Oscillations and waves of cyclic AMP in Dictyostelium: A prototype for spatio-temporal organization and pulsatile intercellular communication

The amoebae Dictyostelium discoideum aggregate after starvation in a wavelike manner in response to periodic pulses of cyclic AMP (cAMP) secreted by cells which behave as aggregation centers. In addition to autonomous oscillations, the cAMP signaling system that controls aggregation is also capable of excitable behavior, which consists in the transient amplification of suprathreshold pulses of extracellular cAMP. Since the first theoretical model for slime mold aggregation proposed by Keller and Segel in 1970, many theoretical studies have addressed various aspects of the mechanism and function of cAMP signaling in Dictyostelium. This paper presents a brief overview of these developments as well as some reminiscences of the author's collaboration with Lee Segel in modeling the dynamics of cAMP relay and oscillations. Considered in turn are models for cAMP signaling in Dictyostelium, the developmental path followed by the cAMP signaling system after starvation, the frequency encoding of cAMP signals, and the origin of concentric or spiral waves of cAMP.     

The cyclic AMP control system in the development of Dictyostelium discoideum. II. An allosteric model.

An allosteric model for the synthesis and release of cAMP by cells of Dictyostelium discoideum is developed, and matched to experimental data on autonomous and refractory periods, delay and release times, and relaying thresholds. In addition, the model predicts recruitment and center inhibition effects, and chemotactic sorting out of cells to form a descending gradient of cAMP content down the slug. This gradient is tied to an opposing gradient in stored reserves which assigns cells with the highest compliment of stored reserves to the prespore region.     

Diffusion-induced morphogenesis in the development of Dictyostelium

A reaction-diffusion model for the regulation of cAMP in Dictyostelium discoideum is analyzed. As a parameter declines with starvation, the model sequentially yields pulse relaying, spiral waves, target patterns, streaming and sorting, directed locomotion, and tissue buckling, closely matching the observed morphogenetic sequence. These morphologies appear through successive bifurcations of a single reaction-diffusion system and do not require the expression of new genetic information.     

A Model Based on Receptor Desensitization for Cyclic AMP Signaling in Dictyostelium Cells

We analyze a model based on receptor modification for the cAMP signaling system that controls aggregation of the slime mold Dictyostelium discoideum after starvation. The model takes into account both the desensitization of the cAMP receptor by reversible phosphorylation and the activation of adenylate cyclase that follows binding of extracellular cAMP to the unmodified receptor. The dynamics of the signaling system is studied in terms of three variables, namely, intracellular and extracellular cAMP, and the fraction of receptor in active state. Using parameter values collected from experimental studies on cAMP signaling and receptor phosphorylation, we show that the model accounts qualitatively and, in a large measure, quantitatively for the various modes of dynamic behavior observed in the experiments: (a) autonomous oscillations of cAMP, (b) relay of suprathreshold cAMP pulses, i.e., excitability, characterized by both an absolute and a relative refractory period, and (c) adaptation to constant cAMP stimuli. A two-variable version of the model is used to demonstrate the link between excitability and oscillations by phase plane analysis. The response of the model to repetitive stimulation allows comprehension, in terms of receptor desensitization, of the role of periodic signaling in Dictyostelium and, more generally, the function of pulsatile patterns of hormone secretion.     

The effect of 2,4-dinitrophenol on Dictyostelium discoideum oscillations.

During aggregation the cellular slime mold $\text{Dictyostelium discoideum}$ emits pulses of cAMP about every 5 minutes. Only a small fraction of the aggregating cells produces the pulses autonomously, while most cells synthesize and release the nucleotide in a chain-reaction response to the autonomous signals (1). We report here that 2,4-dinitrophenol, KCN, and caffeine all inhibit the autonomous cAMP oscillations but do not interfere with the triggered response. Because of this, and other data (2), we question current models of the oscillatory synthesis of cAMP.     

Effects of amino acids and glucose on adenylate cyclase and cell differentiation of Dictyostelium discoideum.

Starvation triggers the differentiation of Dictyostelium discoideum amoebas to aggregation competence. To determine more precisely the nature of the starvation signal, the ability of various components of the growth medium to inhibit differentiation was examined. Changes in adenylate cyclase (the enzyme which generates the cAMP pulses basic to the differentiation process), various physiological and biochemical markers of developing cells, and the ability of amoebas to form specific intercellular contacts were monitored. We show that amino acid mixtures inhibit cell differentiation by preventing the increase of adenylate cyclase activity which normally occurs during the early hours of starvation. High concentrations of glucose also inhibit the differentiation process but at a later stage: The rise in adenylate cyclase still occurs when cells are starved in the presence of sugar, but the enzyme does not appear to function in vivo. Exogenously generated cAMP pulses are not able to bypass the block exerted by amino acids but can bypass the block exerted by glucose. Results support the hypothesis that the presence of amino acids inhibits adenylate cyclase synthesis, while the presence of 3% glucose blocks endogenous activation of adenylate cyclase, perhaps as a consequence of high osmotic pressure.     

Initiation of multicellular differentiation in Dictyostelium discoideum is regulated by coronin A

Many biological systems respond to environmental changes by activating intracellular signaling cascades, resulting in an appropriate response. One such system is represented by the social amoeba Dictyostelium discoideum. When food sources become scarce, these unicellular cells can initiate a cAMP-driven multicellular aggregation program to ensure long-term survival. On starvation, the cells secrete conditioned medium factors that initiate cAMP signal transduction by inducing expression of genes such as cAMP receptors and adenylate cyclase. The mechanisms involved in the activation of the first pulses of cAMP release have been unclear. We here show a crucial role for the evolutionarily conserved protein coronin A in the initiation of the cAMP response. On starvation, coronin A–deficient cells failed to up-regulate the expression of cAMP-regulated genes, thereby failing to initiate development, despite a normal prestarvation response. Of importance, external addition of cAMP to coronin A–deficient cells resulted in normal chemotaxis and aggregate formation, thereby restoring the developmental program and suggesting a functional cAMP relay in the absence of coronin A. These results suggest that coronin A is dispensable for cAMP sensing, chemotaxis, and development per se but is part of a signal transduction cascade essential for system initiation leading to multicellular development in Dictyostelium.     

Transition from an excitable to an oscillatory state in dictyostelium discoideum

Under conditions of starvation, populations of the amoebae Dictyostelium discoideum aggregate are mediated by chemical excitation waves of cAMP. Two types of waves can be observed, either spiral or circular-shaped ones. We investigate transitions from rotating spirals to circular shaped waves (target patterns). Two different experiments demonstrating this phenomenon are presented. In the first case a continuous transition from the spiral type pattern to target waves was observed at the later stages of aggregation. In the second case the transition was induced by annihilation of waves by a spatially homogeneous cAMP pulse. Instead of the originally present spiral waves, oscillating spots bearing target patterns emerged. On the basis of a model for Dictyostelium aggregation, we provide a theoretical explanation for such transitions. It is shown that cell density can be an effective bifurcation parameter. Under certain conditions, the system is shifted from the excitable to the oscillatory state while the frequency of oscillations is proportional to the square root of the cell density. Thus, the regions with the highest cell density during the early stages of the spatial rearrangement of the cells become pacemakers and produce target patterns. The analytic results were confirmed in numerical simulations of the model.     

Arrestins function in cAR1 GPCR-mediated signaling and cAR1 internalization in the development of Dictyostelium discoideum

Oscillation of chemical signals is a common biological phenomenon, but its regulation is poorly understood. At the aggregation stage of Dictyostelium discoideum development, the chemoattractant cAMP is synthesized and released at 6-min intervals, directing cell migration. Although the G protein–coupled cAMP receptor cAR1 and ERK2 are both implicated in regulating the oscillation, the signaling circuit remains unknown. Here we report that D. discoideum arrestins regulate the frequency of cAMP oscillation and may link cAR1 signaling to oscillatory ERK2 activity. Cells lacking arrestins (adcB⁻C⁻) display cAMP oscillations during the aggregation stage that are twice as frequent as for wild- type cells. The adcB⁻C⁻ cells also have a shorter period of transient ERK2 activity and precociously reactivate ERK2 in response to cAMP stimulation. We show that arrestin domain–containing protein C (AdcC) associates with ERK2 and that activation of cAR1 promotes the transient membrane recruitment of AdcC and interaction with cAR1, indicating that arrestins function in cAR1-controlled periodic ERK2 activation and oscillatory cAMP signaling in the aggregation stage of D. discoideum development. In addition, ligand-induced cAR1 internalization is compromised in adcB⁻C⁻ cells, suggesting that arrestins are involved in elimination of high-affinity cAR1 receptors from cell surface after the aggregation stage of multicellular development.     

The development of sporulation competence in Physarum polycephalum.

Plasmodial cells of the slime mold Physarum polycephalum become “competent” for sporulation following a prolonged period of dark starvation in the presence of nicotinamide. Sporulation can then be induced by illumination. Plasmodia are found to release into the medium during starvation one or more cellular products that promote sporulation. These products exert their effect specifically during the dark starvation period, rather than during the final phase of fruiting body construction. The sporulation control factor(s) (SCF) is nondialyzable and can stimulate the development of sporulation competence in the absence of nicotinamide.     

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.     

Folate chemotactic receptors in Dictyostelium discoideum I. Ligand-induced conversion between four receptor states

The presence of three distinct populations of folate binding sites on the surface of Dictyostelium discoideum cells has been reported recently. The A sites (consisting of two subtypes) recognize folic acid, 2-deaminofolic acid and methotrexate with equal affinity and appear to be linked to cAMP synthesis. B and C sites (the latter is composed of two interconvertable subtypes; rapidly equilibrating, C^F, and more slowly equilibrating, C^S) preferentially bind N₁₀-methylfolic acid and folic acid and may both be involved in the chemotactic response (De Wit, R.J.W. and Van Haastert, P.J.M. (1985) Biochim. Biophys. Acta 814, 199–213; De Wit, R.J.W., Bulgakov, R., Pinas, J.E. and Konijn, T.M. (1985) Biochim. Biophys. Acta 814, 214–226). The present study gives two lines of evidence that the B and C sites are interconvertable. (i) Occupancy of the B-sites by N₁₀-methylfolic acid proceeds at a rate identical to that of the association of this ligand to C^S at all concentrations tested. This suggests that association to C^S and formation of occupied B sites share a common pathway. (ii) After preincubation with ligand, removal of free ligand results in the reappearance of unoccupied C^F sites with kinetics identical to the disappearance of occupied B sites. Furthermore, the existence of a third type of C site, which is formed out of C^S and may be converted to B, is proposed. This is based on competition studies using folate analogs with a different affinity for C^S and B.     

Electron transfer and proton pumping under excitation of dark-adapted chloroplasts with flashes of light

Proton release inside thylakoids, which is linked to the action of the water-oxidizing enzyme system, was investigated spectrophotometrically with the dye neutral red under conditions when the external phase was buffered. Under excitation of dark-adapted chloroplasts with four short laser flashes in series, the pattern of proton release as a function of the flash number was recorded and interpreted in the light of the generally accepted scheme for consecutive transitions of the water-oxidizing enzyme system: S₀ → S₁, S₁ → S₂, S₂ → S₃, S₃ → S₄, S₀. It was found that the proton yield after the first flash varied in a reproducible manner, being dependent upon the dark pretreatment given. In terms of the proton-electron reaction during these transitions, the pattern was as follows. In strictly dark-adapted chloroplasts (frozen chloroplasts thawed in darkness and kept for at least 7 min in the dark after dilution), it was fitted well by a stoichiometry of 1:0:1:2. In a less stringently dark-adapted preparation (as above but thawed under light), it was fitted by 0:1:1:2. Mechanistically this is not yet understood. However, it is a first step towards resolving controversy over this pattern among different laboratories. Under conditions where the 1:0:1:2 stoichiometry was observed, proton release was time resolved. Components with half-rise times of 500 and 1000 μs could be correlated with the S₂ → S₃ and S₃ → S₄ transitions, respectively. Proton release during the S₀ → S₁ transition is more rapid, but is less well attributable to the transitions due to error proliferation. A distinct component with a half-rise time of only 100 μs was observed after the second flash. Since it did not fit into the expected kinetics (based on literature data) for the S_i → S_i+1 transitions, we propose that it reflects proton release from a site which is closer to the reaction center of Photosystem (PS) II than the water-splitting enzyme system. This is supported by the observation of rapid proton release under conditions where water oxidation is blocked. Related experiments on the pattern of proton uptake at the reducing side of PS II indicated that protons act as specific counterions for semiquinone anions without binding to them.     

A 3′,5′ Cyclic AMP (cAMP) Phosphodiesterase Modulates cAMP Levels and Optimizes Competence in Haemophilus influenzae Rd

Changes in intracellular 3′,5′ cyclic AMP (cAMP) concentration regulate the development of natural competence in Haemophilus influenzae. In Escherichia coli, cAMP levels are modulated by a cAMP phosphodiesterase encoded by the cpdA gene. We have used several approaches to demonstrate that the homologous icc gene of H. influenzae encodes a functional cAMP phosphodiesterase and that this gene limits intracellular cAMP and thereby influences competence and other cAMP-dependent processes. In E. coli, expression of cloned icc reduced both cAMP-dependent sugar fermentation and β-galactosidase expression, as has been shown for cpdA. In H. influenzae, an icc null mutation increased cAMP-dependent sugar fermentation and competence development in strains where these processes are limited by mutations reducing cAMP synthesis. When endogenous production of cAMP was eliminated by a cya mutation, an icc strain was 10,000-fold more sensitive to exogenous cAMP than an icc⁺ strain. The icc strain showed moderately elevated competence under noninducing conditions, as expected, but had subnormal competence increases at onset of stationary phase in rich medium, and on transfer to a nutrient-limited medium, suggesting that excessive cAMP may interfere with induction. Consistent with this finding, a cya strain cultured in 1 mM cAMP failed to develop maximal competence on transfer to inducing conditions. Thus, by limiting cAMP levels, the H. influenzae cAMP phosphodiesterase may coordinate its responses to nutritional stress, ensuring optimal competence development.     

A link of Ca<Superscript>2+</Superscript> to cAMP oscillations in <Emphasis Type="Italic">Dictyostelium</Emphasis>: the calmodulin antagonist W-7 potentiates cAMP relay and transiently inhibits the acidic Ca<Superscript>2+</Superscript>-store

Background  

During early differentiation of Dictyostelium the attractant cAMP is released periodically to induce aggregation of the cells. Here we pursue the question whether pulsatile cAMP signaling is coupled to a basic Ca²⁺-oscillation.     

Protein Aggregation in E. coli?: Short Term and Long Term Effects of Nutrient Density

During exponential growth some cells of E. coli undergo senescence mediated by asymmetric segregation of damaged components, particularly protein aggregates. We showed previously that functional cell division asymmetry in E. coli was responsive to the nutritional environment. Short term exposure as well as long term selection in low calorie environments led to greater cell division symmetry and decreased frequency of senescent cells as compared to high calorie environments. We show here that long term selection in low nutrient environment decreased protein aggregation as revealed by fluorescence microscopy and proportion of insoluble proteins. Across selection lines protein aggregation was correlated significantly positively with the RNA content, presumably indicating metabolic rate. This suggests that the effects of caloric restriction on cell division symmetry and aging in E. coli may work via altered protein handling mechanisms. The demonstrable effects of long term selection on protein aggregation suggest that protein aggregation is an evolvable phenomenon rather than being a passive inevitable process. The aggregated proteins progressively disappeared on facing starvation indicating degradation and recycling demonstrating that protein aggregation is a reversible process in E. coli.     

Dictyostelium cells produce platelet-activating factor in response to cAMP.

Evidence is provided that Dictyostelium discoideum cells produce 1-O-alkyl-2-delta-acetyl-O-sn-glycero-3-phosphocholine (platelet-activating factor, PAF). D. discoideum PAF has been characterized as being identical with mammalian platelet-activating factor, based on its stimulation of rabbit platelet aggregation, its physicochemical properties and mass spectrum. The basal activity of PAF increases after starvation and during aggregation and declines at the slug stage. PAF is not detected in the extracellular space. Cell treatment with cAMP pulses stimulates a transient accumulation of PAF, probably via activation of a cAMP-dependent acetyltransferase, suggesting a possible involvement of PAF in cAMP-regulated processes in Dictyostelium.     

Juvenile hormone coordinates the regulation of the hemolymph ecdysteroid titer during pupal commitment in Manduca sexta

The timing and magnitude of the pupal commitment peak in the hemolymph ecdysteroid titer of fifth instar Manduca sexta larvae are controlled by the combined effects of prothoracicotropic hormone (PTTH), a prothoracic gland-stimulating factor present in the hemolymph, and the biosynthetic competence of the prothoracic glands themselves. The present data indicate those individual effects are coordinated by juvenile hormone (JH): (1) Treatment of larvae with the JH analog (7S)-hydroprene prevents the normal precommitment drop in the titer of the stimulatory factor; (2) treatment of larvae with (7S)-hydroprene suppresses in a dose- and time-dependent manner the biosynthetic competence of the prothoracic glands; and (3) (7S)-hydroprene acts directly on the brain to inhibit the release of PTTH in vitro. Thus, during Manduca development, a drop in the JH titer early in the fifth instar results in a rapid drop in the titer of the stimulatory factor, the gradual acquisition by prothoracic glands of biosynthetic competence, and lastly, the gated release of PTTH into the hemolymph. The resulting increase in ecdysone synthesis by the prothoracic glands gives rise to the small peak in the ecdysteroid titer that drives pupal commitment.     

Expression of early developmental genes in Dictyostelium discoideum is initiated during exponential growth by an autocrine-dependent mechanism.

Throughout growth, Dictyostelium cells continuously produce an autocrine factor, PSF, that accumulates in proportion to cell density. Production of PSF declines rapidly when cells are shifted to starvation conditions, and the properties of PSF are distinct from those of regulatory factors produced by starving cells. During late exponential growth, PSF induces expression of several early developmental genes, including those for proteins important in cAMP signaling and cell aggregation. Examples are the aggregation stage cAMP receptor (cAR1), the aggregation-specific form of cyclic nucleotide phosphodiesterase, and gp24 (contact sites B). Through PSF, growing cells detect environmental conditions (cell number high, food approaching depletion) that are appropriate for production of the gene products needed to initiate aggregation and development.