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)     

Intracellular cAMP in Dictyostelium discoideum: Characterization of an aggregation-deficient mutant with elevated cAMP pools

Forty aggregation-deficient mutants of Dictyostelium discoideum were screened for changes in intracellular cAMP during the first 10 hr of starvation. The pools in 39 of the mutants remained low and relatively static during this period. However, amoebae of one mutant, strain HC151, exhibited significantly elevated levels of intracellular cAMP during vegetative growth and for several hours after starvation. A more detailed analysis of this mutant indicated that the elevated cAMP pools in these cells are a consequence of the premature appearance and partial activation of an adenylate cyclase. The mutation(s) altering adenylate cyclase regulation in this strain appears to map in linkage group IV. Complementation tests between strain HC151 and another mutant, HH201, which has recently been shown to produce an adenylate cyclase activity precociously [1], indicated that the mutations affecting adenylate cyclase activity in these strains map at different loci. Although both of these mutations behave recessively in heterozygous diploids with respect to gross development, an examination of early cAMP metabolism and terminal spore differentiation in these diploids suggest that these mutations are at least partially expressed during some stage(s) of the developmental cycle.     

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.     

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.     

Cyclic 3'',5''-AMP relay in Dictyostelium discoideum III. The relationship of cAMP synthesis and secretion during the cAMP signaling response

Refinement of a perfusion technique permitted the simultaneous measurement of cAMP-elicited [3H]cAMP secretion and intracellular [3H]cAMP levels in sensitive D. discoideum amoebae. These data were compared with measurements of the rate of [32P]cAMP synthesis by extracts of amoebae sonicated at different times during the cAMP signaling response. cAMP stimulation of intact cells led to a transient activation of adenylate cyclase, which was blocked if 10(-4) M NaN3 was added with the stimulus. During responses elicited by 10(-6) M cAMP, 10(-8) M cAMP, and an increment in cAMP from 10(-8) M to 10(-7) M, the rate of cAMP secretion was proportional to the intracellular cAMP concentration. Removal of a 10(-6) M cAMP stimulus 2 min after the initiation of the response led to a precipitous decline in intracellular cAMP. This decline was more rapid than could be accounted for by secretion alone, suggesting intracellular phosphodiesterase destruction of newly synthesized cAMP. Employing these data and a simple rate equation, estimates of the time-course of the transient activation of adenylate cyclase and the rate constants for cAMP secretion and intracellular phosphodiesterase activity were obtained. The calculated rate of cAMP synthesis rose for approximately 1 to 2 min, peaked, and declined to approach prestimulus levels after 3 to 4 min. This time-course agreed qualitatively with direct measurements of the time-course of activation, indicating that the activation of adenylate cyclase is a major in determining the time-course of the cAMP secretion response.     

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.     

Continuous requirement of cAMP for pre-spore differentiation in Dictyostelium discoideum

Abstract Using a shaking culture system, we have previously shown that both cell contact and cAMP are required for pre-spore differentiation in Dictyostelium discoideum [2]. In the present study, cAMP was removed from the medium by the use of a hydrolysing enzyme after cells had formed agglomerates. This treatment left the agglomerates unchanged, but caused a rapid decrease in the activity of UDP galactose transferase, a pre-spore-specific enzyme. This result indicates that cAMP is required even after agglomerate formation to maintain pre-spore differentiation.     

Intracellular localization of secretable cAMP in relaying Dictyostelium discoideum cells

Dictyostelium discoideum cells synthesize and secrete the chemoattractant cAMP within minutes after chemotactic stimulation. During development, this signal-relay process is instrumental in cell aggregation, pattern formation, and differentiation. Cyclic AMP is known to accumulate inside the cell before secretion. In this study we investigated the subcellular localization of the nascent cAMP. After chemotactic stimulation at 0 degrees C and subsequent accumulation of intracellular cAMP, the newly synthesized chemoattractant could be released by gently opening cells in two different ways. Both methods make the cytosolic compartment accessible, whereas intracellular compartments surrounded by a membrane remain largely intact. The first method involved rapid lysis by forced passage through a 5-micron pore-size Nuclepore filter. The second technique was electropermeabilization under carefully controlled conditions that ensured the formation of small, stable pores in the plasma membrane. These pores allowed the passage of small molecules, such as cAMP, but not of macromolecules. To confirm the selectivity for the plasma membrane of both methods, we showed that a typical vesicular cell compartment, the lysosome, remained intact. Both procedures immediately released all intracellularly accumulated cAMP. We interpret our results as strong evidence for accumulation of nascent cAMP in the cytosolic compartment rather than in a vesicular compartment before it is secreted. This implies that cAMP secretion takes place via a trans-membrane transport mechanism, rather than by exocytosis.     

Singular perturbation analysis of cAMP signalling in Dictyostelium discoideum aggregates

In this paper, we use singular perturbation methods to study the structure of travelling waves for some reaction-diffusion models obtained from the Martiel-Goldbeter and Goldbeter-Segel's models of cAMP signalling in Dictyostelium discoideum. As a consequence, we derive analytic formulae for quantities like wave speed, maximum concentration and other magnitudes in terms of the different biochemical constants that appear in the model.     

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.     

Multiple genes for cell surface cAMP receptors in Dictyostelium discoideum

We have cloned and characterized three genes (CAR1, CAR2, CAR3) encoding potential cell surface, cyclic adenosine 3':5' monophosphate (AMP) receptors from Dictyostelium discoideum. The three proteins are predicted to be substantially similar in amino acid sequence throughout most of their transmembrane (TM) and loop domains but are distinctly different in their carboxyl terminal segments. In addition, all three genes possess an intron which interrupts an equivalent codon of TM3. CAR1 is expressed early in development when the cAMP relay system is being established. As development proceeds multiple size forms of CAR1 RNA are detected which apparently result from differences in their 5'-untranslated regions. Late in development levels of CAR1 RNA decrease. In contrast, CAR2 encodes a single sized RNA which is expressed only during postaggregative development. CAR3 expression is approximately 10% of CAR1 during early development, is maximal during tight aggregate formation but declines thereafter. Only one size class of CAR3 mRNA is detected throughout development. Because RNA for each of the three genes is present in postaggregative cells, it was of interest to determine the cell type distribution of each RNA. Gene-specific probes were hybridized to RNAs isolated from cells of Percoll gradient-enriched prespore and prestalk fractions and relative levels of hybridization compared. CAR1 and CAR3 show approximately the same pattern of accumulation; a 3-4 fold enrichment in prestalk cells. CAR2, however, is highly enriched in prestalk cells, more than 10 fold relative to prespore 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.     

A Dictyostelium discoideum DNA fragment complements a Saccharomyces cerevisiae ura3 mutant

Dictyostelium discoideum DNA fragments have been inserted into the chimeric bacterium-yeast plasmid YEp13. Recombinant plasmids were used to transform yeast using a strain of Saccharomyces cerevisiae deficient in OMP decarboxylase activity. Several clones were selected for growth in uracil-free medium. One clone was further analysed and contains a plasmid with a segment of D. discoideum DNA which complements a yeast ura3 mutation.     

cAMP signal transduction pathways regulating development of Dictyostelium discoideum.

Dictyostelium discoideum development is regulated through receptor/G protein signal transduction using cAMP as a primary extracellular signal. Signaling pathways will be discussed as well as the regulation and function of individual cAMP receptors and G alpha subunits. Finally potential downstream targets including protein kinases and nuclear events will be explored.     

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.     

Specific photoaffinity labeling of the cAMP surface receptor in Dictyostelium discoideum

The recent observation that ammonium sulfate stabilizes cell-surface [3H]cyclic AMP binding in Dictyostelium discoideum (Van Haastert, P., and Kien, E. (1983) J. Biol. Chem. 258, 9636-9642) led us to attempt to identify the surface cAMP receptor by photoaffinity labeling with 8-azido-[32P]cAMP using this stabilization technique. 8-azido-[32P]cAMP specifically labeled a polypeptide which migrates as a closely spaced doublet (Mr = 40,000 to 43,000) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Greater than 60% of the labeled polypeptide was found associated with membranes. This protein was distinguished from the cytosolic regulatory subunit of the cAMP-dependent protein kinase (Mr = 41,000) by differences in developmental regulation, specificity, and subcellular localization. No kinase regulatory subunit was detected in membranes by western blot analysis. Our preliminary observations show that labeling of this doublet correlates closely with cAMP-binding activity, suggesting that it is the surface receptor which mediates chemotaxis and cAMP signaling.     

cAMP regulation of cell differentiation in Dictyostelium discoideum and the role of the cAMP receptor

DNA polymerases and DNA ligases have been studied during development of the amphibian, axolotl. Three forms of DNA polymerase, I, II, and III, with sedimentation coefficients in sucrose of 9, 6, and 3.1 S, respectively, have been found in the axolotl egg. The activity of these three DNA polymerases is unchanged during early embryonic development. The activity of DNA polymerase III then increases significantly, beginning at the tailbud stage, while the activity of DNA polymerase II increases at the larval stage. DNA polymerase I does not show significant variations during this time. On the basis of their catalytic properties, it appears that DNA polymerases I and II are α-type DNA polymerases whereas DNA polymerase III is a β-type enzyme. Two different DNA ligases are found in the axolotl, one showing a sedimentation coefficient in sucrose of 8.2 S (heavy form) and the other, 6 S (light form). The 6 S enzyme is the major DNA ligase activity found in the egg before and after fertilization. Its activity then decreases during embryonic development. It can be observed again, as the only DNA ligase activity, in some adult tissues. The 8.2 S enzyme appears during the first division cycle of the fertilized egg, is present at all stages of embryonic development, and is absent from the adult tissues tested. Properties of the two DNA ligases at different stages of embryonic development have also been compared.     

The spore coat of a fucosylation mutant in Dictyostelium discoideum

Strain HL250 of Dictyostelium discoideum cannot convert GDP-mannose to GDP-fucose, resulting in an inability to fucosylate protein. This affects a group of proteins which are normally fucosylated intracellularly and then secreted via prespore vesicles to become part of the outer lamina of the spore coat. We have found that strain HL250 nevertheless accumulates typical amounts of these proteins, stores them normally in prespore vesicles, and secretes them normally to become a part of the spore coat. However, affected proteins are proteolyzed after germination, the spore coat is more accessible to penetration by a macromolecular probe, and germination is inefficient in older spores. These findings can be explained by a dependence of the integrity of the outer layer of the spore coat on protein-linked fucose.