Trishanku, a novel regulator of cell-type stability and morphogenesis in Dictyostelium discoideum

We have identified a novel gene, trishanku (triA), by random insertional mutagenesis of Dictyostelium discoideum. TriA is a Broad complex Tramtrack bric-a-brac domain-containing protein that is expressed strongly during the late G2 phase of cell cycle and in presumptive spore (prespore (psp)) cells. Disrupting triA destabilizes cell fate and reduces aggregate size; the fruiting body has a thick stalk, a lowered spore: stalk ratio, a sub-terminal spore mass and small, rounded spores. These changes revert when the wild-type triA gene is re-expressed under a constitutive or a psp-specific promoter. By using short- and long-lived reporter proteins, we show that in triA(-) slugs the prestalk (pst)/psp proportion is normal, but that there is inappropriate transdifferentiation between the two cell types. During culmination, regardless of their current fate, all cells with a history of pst gene expression contribute to the stalk, which could account for the altered cell-type proportion in the mutant.     

Differentiation of Various Cell Types during Fruiting Body Formation of Dictyostelium Discoideum*

The processes of differentiation of the presumptive cells (prespore and prestalk cens) into mature spores, stalk and basal-disc cells in Dictyotelium discoideum was investigated. The number of stalk and disc cells in pre-labeled culminating cell masses was estimated by determining the radioactivity of the undissociable fraction separated by filtration from the dissociable fraction containing presumptive cells and spores. Changes in the proportion of amoeboid cells stainable with fluorescein-conjugated antispore serum and encapsulated spores were also followed in the dissociable fraction. Formation of stalk and disc cells began at 17 hr of development and was completed at 26 hr, while formation of morphologically identifiable spores began at 18 hr and was completed at 20 hr, long before completion of stalk formation. At the onset of culmination, unstained cells abruptly increased with an accompanying decrease of stained cells, when unstained rear-guard cells appeared in the hind region. Although some of the rear-guard cells soon differentiated into basal-disc cells, the rest remained amoeboid in the upper part of the spore mass (sorus) after complete formation of a fruiting body. Despite the presence of the amoeboid cells in mature sori, the proportion of the sorus to the stalk and disc of a fruiting body was approximately equal to that of stained (prespore) to unstained (prestalk) cells in a migrating slug.     

THE EFFECTS OF CYCLIC AMP ON DIFFERENTIATION OF A MUTANT DICTYOSTELIUM DISCOIDEUM CAPABLE OF DEVELOPING WITHOUT MORPHOGENESIS

The dev 1510 mutant of Dictyostelium discoideum differs from the wild type in that unaggregated cells are capable of differentiating into either spores or stalk cells depending on the culture conditions (12). Taking advantage of this fact, the effects of cyclic AMP (cAMP) on differentiation of the mutant cells were examined under conditions that prevent normal morphogenesis. In the presence of low concentrations of exogenous cAMP, the cells differentiated into only stalk cells, whereas in the presence of high concentrations they differentiated into only spores. Untreated cells formed stalk cells, but this was inhibited by addition of phosphodiesterase, indicating that it was induced by a low concentration of cAMP which they produced themselves. Cyclic GMP and dibutyryl cAMP also induced spore formation though less effectively, while 5'AMP, ADP and ATP had no effect. During development, the cells increased in sensitivity to cAMP in that spore formation was induced at lower concentration of cAMP after 4 hr of starvation. Treatment of cells that had been starved for 6hr with 10⁻⁴M cAMP for as short a time as 30 min was enough to induce 8% of the cells to form spores.
The effects on cAMP-induced differentiation of chemicals that are known to influence development of the wild type were also examined. Both NH₄Cl and KCl inhibited cAMP-induced stalk formation, but had no effect on spore formation. In the presence of arginine, spore formation was induced at a lower concentration of cAMP with higher efficiency. CaCl₂, LiCl and KF had no effect on cAMP-induced differentiation.     

Bimodal distribution of motility and cell fate in Dictyostelium discoideum

Pre-starvation amoebae of Dictyostelium discoideum exhibit random movements. Starved cells aggregate by directed movements (chemotaxis) towards cyclic AMP and differentiate into live spores or dead stalk cells. Many differences between presumptive spore and stalk cells precede differentiation. We have examined whether cell motility-related factors are also among them. Cell speeds and localisation of motility-related signalling molecules were monitored by live cell imaging and immunostaining (a) in nutrient medium during growth, (b) immediately following transfer to starvation medium and (c) in nutrient medium that was re-introduced after a brief period of starvation. Cells moved randomly under all three conditions but mean speeds increased following transfer from nutrient medium to starvation medium; the transition occurred within 15 min. The distribution of speeds in starvation medium was bimodal: about 20% of the cells moved significantly faster than the remaining 80%. The motility-related molecules F-actin, PTEN and PI3 kinase were distributed differently in slow and fast cells. Among starved cells, the calcium content of slower cells was lower than that of the faster cells. All differences reverted within 15 min after restoration of the nutrient medium. The slow/fast distinction was missing in Polysphondylium pallidum, a cellular slime mould that lacks the presumptive stalk and spore cell classes, and in the trishanku (triA(-)) mutant of D. discoideum, in which the classes exist but are unstable. The transition from growth to starvation triggers a spontaneous and reversible switch in the distribution of D. discoideum cell speeds. Cells whose calcium content is relatively low (known to be presumptive spore cells) move slower than those whose calcium levels are higher (known to be presumptive stalk cells). Slow and fast cells show different distributions of motility-related proteins. The switch is indicative of a bistable mechanism underlying cell motility.     

Different synthetic profiles and developmental fates of prespore versus prestalk proteins of Dictyostelium

Abstract. Depending upon environmental conditions, developing cells of the cellular slime mold Dictyostelium discoideum may enter a slug stage in which the cell mass migrates in response to gradients of light and temperature. This developmental stage has often been used to study the divergent differentiation of the cells that will subsequently form spores and stalk in the mature fruiting body. However, still debated is the extent to which the differentiation evident in slug cells is a precondition for development of the mature cells in fruits. Using two-dimensional gel electrophoresis of polypeptides, we have examined the proteins made by prespore and prestalk cells of migrating slugs and by maturing spore and stalk cells. The data indicate that many of the cell-type specific polypeptides in prespore cells of slugs persist as cell-type specific polypeptides of mature spores. Prestalk slug cells, in contrast, do not contain significant amounts of stalk-specific proteins; these proteins appear only during culmination. The precursor cell types also differ in the times and rates of synthesis of cell-specific proteins: prestalk proteins appear much earlier in development than do the prespore, but never reach the levels of expression that the prespore proteins do later in culmination. These findings may explain the well established ability of prespore cells to regulate their cell type more rapidly than do prestalk cells. There are also implications for our general understanding of what is a 'prestalk' gene product.     

Identification of new differentiation inducing factors from Dictyostelium discoideum

Developing Dictyostelium discoideum amoebae form a stalked fruiting body in which individual cells differentiate into either stalk cells or spores. The major known inducer of stalk cell differentiation is the chlorinated polyketide DIF-1 (1-(3,5-dichloro-2,6-dihydroxy-4-methoxyphenyl)hexan-1-one); however a mutant blocked in the terminal step of DIF-1 biosynthesis still produces one of the prestalk cell subtypes - the pstA cells - as well as some mature stalk cells. We therefore searched for additional stalk cell-inducing factors in the medium supporting development of this mutant. These factors were purified by solvent extraction and HPLC and identified by mass spectroscopy and NMR. The mutant lacked detectable DIF-2 and DIF-3 (the pentanone and deschloro homologues of DIF-1) but four major stalk cell-inducing activities were detected, of which three were identified. Two compounds were predicted intermediates in DIF-1 biosynthesis: the desmethyl, and desmethyl-monochloro analogues of DIF-1 (dM-DIF-1 and Cl-THPH, respectively), supporting the previously proposed pathway of DIF-1 biosynthesis. The third compound was a novel factor and was identified as 4-methyl-5-pentylbenzene-1,3-diol (MPBD) with the structure confirmed by chemical synthesis. To investigate the potential roles of these compounds as signal molecules, their effects on morphological stalk and spore differentiation were examined in cell culture. All three induced morphological stalk cell differentiation. We found that synthetic MPBD also stimulated spore cell differentiation. Now that these factors are known to be produced and released during development, their biological roles can be pursued further.     

ATP, trehalose, glucose and ammonium ion localization in the two cell types of Dictyostelium discoideum

Ultra-microfluorometric techniques were adapted to follow several compounds related to energy metabolism through the developmental cycle of Dictyostelium discoideum. Each compound (ATP, trehalose, glucose, and ammonium ion) was found to be present in stalk and/or spore cells. The accumulation of NH4+ was interpreted as an indication of protein degradation, a source of energy in this organism. During the early stages of differentiation NH4+ was localized only in prestalk cells. However, it accumulated in spore cells during culmination such that levels were comparable in the two cell types by the end of development. Trehalose, an energy source for germinating spores, was found in both cell types but was preferentially degraded in stalk cells late in development. Glucose, the degradation product of trehalose, was localized in prestalk cells and varied inversely with trehalose levels. ATP was not localized in a specific cell type during development. However, ATP declined in stalk cells at an earlier stage of development.     

Genetic evidence that the acyl coenzyme A binding protein AcbA and the serine protease/ABC transporter TagA function together in Dictyostelium discoideum cell differentiation

The acyl coenzyme A (CoA) binding protein AcbA is cleaved to form a peptide (SDF-2) that coordinates spore encapsulation during the morphogenesis of Dictyostelium discoideum fruiting bodies. We present genetic evidence that the misspecification of cell types seen in mutants of the serine protease/ABC transporter TagA results from the loss of normal interactions with AcbA. Developmental phenotypes resulting from aberrant expression of the TagA protease domain, such as the formation of supernumerary tips on aggregates and the production of excess prestalk cells, are suppressed by null mutations in the acbA gene. Phenotypes resulting from the deletion of tagA, such as overexpression of the prestalk gene ecmB and the misexpression of the prespore gene cotB in stalk cells, are also observed in acbA mutants. Moreover, tagA- mutants fail to produce SDF-2 during fruiting body morphogenesis but are able to do so if they are stimulated with exogenous SDF-2. These results indicate that the developmental program depends on TagA and AcbA working in concert with each other during cell type differentiation and suggest that TagA is required for normal SDF-2 signaling during spore encapsulation.     

Folic acid stimulation of the Gα4 G protein-mediated signal transduction pathway inhibits anterior prestalk cell development in Dictyostelium

In Dictyostelium discoideum, several G proteins are known to mediate the transduction of signals that direct chemotactic movement and regulate developmental morphogenesis. The G protein alpha subunit encoded by the Galpha4 gene has been previously shown to be required for chemotactic responses to folic acid, proper developmental morphogenesis, and spore production. In this study, cells overexpressing the wild type Galpha4 gene, due to high copy gene dosage (Galpha4HC), were found to be defective in the ability to form the anterior prestalk cell region, express prespore- and prestalk-cell specific genes, and undergo spore formation. In chimeric organisms, Galpha4HC prespore cell-specific gene expression and spore production were rescued by the presence of wild-type cells, indicating that prespore cell development in Galpha4HC cells is limited by the absence of an intercellular signal. Transplanted wild-type tips were sufficient to rescue Galpha4HC prespore cell development, suggesting that the rescuing signal originates from the anterior prestalk cells. However, the deficiencies in prestalk-specific gene expression were not rescued in the chimeric organisms. Furthermore, Galpha4HC cells were localized to the prespore region of these chimeric organisms and completely excluded from the anterior prestalk region, suggesting that the Galpha4 subunit functions cell-autonomously to prevent anterior prestalk cell development. The presence of exogenous folic acid during vegetative growth and development delayed anterior prestalk cell development in wild-type but not galpha4 null mutant aggregates, indicating that folic acid can inhibit cell-type-specific differentiation by stimulation of the Galpha4-mediated signal transduction pathway. The results of this study suggest that Galpha4-mediated signals can regulate cell-type-specific differentiation by promoting prespore cell development and inhibiting anterior prestalk-cell development.     

Cell Patterning During Slug Migration and Early Culmination in Dictyostelium discoideum

Abstract. The effects of migration and culmination on patterning of presumptive (prespore and prestalk) cells and mature (spore and stalk) cells of D. discoideum were investigated. The ratio of prespore to total cells, as determined by staining with fluorescein-conjugated antispore globulin, was constant (77%) up until 8 h of slug migration, but then decreased to a level (64%) which thereafter remained unchanged during migration. Cells which lost prespore antigen during migration were located in the posterior (prespore) part next to the agar surface.
Upon induction of culmination, however, the ratio of prespore cells quickly increased to the normal level (77%) within 1–2 h. During the transition between migration and culmination prestalk and prespore cells were considerably intermixed within the cell mass, before the normal prestalk-prespore pattern was reestablished at the preculmination (Mexican hat) stage. Spore: stalk ratios within fruiting bodies were normal irrespective of the lengths of slug migration.     

The Effect of Proteases on Gene Expression and Cell Differentiation in Dictyostelium discoideum

We have examined the effects of chymotrypsin or pronase on the differentiation of monolayers of Dictyostelium discoideum amoebae developing in the presence of 1–5 mM cyclic AMP. Using sporogenous mutants, which are capable of forming both spores and stalk cells under these conditions, we have observed that low concentrations of either protease selectively inhibit a late step of spore formation. Higher levels of the proteases act at an earlier time and by a distinct mechanism to reduce the accumulation of the prespore cell specific enzyme UDP galactose polysaccharide transferase while not affecting the appearance of glycogen phosphorylase. The latter is present in both prestalk and prespore cells.     

A MUTANT OF DICTYOSTELIUM DISCOIDEUM CAPABLE OF DIFFERENTIATING WITHOUT MORPHOGENESIS

A mutant which is capable of differentiating into spores and stalk cells without forming a cell aggregate was isolated from the cellular slime mould, Dictyostelium discoideum. The mutant stopped developing at various stages, before formation of mature fruits, and the cells differentiated into spores and stalk cells at whichever stage the development stopped. Unaggregated cells also differentiated into spores or stalk cells, depending on the culture conditions; differentiation into spores predominated in nutrient rich medium, while differentiation into stalk cells predominated in nutrient poor medium. The ratio of spores to stalk cells or of prespores to total cells in cell masses depended on the terminal structures formed; the ratio was unusually high or unusually low in a structure which stopped developing before papilla formation, while the ratio was normal in a structure formed after that stage. When isolated from a cell mass, prespore cells of the mutant did not dedifferentiate or resumed vegetative growth, indicating that they had lost plasticity of differentiation. The conditioned medium in which the mutant cells had grown was effective in inducing differentiation of wild type slug cells into spore-like or stalk-like cells.     

Pattern Formation in Dictyostelium discoideum: Temporal and Spatial Distribution of Prespore Vacuoles

Cell differentiation, cell determination and pattern formation in the pseudoplasmodium of Dictyostelium discoideum , was investigated using the prespore specific vacuole (PV) as a morphological marker. Concomitantly, measurements of cell movement within the pseudoplasmodium were made by tracing radioactively labelled cells. The main results indicate that 1) prespore cells appear first during late aggregation and occur randomly throughout the pseudoplasmodium with the exception of the very tip which stays free of prespore cells throughout development; 2) after late aggregation the number of prespore cells increases over a period of several hours; 3) each prespore cell takes on a progressively more prespore-like character as judged by the increase in number of PVs it contains; 4) establishment of the distribution pattern of prespore and prestalk cells takes place within the first 2 h, mainly by a sorting out mechanism; 5) presumptive spore areas are likely to contain a small proportion of cells lacking PVs (prestalk-cells?) while presumptive stalk cell areas are homogeneous throughout; 6) maintenance of the pattern during migration may be facilitated by a circulation at low level of prestalk cells between prestalk and prespore areas; and 7) during the development of this organism the events of cell determination, cell differentiation and pattern formation overlap substantially in time.     

A linear dominance hierarchy among clones in chimeras of the social amoeba Dictyostelium discoideum

Amoebae from different clones of Dictyostelium discoideum aggregate into a common slug, which migrates towards light for dispersal, then forms a fruiting body consisting of a somatic, dead stalk, holding up a head of living spores. Contributions of two clones in a chimera to spore and stalk are often unequal, with one clone taking advantage of the other's stalk contribution. To determine whether there was a hierarchy of exploitation among clones, we competed all possible pairs among seven clones and measured their relative representation in the prespore and prestalk stages and in the final spore stage. We found a clear linear hierarchy at the final spore stage, but not at earlier stages. These results suggest that there is either a single principal mechanism or additive effects for differential contribution to the spore, and that it involves more than spore/stalk competition.     

Identification of new differentiation inducing factors from Dictyostelium discoideum

Developing Dictyostelium discoideum amoebae form a stalked fruiting body in which individual cells differentiate into either stalk cells or spores. The major known inducer of stalk cell differentiation is the chlorinated polyketide DIF-1 (1-(3,5-dichloro-2,6-dihydroxy-4-methoxyphenyl)hexan-1-one); however a mutant blocked in the terminal step of DIF-1 biosynthesis still produces one of the prestalk cell subtypes – the pstA cells – as well as some mature stalk cells. We therefore searched for additional stalk cell-inducing factors in the medium supporting development of this mutant. These factors were purified by solvent extraction and HPLC and identified by mass spectroscopy and NMR. The mutant lacked detectable DIF-2 and DIF-3 (the pentanone and deschloro homologues of DIF-1) but four major stalk cell-inducing activities were detected, of which three were identified. Two compounds were predicted intermediates in DIF-1 biosynthesis: the desmethyl, and desmethyl-monochloro analogues of DIF-1 (dM-DIF-1 and Cl-THPH, respectively), supporting the previously proposed pathway of DIF-1 biosynthesis. The third compound was a novel factor and was identified as 4-methyl-5-pentylbenzene-1,3-diol (MPBD) with the structure confirmed by chemical synthesis. To investigate the potential roles of these compounds as signal molecules, their effects on morphological stalk and spore differentiation were examined in cell culture. All three induced morphological stalk cell differentiation. We found that synthetic MPBD also stimulated spore cell differentiation. Now that these factors are known to be produced and released during development, their biological roles can be pursued further.     

CulB,a putative ubiquitin ligase subunit,regulates prestalk cell differentiation and morphogenesis in Dictyostelium spp

Dictyostelium amoebae accomplish a starvation-induced developmental process by aggregating into a mound and forming a single fruiting body with terminally differentiated spores and stalk cells. culB was identified as the gene disrupted in a developmental mutant with an aberrant prestalk cell differentiation phenotype. The culB gene product appears to be a homolog of the cullin family of proteins that are known to be involved in ubiquitin-mediated protein degradation. The culB mutants form supernumerary prestalk tips atop each developing mound that result in the formation of multiple small fruiting bodies. The prestalk-specific gene ecmA is expressed precociously in culB mutants, suggesting that prestalk cell differentiation occurs earlier than normal. In addition, when culB mutant cells are mixed with wild-type cells, they display a cell-autonomous propensity to form stalk cells. Thus, CulB appears to ensure that the proper number of prestalk cells differentiate at the appropriate time in development. Activation of cyclic AMP-dependent protein kinase (PKA) by disruption of the regulatory subunit gene (pkaR) or by overexpression of the catalytic subunit gene (pkaC) enhances the prestalk/stalk cell differentiation phenotype of the culB mutant. For example, culB⁻ pkaR⁻ cells form stalk cells without obvious multicellular morphogenesis and are more sensitive to the prestalk O (pstO) cell inducer DIF-1. The sensitized condition of PKA activation reveals that CulB may govern prestalk cell differentiation in Dictyostelium, in part by controlling the sensitivity of cells to DIF-1, possibly by regulating the levels of one or more proteins that are rate limiting for prestalk differentiation.