Protein tyrosine phosphatase PTP1 negatively regulates Dictyostelium STATa and is required for proper cell-type proportioning

The protein tyrosine phosphatase PTP1, which mediates reversible phosphorylation on tyrosine, has been shown to play an important regulatory role during Dictyostelium development. Mutants lacking PTP1 develop more rapidly than normal, while strains that overexpress PTP1 display aberrant morphology. However, the signalling pathways involved have not been characterised. In reexamining these strains, we have found that there is an inverse correlation between levels of PTP1 activity, the extent of tyrosine phosphorylation on Dictyostelium STATa after treatment with cAMP, and the proportion of the slug population exhibiting STATa nuclear enrichment in vivo. This suggests that PTP1 acts to attenuate the tyrosine phosphorylation of STATa and downstream STATa-mediated pathways. Consistent with this, we show that when PTP1 is overexpressed, there is increased expression of a prestalk cell marker at the slug posterior, a phenocopy of STATa null slugs. In ptp1 null strains, STATa tyrosine phosphorylation and nuclear enrichment in the slug anterior is increased. There is also a change in the prestalk to prespore cell ratio. Synergy experiments suggest that this is due to a cell-autonomous defect in forming the subset of prespore cells that are located in the anterior prespore region.     

Function of ammonium transporter A in the initiation of culmination of development in Dictyostelium discoideum

The histidine kinase DhkC controls a phosphorelay involved in regulating the slug versus culmination choice during the multicellular developmental program of Dictyostelium discoideum. When the relay is active, slug migration is favored due to the activation of a cyclic AMP (cAMP) phosphodiesterase and the resultant lowering of the intracellular and extracellular levels of cAMP. Ammonia signaling represents one input into the DhkC phosphorelay, and previous studies indicated that the ammonium transporter C inhibits the relay in response to low ammonia levels. Evidence is presented that another member of the family of ammonium transporters, AmtA, also regulates the slug/culmination choice. Under standard conditions of development, the wild-type strain requires a transitional period of 2 to 3 h to go from fingers to culminants, with some slugs forming and migrating briefly prior to culmination. In contrast, amtA null cells, like cells that lack DhkC, possessed a transitional period of only 1 to 2 h and rarely formed slugs. Disruption of amtA in an amtC null strain overcame the slugger phenotype of that strain and restored its ability to culminate. Strains lacking AmtA were insensitive to the ability of ammonia to promote and prolong slug migration. These findings lead to the proposal that AmtA functions in ammonia sensing as an activator of the DhkC phosphorelay in response to perceived high ammonia levels.     

Temporal and spatial expression of ammonium transporter genes during growth and development of Dictyostelium discoideum

Ammonia is an important signaling molecule involved in the regulation of development in Dictyostelium. During aggregation, ammonia gradients are established, and the ammonia concentration in the immediate environment or within a particular cell throughout development may vary. This is due to the rate of cellular ammonia production, its rate of loss by evaporation to the atmosphere or by diffusion into the substratum, and perhaps to cellular transport by ammonium transporters (AMTs). Recent efforts in genome and cDNA sequencing have identified three ammonium transporters in Dictyostelium. In addition to physically altering the levels of ammonia within cells, AMTs also may play a role in ammonia signaling. As an initial step in identifying such a function, the temporal and spatial expression of the three amt genes is examined. RT-PCR demonstrates that each of the three amt mRNAs is present and relatively constant throughout growth and development. The spatial expression of these three amt genes is examined during multiple stages of Dictyostelium development using in situ hybridization. A distinct and dynamic pattern of expression is seen for the three genes. In general, amtA is expressed heavily in pre-stalk cells in a dynamic way, while amtB and amtC are expressed in pre-spore regions consistently throughout development. AmtC also is expressed in the most anterior tip of fingers and slugs, corresponding to cells that mediate ammonia's effect on the choice between slug migration and culmination. Indeed, amtC null cells have a slugger phenotype, suggesting AmtC functions in the signaling pathway underlying the mechanics of this choice.     

Developmental decisions in Dictyostelium discoideum.

A few hours after the onset of starvation, amoebae of Dictyostelium discoideum start to form multicellular aggregates by chemotaxis to centers that emit periodic cyclic AMP signals. There are two major developmental decisions: first, the aggregates either construct fruiting bodies directly, in a process known as culmination, or they migrate for a period as "slugs." Second, the amoebae differentiate into either prestalk or prespore cells. These are at first randomly distributed within aggregates and then sort out from each other to form polarized structures with the prestalk cells at the apex, before eventually maturing into the stalk cells and spores of fruiting bodies. Developmental gene expression seems to be driven primarily by cyclic AMP signaling between cells, and this review summarizes what is known of the cyclic AMP-based signaling mechanism and of the signal transduction pathways leading from cell surface cyclic AMP receptors to gene expression. Current understanding of the factors controlling the two major developmental choices is emphasized. The weak base ammonia appears to play a key role in preventing culmination by inhibiting activation of cyclic AMP-dependent protein kinase, whereas the prestalk cell-inducing factor DIF-1 is central to the choice of cell differentiation pathway. The mode of action of DIF-1 and of ammonia in the developmental choices is discussed.     

Glutathione is required for growth and prespore cell differentiation in Dictyostelium

Glutathione (GSH) is the most abundant non-protein thiol in eukaryotic cells and acts as reducing equivalent in many cellular processes. We investigated the role of glutathione in Dictyostelium development by disruption of gamma-glutamylcysteine synthetase (GCS), an essential enzyme in glutathione biosynthesis. GCS-null strain showed glutathione auxotrophy and could not grow in medium containing other thiol compounds. The developmental progress of GCS-null strain was determined by GSH concentration contained in preincubated media before development. GCS-null strain preincubated with 0.2 mM GSH was arrested at mound stage or formed bent stalk-like structure during development. GCS-null strain preincubated with more than 0.5 mM GSH formed fruiting body with spores, but spore viability was significantly reduced. In GCS-null strain precultured with 0.2 mM GSH, prestalk-specific gene expression was delayed, while prespore-specific gene and spore-specific gene expressions were not detected. In addition, GCS-null strain precultured with 0.2 mM GSH showed prestalk tendency and extended G1 phase of cell cycle. Since G1 phase cells at starvation differentiate into prestalk cells, developmental defect of GCS-null strain precultured with 0.2 mM GSH may result from altered cell cycle. These results suggest that glutathione itself is essential for growth and differentiation to prespore in Dictyostelium.     

Prespore cell inducing factor, ψ factor,controls both prestalk and prespore gene expression in Dictyostelium development

Prespore cell‐inducing (psi, ψ) factor (PsiA), encoded by the psiA gene of Dictyostelium, is a secreted signal glycoprotein that induces prespore cell differentiation when added to monolayer cultures. In situ hybridization during normal development showed that the psiA gene is highly expressed in scattered cells at the mound stage and in prespore cells at the onset of culmination. The conventional prespore‐cell marker genes, cotC and pspA, were expressed normally in psiA^? and psiA overexpressing strains. Expressions of rnrB and cudA are repressed in the prestalk cells of a wild type slug to render prespore specific pattern. However, a promoter‐reporter fusion gene, rnrB:lacZ, showed an ectopic expression in the prestalk cells of the psiA^? strain while cudA(psp):lacZ did so in those of the psiA overexpressing strain. Overexpression of psiA delayed expression of the prestalk specific gene, ecmB, during development, while knocking out psiA promoted its expression. In addition, overexpression inhibited DIF‐1‐induced stalk formation in monolayer cultures. Together with the known prespore inducing activity, the results indicate that PsiA regulates both prespore and prestalk/stalk cell differentiation. These results indicate that PsiA is also involved in prestalk cell differentiation.     

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.     

The histidine kinase homologue DhkK/Sombrero controls morphogenesis in Dictyostelium

A key event in Dictyostelium development is the formation of the Mexican hat. This corresponds to a commitment step in morphogenesis that irreversibly signals progression from the slug stage to the fruiting body. We describe the characterization of the dhkK gene that controls this morphogenetic step. Null mutants of dhkK repeatedly attempt, and fail, to undergo morphogenesis from the slug to the Mexican hat, causing them to exhibit a "slugger" phenotype, which cannot be corrected by co-development with wild-type cells. The dhkK gene encodes a putative receptor histidine kinase whose expression is enriched in prestalk cells in the slug. Uniquely for a histidine kinase, DhkK is located in the nuclear envelope. Entry into culmination requires the DhkK response regulator domain, which appears to directly regulate cyclic AMP signaling.     

The function of PP2A/B56 in non-metazoan multicellular development

DB56, the Dictyostelium B56 homolog, displayed high sequence homology to other eukaryotic B56 subunits of the PP2A and a specific association with the PP2A catalytic subunit. Cells lacking DB56, psrA(-), displayed higher PP2A phosphatase activity compared with the wild type, approximately 10 hr of delayed expression of ecmA and ecmB prestalk markers, and inefficient culmination. The prespore marker cotB declined as wild-type cells culminate, but no such decline was observed from psrA(-) cells. Interestingly, psrA(-) cells exhibited higher GSK3 kinase activity. Furthermore, the expression of the dominant negative GSK3 mutant (K84/85M) in psrA(-) cells improved both prestalk and prespore expression patterns similarly to wild-type cells. However, culmination was not restored in psrA(-) cells expressing dominant negative GSK3, which suggests that PP2A/DB56 has an extra target during terminal differentiation. This report shows that PP2A/DB56 controls not only metazoan development, but also non-metazoan cell fate decision processes.     

An analysis of culmination in Dictyostelium using prestalk and stalk-specific cell autonomous markers

The ecmA (pDd63) and ecmB (pDd56) genes encode extracellular matrix proteins of the slime sheath and stalk tube of Dictyostelium discoideum. Using fusion genes containing the promoter of one or other gene coupled to an immunologically detectable reporter, we previously identified two classes of prestalk cells in the tip of the migrating slug; a central core of pstB cells, which express the ecmB gene, surrounded by pstA cells, which express the ecmA gene. PstB cells lie at the position where stalk tube formation is initiated at culmination and we show that they act as its founders. As culmination proceeds, pstA cells transform into pstB cells by activating the ecmB gene as they enter the stalk tube. The prespore region of the slug contains a population of cells, termed anterior-like cells (ALC), which have the characteristics of prestalk cells. We show that the ecmA and ecmB genes are expressed at a low level in ALC during slug migration and that their expression in these cells is greatly elevated during culmination. Previous observations have shown that ALC sort to surround the prespore cells during culmination (Sternfeld and David, 1982 Devl Biol. 93, 111-118) and we find just such a distribution for pstB cells. We believe that the ecmB protein plays a structural role in the stalk tube and its presence, as a cradle around the spore head, suggests that it may play a further function, perhaps in ensuring integrity of the spore mass during elevation. If this interpretation is correct, then a primary role of anterior-like cells may be to form these structures at culmination. We previously identified a third class of prestalk cells, pstO cells, which lie behind pstA cells in the slug anterior and which appeared to express neither the ecmA nor the ecmB gene. Using B-galactosidase fusion constructs, which give more sensitive detection of gene expression, we now find that these cells express the ecmA gene but at a much lower level than pstA cells. We also show that expression of the ecmA gene becomes uniformly high throughout the prestalk zone when slugs are allowed to migrate in the light. Overhead light favours culmination and it may be that increased expression of the ecmA gene in the pst 'O' region is a preparatory step in the process.