Prespore Cells Research Articles

Inducible nuclear translocation of a STAT protein in Dictyostelium prespore cells: implications for morphogenesis and cell-type regulation.

Dd-STATa, the Dictyostelium STAT (signal transducer and activator of transcription) protein, is selectively localised in the nuclei of a small subset of prestalk cells located in the slug tip. Injection of cAMP into the extracellular spaces in the rear of the slug induces rapid nuclear translocation of a Dd-GFP:STATa fusion protein in prespore cells surrounding the site of injection. This suggests that cAMP signals that emanate from the tip direct the localised nuclear accumulation of Dd-STATa. It also shows that prespore cells are competent to respond to cAMP, by Dd-STATa activation, and it implies that cAMP signalling is in some way limiting in the rear of the slug. Co-injection of a specific inhibitor of the cAR1 serpentine cAMP receptor almost completely prevents the cAMP-induced nuclear translocation, showing that most or all of the cAMP signal is transduced by cAR1. Dd-GFP:STATa also rapidly translocates into the nuclei of cells adjoining the front and back cut edges when a slug is bisected. Less severe mechanical disturbances, such as pricking the rear of a slug with an unfilled micropipette, also cause a more limited nuclear translocation of Dd-GFP:STATa. We propose that these signalling events form part of a repair mechanism that is activated when the migrating slug suffers mechanical damage.

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.

The carboxyl terminus of Dictyostelium discoideum protein 1I encodes a functional glycosyl-phosphatidylinositol signal sequence

The 1I gene is expressed in the prespore cells of culminating Dictyostelium discoideum. The open reading frame of 1I cDNA encodes a protein of 155 amino acids with hydrophobic segments at both its NH 2- and COOH-termini that are indicative of a glycosyl-phosphatidylinositol (GPI)-anchored protein. A hexaHis-tagged form of 1I expressed in D. discoideum cells appeared on Western blot analysis as a doublet of 27 and 24 kDa, with a minor polypeptide of 22 kDa. None of the polypeptides were released from the cell surface with bacterial phosphatidylinositol-specific phospholipase C, although all three were released upon nitrous acid treatment, indicating the presence of a phospholipase-resistant GPI anchor. Further evidence for the C-terminal sequence of 1I acting as a GPI attachment signal was obtained by replacing the GPI anchor signal sequence of porcine membrane dipeptidase with that from 1I. Two constructs of dipeptidase with the 1I GPI signal sequence were constructed, one of which included an additional six amino acids in the hydrophilic spacer. Both of the resultant constructs were targeted to the surface of COS cells and were GPI-anchored as shown by digestion with phospholipase C, indicating that the Dictyostelium GPI signal sequence is functional in mammalian cells. Site-specific antibodies recognising epitopes either side of the expected GPI anchor attachment site were used to determine the site of GPI anchor attachment in the constructs. These parallel approaches show that the C-terminal signal sequence of 1I can direct the addition of a GPI anchor.

How amoeboids self-organize into a fruiting body: multicellular coordination in Dictyostelium discoideum.

When individual amoebae of the cellular slime mold Dictyostelium discoideum are starving, they aggregate to form a multicellular migrating slug, which moves toward a region suitable for culmination. The culmination of the morphogenesis involves complex cell movements that transform a mound of cells into a globule of spores on a slender stalk. The movement has been likened to a "reverse fountain," whereby prestalk cells in the upper part form a stalk that moves downwards and anchors to the substratum, while prespore cells in the lower part move upwards to form the spore head. So far, however, no satisfactory explanation has been produced for this process. Using a computer simulation that we developed, we now demonstrate that the processes that are essential during the earlier stages of the morphogenesis are in fact sufficient to produce the dynamics of the culmination stage. These processes are cAMP signaling, differential adhesion, cell differentiation, and production of extracellular matrix. Our model clarifies the processes that generate the observed cell movements. More specifically, we show that periodic upward movements, caused by chemotactic motion, are essential for successful culmination, because the pressure waves they induce squeeze the stalk downwards through the cell mass. The mechanisms revealed by our model have a number of self-organizing and self-correcting properties and can account for many previously unconnected and unexplained experimental observations.

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.

Calcium stores in differentiated Dictyostelium discoideum: prespore cells sequester calcium more efficiently than prestalk cells

Dictyostelium discoideum pseudoplasmodia exhibit a gradient of the cytosolic free Ca2+-concentration ([Ca2+]i) along their anterior–posterior axis involved in cell-type specific differentiation. [Ca2+]iis high in prestalk and low in prespore cells. We determined the content and localization of calcium and other elements in cryosectioned cells of pseudoplasmodia and fruiting bodies by X-ray microanalysis. Granular stores rich in Ca, Mg and P were ident ified. Average Ca was higher in prespore than prestalk granules (225vs 111mmol/kg dry weight). Total Ca stored in granules was also higher in prespore than prestalk cells. The amount of P and S in granules differed between the two cell types indicating different store composition. In spores mean granular Ca was 120mmol/kg dry weight. Stalk cells had smaller granules with 360mmol Ca/kg dry weight. Complementary to microanalysis, vesicular Ca2+-fluxes were studied in fractionated cell homogenates. The rate of Ca2+-uptake was higher in pellet fractions of prespore than prestalk amoebae (4.7 vs 3.4nmol/minemg). Ca2+-release was greater in supernatant fractions from prestalk than prespore cells (16.5vs 7.7nmol/108cells). In summary, prestalk and prespore cells possess qualitatively different, high-capacity stores containing distinct amounts of Ca and probably being involved in regulation of the anterior–posterior [Ca2+]i-gradient.

A genetic interaction between a ubiquitin-like protein and ubiquitin-mediated proteolysis in Dictyostelium discoideum

A ubiquitination factor, NosA, is essential for cellular differentiation in Dictyostelium discoideum. In the absence of nosA, development is blocked, resulting in a developmental arrest at the tight-aggregate stage, when cells differentiate into two precursor cell types, prespore and prestalk cells. Development is restored when a second gene, encoding the ubiquitin-like protein SonA, is inactivated in nosA-mutant cells. SonA has homology over its entire length to Dsk2 from Saccharomyces cerevisiae, a ubiquitin-like protein that is involved in the assembly of the spindle pole body. Dsk2 and SonA are both stable proteins that do not seem to be subjected to degradation via the ubiquitin pathway. SonA does not become ubiquitinated and the intracellular levels of SonA are not affected by the absence of NosA. The high degree of suppression suggests that SonA rescues most or all of the defects caused by the absence of nosA. We propose that NosA and SonA act in concert to control the activity of a developmental regulator that must be deactivated for cells to cross a developmental boundary.

Rapid patterning in 2-D cultures of Dictyostelium cells and its relationship to zonal differentiation.

Rapid patterning has been observed in confined 2-D cultures of Dictyostelium discoideum Ax-2 cells as an outer dark zone and a inner light zone. The width of outer zone was usually approximately100 microm, irrespective of the size of cell masses under atmospheric conditions. The width of the outer zone, however, changed depending on external O2 concentrations and reached up to 250 microm at 100% O2. A clear regional difference in tetramethyl rhodamine methyl ester (TMRM) staining was noticed between the outer zone and the inner zone: the inner zone was more strongly stained with TMRM than the outer zone, which faced the air. Using inhibitors of oxidative phosphorylation (dinitrophenol (DNP) or NaN3) and a specific inhibitor of CN-resistant respiration (benzohydroxamic acid (BHAM)), it has been demonstrated that the outer zone is basically formed by the O2 threshold for oxidative phosphorylation, while the inner cells mainly perform cyanide-resistant respiration. When cells around the early mound stage (just before prestalk and prespore differentiation) were cultured as 2-D cell masses, ecmA-expressing cells (pstA cells), ecmB-expressing cells (pstB cells) and D19-expressing cells (prespore; psp cells), arose in a position-dependent manner in the outer zone. In the inner zone, cell motility seemed to be markedly impaired and neither prestalk nor prespore differentiation occurred. In addition, once-differentiated prespore cells were found to dedifferentiate rapidly in the inner zone. The reason for dedifferentiation as well as for failure of cells to differentiate in the inner zone is discussed with reference to O2 radicals.

Cell-Fate Choice in Dictyostelium: Intrinsic Biases Modulate Sensitivity to DIF Signaling

Cell fate in Dictyostelium development depends on intrinsic differences between cells, dating from their growth period, and on cell interactions occurring during development. We have sought for a mechanism linking these two influences on cell fate. First, we confirmed earlier work showing that the vegetative differences are biases, not commitments, since cells that are stalky-biased when developed with one partner are sporey with another. Then we tested the idea that these biases operate by modulating the sensitivity of cells to the signals controlling cell fate during development. Cells grown without glucose are stalky-biased when developed with cells grown with glucose. We find, using monolayer culture conditions, that they are more sensitive to each of the stalk-inducing signals, DIFs 1–3. Mixing experiments show that this bias is a cell-intrinsic property. Cells initiating development early in the cell cycle are stalky compared to those initiating development later in the cycle. Likewise, they are more sensitive to DIF-1. Assays of standard markers for prestalk and prespore cell differentiation reveal similar differences in DIF-1 sensitivity between biased cells; DIF-1 dechlorinase (an early prestalk cell marker enzyme) behaves in a consistent manner. We propose that cell-fate biases are manifest as differences in sensitivity to DIF.

Identification of STKA-dependent genes in Dictyostelium discoideum

During culmination of Dictyostelium aggregates, prespore and prestalk cells undergo terminal differentiation to form spores and a cellular stalk. Disruption of the cell-fate gene stkA leads to a phenotype in which all the cells destined to become spores end up as stalk cells. ‘Stalky’ mutants express normal levels of prespore cell transcripts but fail to produce the culmination-stage spore transcript spiA. The stkA gene encodes a putative GATA-type transcription factor (STKA). In order to identify possible downstream targets of STKA we used the technique of mRNA differential display and isolated four cDNA fragments that hybridise to mRNAs present during the later stages of development. All four gene tags were cloned and sequenced. mRNAs represented by these four sequence tags do not accumulate during culmination of ‘stalky’ cells and therefore must be specific to the spore pathway. By screening a cDNA library, longer cDNAs for all four were cloned and sequenced. Three of these contained complete protein-coding regions while only a partial cDNA was recovered for the fourth. One of the corresponding proteins has significant homology to a surface zinc metalloproteinase (GP63) of the protozoan parasite Leishmania, while another is closely related to a human pre-RNA binding protein (hnRNP R).

Protostelids of Macquarie Island

This is the first report of protostelids from Macquarie Island a small, oceanic island located 1000 km southeast of Tasmania, just north of the Antarctic Convergence. It is the southernmost locality from which they have been recovered. Litter samples collected under stands of Pleurophyllum hookeri and of Acaena spp. were plated out using standard techniques for protostelid identification. Six species were identified by sporocarp morphology using light microscopy, Protostelium mycophaga, P. nocturnum, Tychosporium acutostipes, Schizoplasmodiopsis pseudoendospora, Sc. amoeboidea, and Soliformovum irregularis. Amoebal and prespore cell morphology were used whenever necessary to corroborate identification. It is of interest that most of these six species rarely occur in litter in temperate ecosystems and that the Macquarie Island samples lack any typical litter species such as Nematostelium spp.

A cell type-specific effect of calcium on pattern formation and differentiation in dictyostelium discoideum.

Spatial gradients of sequestered and free cellular calcium (Ca2+) exist in the slug of Dictyostelium discoideum (Maeda and Maeda, 1973; Tirlapur et al., 1991; Azhar et al., 1995; Cubitt et al., 1995). When we vary intracellular Ca2+ with the help of calcium buffers and the ionophore Br-A23187, there are striking effects on slug morphology, patterning and cell differentiation. In the presence of a calcium ionophore, high external Ca2+ levels lead to an increase of intracellular sequestered and free Ca2+, the formation of long slugs, a decrease in the fraction of genetically defined prespore cells and 'stalky' fruiting bodies. Conversely, a lowering of external Ca2+ levels results in a decrease of intracellular Ca2+, the formation of short slugs, an increase in the prespore fraction and 'spory' fruiting bodies. We infer that Ca2+ plays a significant morphogenetic role in D. discoideum development, by selectively promoting the prestalk pathway relative to the prespore pathway.

The control of chemotactic cell movement during Dictyostelium morphogenesis.

Differential cell movement is an important mechanism in the development and morphogenesis of many organisms. In many cases there are indications that chemotaxis is a key mechanism controlling differential cell movement. This can be particularly well studied in the starvation-induced multicellular development of the social amoeba Dictyostelium discoideum. Upon starvation, up to 10(5) individual amoebae aggregate to form a fruiting body The cells aggregate by chemotaxis in response to propagating waves of cAMP, initiated by an aggregation centre. During their chemotactic aggregation the cells start to differentiate into prestalk and prespore cells, precursors to the stalk and spores that form the fruiting body. These cells enter the aggregate in a random order but then sort out to form a simple axial pattern in the slug. Our experiments strongly suggest that the multicellular aggregates (mounds) and slugs are also organized by propagating cAMP waves and, furthermore, that cell-type-specific differences in signalling and chemotaxis result in cell sorting, slug formation and movement.

Differential developmental expression and cell type specificity of Dictyostelium catalases and their response to oxidative stress and UV-light

Cells of Dictyostelium discoideum are highly resistant to DNA damaging agents such as UV-light, γ-radiation and chemicals. The genes encoding nucleotide excision repair (NER) and base excision repair (BER) enzymes are rapidly upregulated in response to UV-irradiation and DNA-damaging chemicals, suggesting that this is at least partially responsible for the resistance of this organism to these agents. Although Dictyostelium is also unusually resistant to high concentrations of H 2O 2, little is known about the response of this organism to oxidative stress. To determine if transcriptional upregulation is a common mechanism for responding to DNA-damaging agents, we have studied the Dictyostelium catalase and Cu/Zn superoxide dismutase antioxidant enzymes. We show that there are two catalase genes and that each is differentially regulated both temporally and spatially during multicellular development. The catA gene is expressed throughout growth and development and its corresponding enzyme is maintained at a steady level. In contrast, the catB gene encodes a larger protein and is only expressed during the final stages of morphogenesis. Cell type fractionation showed that the CatB enzyme is exclusively localized to the prespore cells and the CatA enzyme is found exclusively in the prestalk cells. Each enzyme has a different subcellular localization. The unique developmental timing and cell type distribution suggest that the role for catB in cell differentiation is to protect the dormant spores from oxidative damage. We found that exposure to H 2O 2 does not result in the induction of the catalase, superoxide dismutase, NER or BER mRNAs. A mutant with greatly reduced levels of catA mRNA and enzyme has greatly increased sensitivity to H 2O 2 but normal sensitivity to UV. These results indicate that the natural resistance to oxidative stress is not due to an ability to rapidly raise the level of antioxidant or DNA repair enzymes and that the response to UV-light is independent from the response to reactive oxygen compounds.

Analysis of the promoter of the cudA gene reveals novel mechanisms of Dictyostelium cell type differentiation.

The cudA gene encodes a nuclear protein that is essential for normal multicellular development. At the slug stage cudA is expressed in the prespore cells and in a sub-region of the prestalk zone. We show that cap site distal promoter sequences direct cudA expression in prespore cells, while proximal sequences direct expression in the prestalk sub-region. The promoter domain that directs prespore-specific transcription consists of a positively acting region, that has the potential to direct expression in all cells within the slug, and a negatively acting region that prevents expression in the prestalk cells. Dd-STATa is the STAT protein that regulates commitment to stalk cell gene expression, where it is known to function as a transcriptional repressor. We show that Dd-STATa binds in vitro to the positively acting part of the prespore domain of the cudA promoter. However, Dd-STATa cannot be utilised for this purpose in vivo, because analysis of a Dd-STATa null mutant strain shows that Dd-STATa is not necessary for cudA transcription in prespore cells. In contrast, the part of the cudA promoter that directs prestalk-specific expression contains a binding site for Dd-STATa that is essential for its biological activity. Dd-STATa appears therefore to serve as a direct activator of cudA transcription in prestalk cells, while a protein with a DNA binding specificity highly related to that of Dd-STATa is utilised to activate cudA transcription in prespore cells.

Cell type specificity and mechanism of control of a gene may be reverted in different strains of Dictyostelium discoideum

Twelve genes which are expressed exclusively in pre-spore cells of Dictyostelium strain AX3 are expressed exclusively in pre-stalk cells of strain AX2. One gene has the opposite behavior: it is expressed in pre-stalk cells in AX3 and in pre-spore cells in AX2. The change in cell type specificity involves a change in the mechanism of control of gene expression. When they are expressed in pre-stalk cells, genes are controlled at the level of transcription, whilst in pre-spore cells, they are controlled at the level of mRNA stability. Genes expressed in pre-stalk cells in strain AX2, fused with an AX2 pre-spore specific promoter, become regulated at the level of mRNA stability. These findings indicate that at least a group of pre-stalk mRNAs possess the cis-destabilizing element typical of pre-spore mRNAs, though they are not destabilized in disaggregated cells. This is due to the fact that ribosomal protein S6, phosphorylation of which is responsible for controlling the stability of pre-spore mRNAs, is not dephosphorylated in disaggregated pre-stalk cells. These cells lack an S6 phosphatase activity which has been purified from disaggregated pre-spore cells.

Evidence That a Cell-Type-Specific Efflux Pump Regulates Cell Differentiation in Dictyostelium

We have identified a cellular efflux pump, RhT, with the properties of an MDR transporter—a type of ATP-binding cassette transporter whose substrates include small hydrophobic molecules. RhT transports rhodamine 123 (Rh123) and is inhibited by low temperature, energy poisons, and several MDR transport inhibitors, such as verapamil. All vegetative cells have RhT activity, but during development prestalk cells lose RhT activity while prespore cells retain it. We also identified several RhT inhibitors. The most effective inhibitor is the stalk cell-inducing chlorinated alkyl phenone, DIF-1. The RhT inhibitors disrupted development, to varying degrees, and induced stalk cell formation in submerged culture. The inhibitors displayed the same rank order of pharmacological efficacy for stalk cell induction as they did for Rh123 transport inhibition. We also found that cerulenin, a specific inhibitor of DIF-1 biosynthesis (R. R. Kay, 1998, J. Biol. Chem. 273, 2669–2675), abolished the induction of stalk cells by each of the RhT inhibitors, and this effect could be reversed by DIF-1. Thus, DIF-1 synthesis appears to be required for the induction of stalk cells by the RhT inhibitors. Since DIF-1 is the most potent inhibitor of RhT activity, and thus a likely transport substrate itself, we propose that RhT inhibitors induce stalk cell differentiation by blocking DIF-1 export, causing DIF-1 to build up within cells. Our results provide evidence for a prespore-specific efflux pump that regulates cell fate determination, perhaps by regulating the cellular concentration of DIF-1.

The Regulative Capacity of Prespore Amoebae as Demonstrated by Fluorescence-Activated Cell Sorting and Green Fluorescent Protein

The ability of prespore Dictyostelium discoideum amoebae to undergo redifferentiation so as to reestablish normal spore/stalk proportioning has been demonstrated in various ways over the years, beginning with the classic microdissection work of K. Raper. The discovery of anterior-like cells in the slug posterior, however, cast doubt on that ability, and more recent experiments using a cell-specific toxin suggested that prespore redifferentiation may not in fact occur. To reexamine this question, we performed fluorescence-activated cell sorting (FACS) upon amoebae expressing a mutated green fluorescent protein gene (S65T-GFP) under the control of a prespore-specific (PsA) promoter. FACS produced prespore cell populations with purities, measured by GFP expression, as high as 99.5%. Sorted GFP+ cells were developmentally competent and produced normally proportioned fruits, indistinguishable from those of “sham-sorted” (permissively gated, mixed GFP+ and GFP−) amoebae. This result confirms the developmental totipotency of prespore amoebae.

DIF signalling and cell fate

The DIFs are a family of secreted chlorinated molecules that control cell fate during development ofDictyostelium cells in culture and probably during normal development too. They induce stalk cell differentiation and suppress spore cell formation. The biosynthetic and inactivation pathways of DIF-1 (the major bioactivity) have been worked out. DIF-1 is probably synthesised in prespore cells and inactivated in prestalk cells, by dechlorination. Thus, each cell type tends to alter DIF-1 level so as to favour differentiation of the other cell type. This relationship leads to a model for cell-type proportioning during normal development.

A linking function for the cellulose-binding protein SP85 in the spore coat of Dictyostelium discoideum.

SP85 is a multidomain protein of the Dictyostelium spore coat whose C-terminal region binds cellulose in vitro. To map domains critical for localizing SP85 and for binding to other proteins in vivo, its N- and C-terminal regions, and a hybrid fusion of the N- and C-regions, were expressed in prespore cells. Immunofluorescence showed that only the N-terminal region and the N/C-hybrid accumulated in prespore vesicles, where coat proteins are normally stored prior to secretion. In contrast, only the C-terminal region and N/C-hybrid were incorporated into the coat after secretion. To determine if SP85 is important for the incorporation of other coat proteins, an SP85-null strain was created and found to mislocalize the coat protein SP65 to the interspore matrix. In vitro binding studies demonstrated that the SP85 C-terminal region bound SP65 and cellulose simultaneously, and SP65 incorporation was rescued in vivo by the C-terminal region. SP85-null spores showed increased latent permeability to a fluorescent lectin probe and accelerated germination times, and decreased buoyant density of their coats, suggesting that coat barrier functions were compromised. Dominant negative reductions in barrier functions also resulted from expression of the SP85 terminal regions, suggesting that a linking activity was important for SP85's function. Thus, separate domains of SP85 specify prespore vesicle compartmentalization and coat incorporation, and additional domains link SP65 to the coat and simultaneously interact with other binding partners which contribute to coat barrier functions.