-) cells

The disruption of the gene encoding the Dictyostelium Ras subfamily protein, RasC results in a strain that fails to aggregate with defects in both cAMP signal relay and chemotaxis. Restriction enzyme mediated integration disruption of a second gene in the rasC(-) strain resulted in cells that were capable of forming multicellular structures in plaques on bacterial lawns. The disrupted gene, designated pikD(1), encodes a member of the phosphatidyl-inositol-4-kinase beta subfamily. Although the rasC(-)/pikD(1) cells were capable of progressing through early development, when starved on a plastic surface under submerged conditions, they did not form aggregation streams or exhibit pulsatile motion. The rasC(-)/pikD(1) cells were extremely efficient in their ability to chemotax to cAMP in a spatial gradient, although the reduced phosphorylation of PKB in response to cAMP observed in rasC(-) cells, was unchanged. In addition, the activation of adenylyl cyclase, which was greatly reduced in the rasC(-) cells, was only minimally increased in the rasC(-)/pikD(1) strain. Thus, although the rasC(-)/pikD(-) cells were capable of associating to form multicellular structures, normal cell signaling was clearly not restored. The disruption of the pikD gene in a wild type background resulted in a strain that was delayed in aggregation and formed large aggregation streams, when starved on a plastic surface under submerged conditions. This strain also exhibited a slight defect in terminal development. In conclusion, disruption of the pikD gene in a rasC(-) strain resulted in cells that were capable of forming multicellular structures, but which did so in the absence of normal signaling and aggregation stream formation.     

Replacement of threonine residues by serine and alanine in a phosphorylatable heavy chain fragment of Dictyostelium myosin II

The target sites of soluble myosin heavy chain kinases partially purified from growth phase or aggregation competent cells of Dictyostelium discoideum were identified by the use of normal and mutated fragments of the myosin heavy chain. The kinases from both developmental stages phosphorylated two previously established threonine residues, as well as an additional one. The newly identified site is located within the putative core region of the coiled-coil formed by the myosin tail. A lysine following the phosphorylated threonine residue is the only common feature of the sequences around these sites. The kinases, which specifically phosphorylate threonine residues in wild-type myosin, did accept serine if it was in the right structural context.     

RasG regulates discoidin gene expression during Dictyostelium growth

Activated rasG, rasG(G12T), was expressed in Dictyostelium cells under the control of the folate-repressible discoidin promoter (pVEII-rasG(G12T)) and found to have a unique pattern of expression when cells were transferred to folate-deficient media: an initial increase of RasG(G12T) resulting from the removal of folate, followed by a rapid decline while cells were still in the early exponential phase of growth. Discoidin levels were considerably lower and declined more rapidly in the pVEII-rasG(G12T) transformant than they did in the wild type, suggesting that RasG(G12T) represses discoidin expression. This was independently confirmed by placing the rasG(G12T) gene under the control of the ribonucleotide reductase (rnrB) promoter. Exposure of cells to 10 mM methyl methanesulfonate (MMS) rapidly generated RasG(G12T) and this was accompanied by an equally rapid decrease in discoidin mRNA levels. rasG null cells also contained decreased levels of discoidin under all conditions tested, indicating that RasG is essential for optimum discoidin expression. However, rasG null cells showed normal regulation of discoidin expression in response to PSF, CMF, folate, bacteria, and axenic media, indicating that RasG is not necessary for any of these responses. These results reveal a role for RasG in regulating discoidin gene expression and add a further level of complexity to the regulation of the discoidin promoter.     

Dictyostelium macroautophagy mutants vary in the severity of their developmental defects

Macroautophagy is the major mechanism that eukaryotes use to recycle cellular components during stressful conditions. We have shown previously that the Atg12-Atg5 conjugation system, required for autophagosome formation in yeast, is necessary for Dictyostelium development. A second conjugation reaction, Aut7/Atg8 lipidation with phosphatidylethanolamine, as well as a protein kinase complex and a phosphatidylinositol 3-kinase complex are also required for macroautophagy in yeast. In this study, we characterize mutations in the putative Dictyostelium discoideum orthologues of budding yeast genes that are involved in one of each of these functions, ATG1, ATG6, and ATG8. All three genes are required for macroautophagy in Dictyostelium. Mutant amoebae display reduced survival during nitrogen starvation and reduced protein degradation during development. Mutations in the three genes produce aberrant development with defects of varying severity. As with other Dictyostelium macroautophagy mutants, development of atg1-1, atg6(-), and atg8(-) is more aberrant in plaques on bacterial lawns than on nitrocellulose filters. The most severe defect is observed in the atg1-1 mutant, which does not aggregate on bacterial lawns and arrests as loose mounds on nitrocellulose filters. The atg6(-) and atg8(-) mutants display almost normal development on nitrocellulose filters, producing multi-tipped aggregates that mature into small fruiting bodies. The distribution of a green fluorescent protein fusion of the autophagosome marker, Atg8, is aberrant in both atg1-1 and atg6(-) mutants.     

Analysis of a spatially regulated phosphotyrosine phosphatase identifies tyrosine phosphorylation as a key regulatory pathway in Dictyostelium.

We have cloned a Dictyostelium phosphotyrosine phosphatase (PTP1) with a catalytic domain showing approximately 38%-50% amino acid identity to those of other PTPs. PTP1 contains an approximately 99 amino acid insert and bacterially produced PTP1 possesses PTP activity. PTP1 is expressed at a very low level in vegetative cells, induced by 4 hr, and maximally expressed at the tight aggregate stage. PTP1-lacZ studies indicate that PTP1 is spatially localized to prestalk and anterior-like cell types. PTP1 gene disruptants show accelerated development, whereas strains overexpressing PTP1 to a high level fail to aggregate. Strains overexpressing moderate levels exhibit severe morphological defects following aggregation, including multiply tipped aggregates and morphologically aberrant fruiting bodies. Western blot analysis using anti-phosphotyrosine antibodies shows specific changes in the mutant strains when compared with wild-type cells. The results indicate that reversible protein-tyrosine phosphorylation and PTP1 play important regulatory roles during Dictyostelium development.     

Rap1 Overexpression Reveals That Activated RasD Induces Separable Defects duringDictyosteliumDevelopment

One of theDictyostelium rasgenes,rasD,is expressed preferentially in prestalk cells at the slug stage of development and overexpression of this gene containing a G12T activating mutation causes the formation of aberrant multitipped aggregates that are blocked from further development (Reymondet al.,1986,Nature,323, 340–343). The ability of theDictyostelium rap1gene to suppress this abnormal developmental phenotype was investigated. Therap1gene and G12V activated and G10V negative mutant forms of therap1gene were independently linked to therasDpromoter and each construct used to transform M1, aDictyosteliumcell line expressing RasD[G12T]. Transformants of M1 that expressed Rap1 or Rap1[G12V] protein still formed multitipped aggregates, but most tips were able to complete development and form fruiting bodies. Cell lines showing this modified phenotype were designated ME (multitipped escape). Therap1[G10V] construct did not modify the M1 phenotype. These data suggest that overexpression of RasD[G12T] has two effects, the formation of a multitipped aggregate and a block in subsequent differentiation and that the expression of Rap1 or Rap1[G12V] reverses only the latter. Differentiation of ME cells in low density monolayers showed the identical low level of stalk and spore cell formation seen for M1 cells under the same conditions. Thus the cell autonomous defect in monolayer differentiation induced in the M1 strain was not corrected in the ME strain. Cell type-specific gene expression during the development of M1 cells is dramatically altered: prestalk cell-specific gene expression is greatly enhanced, whereas prespore-specific gene expression is almost suppressed (Louiset al.,1997,Mol. Biol. Cell,8, 303–312). During the development of ME cells,ecmA mRNA levels were restored to those seen for Ax3, andtagB mRNA levels were also markedly reduced, although not to Ax3 levels.cotCexpression in ME cells was enhanced severalfold relative to M1, although levels were still lower than those observed during the development of Ax3. The low expression ofcar1mRNA during early development of the M1 strain remained low during the development of ME cells. These data are consistent with the idea that the expression of RasD[G12T] affects two independent and temporally separated events and that only the later defect is reversed byrap1.     

Overproduction of the regulatory subunit of the cAMP-dependent protein kinase blocks the differentiation of Dictyostelium discoideum.

During the aggregation of Dictyostelium discoideum extracellular cAMP is known to act as a chemotractant and as an inducer of cellular differentiation. However, its intracellular role as a second messenger remains obscure. We have constructed a fusion gene consisting of the cDNA encoding the regulatory subunit (R) of the cAMP-dependent protein kinase fused to the promoter and N-terminal-proximal sequences of a Dictyostelium actin gene. Stable transformants, containing multiple copies of this gene, overproduce the R subunit which accumulates prematurely relative to the endogenous protein. These transformants fail to aggregate. Detailed analysis has shown that they are blocked at interphase, the period prior to aggregation, and that they are severely defective in most responses to cAMP including the induction of gene expression. Our observations suggest that intracellular cAMP acts, presumably by activation of the catalytic subunit of the cAMP-dependent protein kinase, to facilitate early development.     

Functional analysis of the signal-sequence processing site of yeast acid phosphatase

A systematic study of the signal peptidase cleavage site of the main cell-wall-repressible Saccharomyces cerevisiae acid phosphatase encoded by the PHO5 gene is presented. The last amino acid of the signal sequence, the chromosomally encoded alanine of the wild-type gene, was changed by any of 19 other amino acids in the chromosomal DNA by using in vitro mutagenesis in Escherichia coli and the technique of gene replacement. Processing and secretion are normal when the amino acid at this position is a small neutral amino acid, i.e. alanine, glycine, cysteine, serine or threonine. Processing glycosylation, and secretion of regulated acid phosphatase are distinctly affected with other amino acid substitutions and core-glycosylated protein accumulates in the cell. Surprisingly, PHO5 protein is still secreted to the cell wall and into the growth medium but at a lower rate and without cleavage of the signal sequence. The same features are exhibited by a mutated acid phosphatase with a deletion of four amino acids at the end of the signal peptide (-7 to -4 relative to the processing site) thus preserving the important -3 to -1 region.     

Ligand-independent reduction of cAMP receptors in Dictyostelium discoideum cells over-expressing a mutated ras gene.

Drug-resistance selection in Dictyostelium discoideum transformants resulted in up to eight-times-higher ras protein levels. Over-production of the wild-type ras protein did not lead to an aberrant phenotype. Increased levels of the mutated [G12T]ras protein, however, were correlated with severe deficiencies in aggregation and development. This aberrant phenotype is associated with reduced cAMP binding, due to a lower number of cell-surface receptors. We show that both RNA and cAMP-receptor-protein levels are reduced. These results indicate that ras in Dictyostelium discoideum seems to be involved in regulating cAMP-receptor-gene expression.     

Factors affecting the electroporation of Bacillus subtilis

Bacillus subtilis 168 trp ^- was found to be transformable with the tetracycline resistance plasmid pAB124 by electroporation of whole cells, inconsistently and at very low frequencies. Supplementation of the growth medium with glycine, or particularly DL-threonine, produced cells that could be electrotransformed much more efficiently at frequencies up to 2.5 X 10³ transformants per μg plasmid DNA. Transformation was optimal with cells grown in medium containing a racemic mixture of the D- and L-isomers of threonine, and no transformants were obtained when pure forms of the D- and L-threonine isomers were used. The cell walls of B. subtilis grown in the presence or absence of D-, L- and DL-threonine had a similar amino acid composition which did not include threonine. A more complex biochemical explanation of the enhancement of electroporation by growth in DL-threonine is likely, and this is discussed. Lysozyme treatments to weaken the cell wall and possibly mimic the effect of DL-threonine did not yield any transformants. The effects of buffer composition and culture incubation time were also determined and the electroporation protocol optimized accordingly. The response of a range of other B. subtilis strains to electroporation by the method produced was found to be variable. In all cases, transformation was verified by recovery of the plasmid DNA from putative transformants.     

Utilization of l-threonine by a species of Arthrobacter. A novel catabolic role for `aminoacetone synthase''

1. A species of Arthrobacter (designated Arthrobacter 9759) was isolated from soil by its ability to grow aerobically on l-threonine as sole source of carbon atoms, nitrogen atoms and energy; the organism also grew well on other sources of carbon atoms including glycine, but no growth was obtainable on aminoacetone or dl-1-aminopropan-2-ol. 2. During growth on threonine, (14)C from l-[U-(14)C]threonine was rapidly incorporated into glycine and citrate, and thereafter into serine, alanine, aspartate and glutamate. 3. With extracts of threonine-grown cells supplied with l-[U-(14)C]threonine, evidence was obtained of the NAD and CoA-dependent catabolism of l-threonine to produce acetyl-CoA plus glycine. Short-term incorporation studies in which [2-(14)C]acetate and [2-(14)C]glycine were supplied (a) to cultures growing on threonine, and (b) to extracts of threonine-grown cells, showed that the acetyl-CoA was metabolized via the tricarboxylic acid cycle and glyoxylate cycle whereas the glycine was converted into pyruvate via the folate-dependent ;serine pathway'. 4. The threonine-grown organism contained ;biosynthetic' threonine dehydratase and a potent NAD-linked l-threonine dehydrogenase but possessed no l-threonine aldolase activity. 5. Evidence was obtained that the acetyl-CoA and glycine produced from l-threonine had their immediate origin in the alpha-amino-beta-oxobutyrate formed by the threonine dehydrogenase; the CoA-dependent cleavage of this compound was catalysed by an alpha-amino-beta-oxobutyrate CoA-ligase, which was identified with ;aminoacetone synthase'. A continuous spectrophotometric assay of this enzyme was developed, and it was found to be inducibly synthesized only during growth on threonine and not during growth on acetate plus glycine. 6. By using a reconstituted mixture of separately purified l-threonine dehydrogenase and alpha-amino-beta-oxobutyrate CoA-ligase (i.e. ;aminoacetone synthase'), l-[U-(14)C]threonine was broken down to [(14)C]glycine plus [(14)C]acetyl-CoA (trapped as [(14)C]citrate). 7. There was no evidence of aminoacetone metabolism by Arthrobacter 9759 even though a small amount of this amino ketone appeared in the culture medium during growth on threonine.     

A developmentally regulated, putative serine/threonine protein kinase is essential for development in Dictyostelium.

Using PCR technology, we have cloned parts of three developmentally regulated putative serine/threonine kinases from Dictyostelium. All show significant homology to members of the cAMP-dependent protein kinase A/protein kinase C subfamilies. A genomic clone encoding one of these, DdPK3, has been isolated and sequenced. The open reading frame encodes a protein of 648 amino acids with the conserved kinase domain in the C-terminal half. The protein encoded by this gene is unusual in that it contains long homopolymer runs in the N-terminal half of the protein, including a long run of 88 amino acids in which 73 are glutamine residues. To examine the function of DdPK3, a gene disruption was created via homologous recombination. Ddpk3- cells do not aggregate by themselves but will co-aggregate with wild-type cells. However, after aggregation these cells are 'sloughed off' and do not proceed further through development, but are found as a discrete mass alongside the fruiting body formed by the wild-type cells. Analysis of signal transduction pathways indicates that cAMP pulse-induced expression of aggregation stage-specific genes is normal in Ddpk3- cells, as is induction of the prestalk gene Ddras in single cell assays. However, cAMP induction of the late promoters of cAMP receptor cAR1 and of two prespore-specific genes is absent under similar conditions. These cells show normal activation of adenylate cyclase and normal phosphorylation of the G alpha protein G alpha 2 in response to cAMP. The possible role of DdPK3 in Dictyostelium development is discussed.     

The role of Src family kinases in egg activation

Solitary amoebae of Dictyostelium discoideum are frequently exposed to stressful conditions in nature, and their multicellular development is one response to environmental stress. Here we analyzed an aggregation stage abundant gene, krsA, homologous to human krs1 (kinase responsive to stress 1) to understand the mechanisms for the initiation of development and cell fate determination. The krsA- cells exhibited reduced viability under hyperosmotic conditions. They produced smaller aggregates on membrane filters and did not form aggregation streams on a plastic surface under submerged starvation conditions, but were normal in sexual development. During early asexual development, the expression of cAMP-related genes peaked earlier in the knockout mutants. Neither cAMP oscillation in starved cells nor an increase in the cAMP level following osmotic stress was observed in krsA-. The nuclear export signal, as well as the kinase domain, in KrsA was necessary for stream formation. These results strongly suggest that krsA is involved in cAMP relay, and that signaling pathways for multicellular development have evolved in unison with the stress response.