Aggregation in Dictyostelium discoideum

Cultured peritoneal macrophages have previously been shown to release a potent mitogen for mesenchymal cells. Peritoneal macrophages are derived from peripheral blood monocytes, one of the principal inflammatory cells associated with numerous tissue responses to injury. Cultured human monocytes can be activated by endotoxin or concanavalin A to secrete a potent growth factor(s) that is active on human smooth muscle cells, human fibroblasts and 3T3 cells. The optimal conditions for activation of monocyte release of this monocyte-derived growth factor(s) (MDGF) were to expose 5-day-old monocyte cultures (initially plated at 6.8 × 10⁵ cells/ml medium) to 10 μg/ml endotoxin or 6 μg/ml concanavalin A for approximately 20 hr. Monocytes can secrete MDGF into serum-free medium supplemented with 0.15% bovine serum albumin. MDGF stimulates both DNA synthesis and increase in cell number and is trypsin-sensitive, heat labile and nondialyzable. The relationship of MDGF to other monocyte products and its potential importance in wound repair and atherogenesis are discussed.     

Aggregation during sexual development in Dictyostelium discoideum.

A microcinematographic analysis of the behaviour and movements of cells and cell masses in mated cultures (NC4 X VI2) of Dictyostelium discoideum indicates that a chemotactic process directs cell aggregation during macrocyst development. Zygote giant cells form before aggregation begins and act as the aggregation centres. Young multicellular macrocyst stages are sources of cyclic AMP, and amoebae from macrocyst cultures orient chemotactically to cyclic AMP. The data, coupled with other characteristics such as pulsatile streaming, suggest that the aggregation process leading to macrycyst development is the same as that occurring during fruit construction. Other aspects of sexual development are also discussed. Based upon these data, we propose a model for the sequence of events leading to macrocyst development in D. discoideum.     

Cell type specific inhibition of cAMP phosphodiesterase activity during terminal differential in Dictyostelium discoideum

Cyclic AMP phosphodiesterase (PDE) activity reaches a peak during the aggregation stage of development where it functions to regulate extracellular levels of cAMP. During the subsequent differentiation of the two cell types at the culmination stage, the activity reappears but only in stalk cells. We found that extracts from the culmination stage contained PDE which could be activated by preincubation with Mg2+ and dithiothreitol (DTT), a treatment which is known to release an endogenous inhibitor from the aggregation stage enzyme. When the culmination stage extracts were subjected to chromatography on Biogel P300, two peaks of activity were eluted, PDE-I (Mr greater than 260,000) and PDE-II (Mr 100,000). Treatment of the fractions with Mg-DTT did not affect the low-molecular-weight enzyme but caused activation of the high-molecular-weight enzyme and the appearance of a third, intermediate form. Kinetic analysis of the two peaks revealed Km values for cAMP of 2 mM and 10 microM for PDE-I and PDE-II, respectively. We tested the possibility that these forms of the enzyme might be distributed differently in the two cell types by measuring the Km for cAMP and the effect of Mg-DTT treatment on isolated sections of stalk and spore cells. The spore sections contained a high Km form of the enzyme (0.3 mM) which was activated by preincubation with Mg . DTT whereas stalk sections contained a low Km form (3 microM) which was not affected by the activation treatment. We conclude that both cell types contain enzyme protein and that the apparent localization of PDE activity in stalk cells is due to the inhibition of activity in spore cells.     

Measurements of metabolites during cAMP oscillations of Dictyostelium discoideum

During aggregation the cellular slime mold Dictyostelium discoideum synthesizes and releases pulses of cAMP about every five minutes. Current models proposed to explain this phenomenon postulate that oscillating levels of some key intracellular metabolite control the oscillatory synthesis of cAMP. We have assayed the levels of likely candidates for this metabolite during a cAMP oscillation, but have found them to remain constant. Compounds measured include ATP, GTP, glucose-1-phosphate, glucose-6-phosphate, isocitrate, α-ketoglutarate, amino acids, and other aminated metabolites. On the basis of this negative data, as well as results described elsewhere (Geller and Brenner, 1978), we question whether the proposed models are correct, and discuss several alternatives.     

Adaptation in the motility response to cAMP in Dictyostelium discoideum

When developing amebae of Dictyostelium discoideum are treated with constant concentrations of cAMP above 10(-8)M, the average rate of motility is depressed, with maximum inhibition at roughly 10(-6)M. It is demonstrated that shifting the concentration of cAMP from 0 M to concentrations ranging from 10(-8) to 10(-6)M in a perfusion chamber results in the immediate inhibition of motility. After shifting from 0 M to 10(-8) or 10(-7)M, the rate of cell motility remains low, then rebounds to a higher level, exhibiting a standard adaptation response. No adaptation is exhibited after a shift from 0 M to 10(-6)M, a concentration resulting in maximum inhibition. It is demonstrated that the level of inhibition and the extent of the adaptation period are dependent upon the concentration of cAMP after the shift, and that submaximal inhibition is additive. The characteristics of adaptation in this motility response are very similar to the characteristics of adaptation for the relay system and phosphorylation of the putative cAMP receptor.     

Cell adhesion during the migratory slug stage of Dictyostelium discoideum

Prespore-specific Antigen (PsA) is selectively expressed on the surface of prespore cells at the multicellular migratory slug stage of Dictyostelium discoideum development. It is a developmentally regulated glycoprotein that is anchored to the cell membrane through a glycosyl phosphatidylinositol (GPI) anchor. We present the results of an in vitro immunological investigation of the hypothesis that PsA functions as a cell adhesion molecule (CAM), and of a ligand-binding assay indicating that PsA has cell membrane binding partner(s). This is the first evidence to implicate a direct role for a putative CAM in cell-cell adhesion during the multicellular migratory slug stage of D. discoideum development. Cell-cell adhesion assays were carried out in the presence or absence of the monoclonal antibody (mAb) MUD1 that has a single antigenic determinant: a peptide epitope on PsA. These assays showed specific inhibition of cell-cell adhesion by MUD1. Further, it was found that a purified recombinant form of PsA (rPsA), can neutralize the inhibitory effect of MUD1; the inhibitory effect on cell-cell adhesion is primarily due to the blocking of PsA by the mAb. The resistance of aggregates to dissociation in the presence of 10 mM EDTA (ethylenediamintetraacetic acid) indicates that PsA mediates EDTA-stable cell-cell contacts, and that PsA-mediated cell adhesion is likely to be independent of divalent cations such as Ca(2+) or Mg(2+).     

Ligand-induced phosphorylation of the cAMP receptor from Dictyostelium discoideum

The cell surface cAMP receptor of Dictyostelium discoideum exists as a doublet of low (D) and high (R) electrophoretic mobility forms, both of which are phosphorylated in vivo. The R form is phosphorylated in a ligand-independent manner, while conversion of the R to D forms, induced by the chemoattractant, is accompanied by at least a 4-fold increase in the level of phosphorylation. When cells are stimulated with saturating levels of cAMP, increased phosphorylation is detectable within 5 s and reaches maximum levels by 5 min with a t1/2 of 45 s. Dephosphorylation of receptor, initiated by removal of the stimulus, is detectable within 30 s, has a half-time of 2 min, and reaches a plateau by 20 min. At half-maximal occupancy, phosphorylation occurred more slowly than at saturation, t1/2 = 1.5 min, and remained at intermediate levels until the cAMP concentration was increased. Accompanying electrophoretic mobility shifts occurred in all cases with similar, though not identical, kinetics. Both phosphorylation and mobility shift were half-maximal at 5 nM cAMP and saturated at 100 nM. Estimation of the specific activity of each receptor form indicates that not all sites are phosphorylated during the R to D transition; at least half of the sites are phosphorylated after the transition is completed. The rate of incorporation of phosphates into the receptor, held in the D form by cAMP, was less than one-third the rate of ligand-induced incorporation starting with the R form and was approximately twice the basal rate of incorporation. These results are compatible with ligand-induced receptor phosphorylation being an early event in the adaptation of other cAMP-induced responses.     

Cell Differentiation in Dictyostelium discoideum

We have shown previously that amoebae of D. discoideum strain V12 M2 starved at low density in the presence of cyclic AMP fail to form either stalk cells or prespore cells; a low molecular weight factor released by cells at high density promotes stalk formation under these conditions but formation of prespore cells requires 'cell contact'. Here we summarise evidence that:
1. Elevated intracellular cyclic AMP levels are required for all developmental gene expression beyond the preaggregative phase, and ammonia antagonises this expression in some way. However, the action of ammonia is not pathway specific.
2.'Cell contact' is a specific requirement for entry into the prespore pathway of gene expression since isolated cells provided with cyclic AMP synthesise much reduced amounts of the presporespecific enzyme uridine diphosphate (UDP) galactose polysaccharide transferase but normal amounts of the pathway-indifferent enzyme glycogen phosphorylase.
3. The 'cell contact' mechanism is uniquely sensitive to low concentrations of pronase. This protease selectively inhibits transferase synthesis and blocks in vitro spore differentiation (in a spore-forming mutant). It does not prevent chemotactic aggregation, stream formation, or stalk cell formation in the presence of cyclic AMP.     

The cAMP signal from Dictyostelium discoideum amoebae.

Dictyostelium discoideum cells were allowed to differentiate on agar for 600 min at room temperature. All of the cells were then competent to relay or amplify a cAMP signal, but none to produce a cAMP signal autonomously. The cells were stimulated with cAMP concentrations ranging from 10^?9 to 3.5 × 10^?7M. Populations of 10⁶ cells could amplify an initial cAMP concentration of 2.5 × 10^?9M with a low probability, while an initial cAMP concentration of 5 × 10^?8M always induced a response. An initial cAMP concentration of 1.2 × 10^?7M induced the maximum cellular release of cAMP observed; this corresponded to 3 × 10⁷ molecules per cell. No cellular release of cAMP was detected for initial cAMP concentrations of 3 × 10^?7M or more. The amplification of a 10^?7M cAMP stimulus was complete within 8 sec, indicating the pulsatile nature of the cellular release of cAMP. The phosphodiesterase (PDE) activities of D. discoideum cells were measured over a wide range of cell densities. At densities above 7.5 × 10⁴ cells/cm², both cell-bound and extracellular (ePDE) activities declined, per cell, as cell density increased. These results are compared to ePDE activities derived from critical density measurements. We found that PDE activities were in the range of 10^?13–10^?14 moles of cAMP converted/cell/min under culture conditions consistent with normal aggregation.     

Cell-type-specific responsiveness to cAMP in cell differentiation of Dictyostelium discoideum.

In Dictyostelium discoideum, both prespore and prestalk differentiation require extracellular cAMP. We investigated the difference in inducibility of the two cell types by cAMP. Previous studies indicate that cAMP added in the early stage of development inhibits prespore differentiation, and this was confirmed using three species of prespore specific mRNAs. By contrast, early treatment with cAMP did not inhibit, but induced the expression of prestalk-specific mRNA. These results indicate that differentiation pathways of the two cell types have different processes in the early stage of development.     

Cell Volume Determinations of Dictyostelium discoideum

The intercellular water present in pellets of centrifuged cell suspensions of the slime mold Dictyostelium discoideum was measured at four stages of differentiation.     

Characterization of a cAMP-stimulated cAMP phosphodiesterase in Dictyostelium discoideum

A cyclic nucleotide phosphodiesterase, PdeE, that harbors two cyclic nucleotide binding motifs and a binuclear Zn(2+)-binding domain was characterized in Dictyostelium. In other eukaryotes, the Dictyostelium domain shows greatest homology to the 73-kDa subunit of the pre-mRNA cleavage and polyadenylation specificity factor. The Dictyostelium PdeE gene is expressed at its highest levels during aggregation, and its disruption causes the loss of a cAMP-phosphodiesterase activity. The pdeE null mutants show a normal cAMP-induced cGMP response and a 1.5-fold increase of cAMP-induced cAMP relay. Overexpression of a PdeE-yellow fluorescent protein (YFP) fusion construct causes inhibition of aggregation and loss of the cAMP relay response, but the cells can aggregate in synergy with wild-type cells. The PdeE-YFP fusion protein was partially purified by immunoprecipitation and biochemically characterized. PdeE and its Dictyostelium ortholog, PdeD, are both maximally active at pH 7.0. Both enzymes require bivalent cations for activity. The common cofactors Zn(2+) and Mg(2+) activated PdeE and PdeD maximally at 10 mm, whereas Mn(2+) activated the enzymes to 4-fold higher levels, with half-maximal activation between 10 and 100 microm. PdeE is an allosteric enzyme, which is approximately 4-fold activated by cAMP, with half-maximal activation occurring at about 10 microm and an apparent K(m) of approximately 1 mm. cGMP is degraded at a 6-fold lower rate than cAMP. Neither cGMP nor 8-Br-cAMP are efficient activators of PdeE activity.     

The Social Amoeba Dictyostelium discoideum Is Highly Resistant to Polyglutamine Aggregation

The expression, misfolding, and aggregation of long repetitive amino acid tracts are a major contributing factor in a number of neurodegenerative diseases, including C9ORF72 amyotrophic lateral sclerosis/frontotemporal dementia, fragile X tremor ataxia syndrome, myotonic dystrophy type 1, spinocerebellar ataxia type 8, and the nine polyglutamine diseases. Protein aggregation is a hallmark of each of these diseases. In model organisms, including yeast, worms, flies, mice, rats, and human cells, expression of proteins with the long repetitive amino acid tracts associated with these diseases recapitulates the protein aggregation that occurs in human disease. Here we show that the model organism Dictyostelium discoideum has evolved to normally encode long polyglutamine tracts and express these proteins in a soluble form. We also show that Dictyostelium has the capacity to suppress aggregation of a polyglutamine-expanded Huntingtin construct that aggregates in other model organisms tested. Together, these data identify Dictyostelium as a novel model organism with the capacity to suppress aggregation of proteins with long polyglutamine tracts.     

Cell-cell contact mediates cAMP secretion in Dictyostelium discoideum.

Cyclic adenosine 3':5' monophosphate (cAMP) and cell-cell contact regulate developmental gene expression in Dictyostelium discoideum. Developing D. discoideum amoebae synthesize and secrete cAMP following the binding of cAMP to their surface cAMP receptor, a response called cAMP signaling. We have demonstrated two responses of developing D. discoideum amoebae to cell-cell contact. Cell-cell contact elicits cAMP secretion and alters the amount of cAMP secreted in a subsequent cAMP signaling response. Depending upon experimental conditions, bacterial-amoebal contact and amoebal-amoebal contact can enhance or diminish the amount of cAMP secreted during a subsequent cAMP signaling response. We have hypothesized that cell-cell contact regulates D. discoideum development by altering cellular and extracellular levels of cAMP. To begin testing this hypothesis, these responses were further characterized. The two responses to cell-cell contact are independent, i.e., they can each occur in the absence of the other. The responses to cell-cell contact also have unique temperature dependences when compared to each other, cAMP signaling, and phagocytosis. This suggests that these four responses have unique steps in their transduction mechanisms. The secretion of cAMP in response to cell-cell contact appears to be a non-specific response; contact between D. discoideum amoebae and Enterobacter aerogenes, latex beads, or other amoebae elicits cAMP secretion. Despite the apparent similarities of the effects of bacterial-amoebal and amoebal-amoebal contact on the cAMP signaling response, this contact-induced response appears to be specific. Latex beads addition does not alter the magnitude of a subsequent cAMP signaling response.(ABSTRACT TRUNCATED AT 250 WORDS)     

Reduced cAMP secretion in Dictyostelium discoideum mutant HB3

Extracellular cAMP induces the intracellular synthesis and subsequent secretion of cAMP in Dictyostelium discoideum (relay). cAMP relay was strongly diminished in mutant HB3 which shows abnormal development by making very small fruiting bodies. Extracellular cAMP binds to receptors on the surface of mutant cells and induces the rapid activation of adenylate cyclase. Intracellular cAMP rises to a concentration as high as that in wild-type cells but only a very small amount of cAMP is secreted. cAMP secretion in wild-type cells starts immediately after cAMP production, and is proportional to the intracellular cAMP concentration. In the mutant cells cAMP secretion starts a few minutes after cAMP production; by that time most of the intracellular cAMP is already degraded by phosphodiesterase and little cAMP is available for secretion. We conclude that mutant HB3 has a defect in the mechanism by which Dictyostelium cells secrete cAMP.     

Chemoresponsiveness to cAMP and folic acid during growth, development, and dedifferentiation in Dictyostelium discoideum

Chemoresponsiveness to cAMP and to folic acid are monitored in growing, developing, and dedifferentiating amebae of the cellular slime mold Dictyostelium discoideum. Two semiquantitative assays are employed, one measuring the directed movement of cells up a gradient of chemoattractant ('chemotaxis' assay) and the other measuring the outward spreading of cells in response to a chemical stimulant distributed equally throughout the substratum ('spreading' assay). Vegetative amebae possess relatively insignificant levels of chemotactic responsiveness to cAMP. Six h after the initiation of development, at approximately the same time as the onset of aggregation, cells rapidly acquire chemotactic responsiveness to cAMP. During 'erasure', a dedifferentiation induced by resuspending aggregating cells in fresh nutrient medium, chemotactic responsiveness to cAMP is lost just after the erasure event. By the same chemotactic assay, it is demonstrated that vegetative amebae possess a significant level of chemotactic responsiveness to folic acid. Two h after the initiation of development, cells completely lose chemotactic responsiveness to folic acid. During erasure, cells reacquire chemotactic responsiveness to folic acid at approximately the same time that they lose responsiveness to cAMP. Dramatically different results are obtained by the spreading assay. When cells lose chemotactic responsiveness to folic acid early in development and when erasing cells lose chemotactic responsiveness to cAMP, they retain the spreading response to the two stimulants, respectively. The different results obtained for chemoreception employing the two assays are discussed in terms of molecular mechanisms, and a testable hypothesis is proposed for the possible roles of chemoresponsiveness and erasure in late morphogenesis.