Contact alters cAMP metabolism in aggregation-competent Dictyostelium amoebae

cAMP and cell-cell contact are involved in the coordination of differentiation and morphogenesis in Dictyostelium discoideum. The experiments described in this paper establish a relationship between cAMP and cell-cell contact. Contact between Enterobacter aerogenes and aggregation-competent Dictyostelium amoebae and contact between Dictyostelium amoebae themselves results in the transient secretion of cAMP and an alteration in the amount of cAMP secreted in response to subsequent stimulation by cAMP, i.e., an alteration in magnitude of a cAMP relay response. The subsequent cAMP relay response can be enhanced or diminished depending upon the number of contacts formed and the concentration of cAMP present at the time of contact. Latex beads are capable of evoking cAMP secretion. However, the bead/amoebal contact is unable to alter the magnitude of a subsequent response to cAMP. This suggests that a nonspecific interaction via cell-cell contact elicits transient cAMP secretion in aggregation-competent Dictyostelium amoebae. The two responses to cell-cell contact are distinct from each other and distinct from the cAMP relay response. 1) The dose-response curves for the responses to Enterobacter contact are clearly different. 2) Contact with latex beads can elicit cAMP secretion but not alter the magnitude of a subsequent cAMP relay response. 3) The temperature dependences of the contact-induced responses and the cAMP relay response show that only the contact-induced cAMP secretion is inhibited at 12 and 15 degrees C, while only the cAMP relay response is inhibited at 28 degrees C. A 4-second application of cAMP at the time that contact is initiated enhances both contact-induced responses. Whether the relationship between these two developmental regulators is important for the regulation of Dictyostelium development has yet to be established.     

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)     

Adenylyl cyclase expression and regulation during the differentiation of Dictyostelium discoideum

Cyclic AMP metabolism is essential for the survival of the social amoebae Dictyostelium discoideum. Three distinct adenylyl cyclases are expressed and required for the normal development of this simple eukaryote. The adenylyl cyclase expressed during aggregation, ACA, is related to the mammalian and Drosophila G protein-coupled enzymes and is responsible for the synthesis of cAMP that is required for cell-cell signaling in early development. ACB harbors histidine kinase and response-regulator domains and is required for terminal differentiation. Finally, the adenylyl cyclase expressed during germination, ACG, acts as an osmosensor and is involved in controlling spore germination. Together, these enzymes generate the various levels of cAMP that are required for D. discoideum to transition from uni- to multi-cellularity. This review will highlight the properties of these enzymes and describe the signaling cascades that lead to their activation.     

Visualizing PI3 kinase-mediated cell-cell signaling during Dictyostelium development

BACKGROUND: Starving amoebae of Dictyostelium discoideum communicate by relaying extracellular cAMP signals, which direct chemotactic movement, resulting in the aggregation of thousands of cells into multicellular aggregates. Both cAMP relay and chemotaxis require the activation of PI3 kinase signaling. The spatiotemporal dynamics of PI3 kinase signaling can be followed in individual cells via the cAMP-induced membrane recruitment of a GFP-tagged PH domain-containing protein, CRAC, which is required for the activation of adenylylcyclase.RESULTS: We show that polarized periodic CRAC-GFP translocation occurs during the aggregation and mound stages of development in response to periodic cAMP signals. The duration of CRAC translocation to the membrane is determined by the duration of the rising phase of the cAMP signal. The system shows rapid adaptation and responds to the rate of change of the extracellular cAMP concentration. When the cells are in close contact, it takes 10 s for the signal to propagate from one cell to the next. In slugs, all cells show a permanent polarized PI3 kinase signaling in their leading edge, which is dependent on cell-cell contact.CONCLUSIONS: Measuring the redistribution of GFP-tagged CRAC has enabled us to study the dynamics of PI3 kinase-mediated cell-cell communication at the individual cell level in the multicellular stages of Dictyostelium development. This approach should also be useful to study the interactions between cell-cell signaling, cell polarization, and movement in the development of other organisms.     

A cell number-counting factor regulates group size in Dictyostelium by differentially modulating cAMP-induced cAMP and cGMP pulse sizes

A secreted counting factor (CF), regulates the size of Dictyostelium discoideum fruiting bodies in part by regulating cell-cell adhesion. Aggregation and the expression of adhesion molecules are mediated by relayed pulses of cAMP. Cells also respond to cAMP with a short cGMP pulse. We find that CF slowly down-regulates the cAMP-induced cGMP pulse by inhibiting guanylyl cyclase activity. A 1-min exposure of cells to purified CF increases the cAMP-induced cAMP pulse. CF does not affect the cAMP receptor or its interaction with its associated G proteins or the translocation of the cytosolic regulator of adenylyl cyclase to the membrane in response to cAMP. Pulsing streaming wild-type cells with a high concentration of cAMP results in the formation of small groups, whereas reducing cAMP pulse size with exogenous cAMP phosphodiesterase during stream formation causes cells to form large groups. Altering the extracellular cAMP pulse size does not phenocopy the effects of CF on the cAMP-induced cGMP pulse size or cell-cell adhesion, indicating that CF does not regulate cGMP pulses and adhesion via CF's effects on cAMP pulses. The results suggest that regulating cell-cell adhesion, the cGMP pulse size, or the cAMP pulse size can control group size and that CF regulates all three of these independently.     

Direct biochemical measurements of signal relay during Dictyostelium development

Upon starvation, individual Dictyostelium discoideum cells enter a developmental program that leads to collective migration and the formation of a multicellular organism. The process is mediated by extracellular cAMP binding to the G protein-coupled cAMP receptor 1, which initiates a signaling cascade leading to the activation of adenylyl cyclase A (ACA), the synthesis and secretion of additional cAMP, and an autocrine and paracrine activation loop. The release of cAMP allows neighboring cells to polarize and migrate directionally and form characteristic chains of cells called streams. We now report that cAMP relay can be measured biochemically by assessing ACA, ERK2, and TORC2 activities at successive time points in development after stimulating cells with subsaturating concentrations of cAMP. We also find that the activation profiles of ACA, ERK2, and TORC2 change in the course of development, with later developed cells showing a loss of sensitivity to the relayed signal. We examined mutants in PKA activity that have been associated with precocious development and find that this loss in responsiveness occurs earlier in these mutants. Remarkably, we show that this loss in sensitivity correlates with a switch in migration patterns as cells transition from streams to aggregates. We propose that as cells proceed through development, the cAMP-induced desensitization and down-regulation of cAMP receptor 1 impacts the sensitivities of chemotactic signaling cascades leading to changes in migration patterns.     

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.     

The cyclase-associated protein CAP as regulator of cell polarity and cAMP signaling in Dictyostelium

Cyclase-associated protein (CAP) is an evolutionarily conserved regulator of the G-actin/F-actin ratio and, in yeast, is involved in regulating the adenylyl cyclase activity. We show that cell polarization, F-actin organization, and phototaxis are altered in a Dictyostelium CAP knockout mutant. Furthermore, in complementation assays we determined the roles of the individual domains in signaling and regulation of the actin cytoskeleton. We studied in detail the adenylyl cyclase activity and found that the mutant cells have normal levels of the aggregation phase-specific adenylyl cyclase and that receptor-mediated activation is intact. However, cAMP relay that is responsible for the generation of propagating cAMP waves that control the chemotactic aggregation of starving Dictyostelium cells was altered, and the cAMP-induced cGMP production was significantly reduced. The data suggest an interaction of CAP with adenylyl cyclase in Dictyostelium and an influence on signaling pathways directly as well as through its function as a regulatory component of the cytoskeleton.     

Inhibition by EDTA and enhancement by divalent cations or polyamines of the dithiothreitol-induced activation of adenylate cyclase in the cellular slime mold, Dictyostelium discoideum.

Binding of cAMP to cell surface receptors evokes the transient activation of of adenylate cyclase in Dictyostelium discoideum. Dithiothreitol is also known as an activator of this enzyme. We found that the dithiothreitol-induced activation was specifically enhanced by extracellular polyamines or divalent cations. Furthermore, EDTA, a chelating agent of divalent cations, completely inhibited the dithiothreitol-induced activation of adenylate cyclase while EDTA did not inhibit the cAMP-induced activation. The inhibition was nullified by addition of polyamines or divalent cations. These results suggest that extracellular polyamines and divalent cations play a specific role in the dithiothreitol-induced activation of adenylate cyclase.     

Down-regulation of cell surface cyclic AMP receptors and desensitization of cyclic AMP-stimulated adenylate cyclase by cyclic AMP in Dictyostelium discoideum. Kinetics and concentration dependence

cAMP binds to Dictyostelium discoideum surface receptors and induces a transient activation of adenylatecyclase, which is followed by desensitization. cAMP also induces a loss of detectable surface receptors (down-regulation). Cells were incubated with constant cAMP concentrations, washed free of cAMP, and cAMP binding to surface receptors and cAMP-induced activation of adenylate cyclase were measured. cAMP could induce maximally 65% loss of binding activity and complete desensitization of cAMP-stimulated adenylate cyclase activity. Half-maximal effects for down-regulation were observed at 50 nM cAMP and for desensitization at 5 nM cAMP. Down-regulation was rapid with half-times of 4, 2.5, and 1 min at 0.1, 1, and 10 microM cAMP, respectively. Similar kinetic data have been reported for desensitization (Dinauer, M.C., Steck, T.L., and Devreotes, P.N. (1980) J. Cell Biol. 86, 554-561). Down-regulation and desensitization were not reversible at 0 degrees C. Down-regulation reversed slowly at 20 degrees C with a half-time of about 1 h. Resensitization of adenylate cyclase was biphasic showing half-times of 4 min and about 1 h, respectively; the contribution of the rapidly resensitizing component was diminished when down-regulation of receptors was enhanced. These results suggest that cAMP-induced down-regulation of receptors and desensitization of adenylate cyclase stimulation proceed by at least two steps. One step is rapidly reversible, occurs at low cAMP concentrations, and induces desensitization without down-regulation, while the second step is slowly reversible, requires higher cAMP concentrations, and also induces down-regulation.     

Reducing reagent-induced activation of adenylate cyclase in the cellular slime mold, Dictyostelium discoideum.

Binding of an intrinsic agonist (cAMP) to specific receptors on the cell surface induces transmembrane signals for activation and desensitization (adaptation and down regulation) of adenylate cyclase in the cellular slime mold, Dictyostelium discoideum. It is generally believed that dithiothreitol (DTT) induces the activation through interaction between the receptor and gradually accumulated cAMP, since DTT is known to inhibit cAMP-phosphodiesterase which degrades cAMP. In the present paper, we investigated the mechanism of activation of adenylate cyclase by the thiol-reducing agents, DTT and 2,3-dimercapto-1-propanol (BAL). We found that BAL activated adenylate cyclase transiently even under conditions where the intrinsic agonist supersaturated the cAMP-receptors and competitively inhibited phosphodiesterase. This result is inconsistent with the generally accepted notion. We conclude that BAL has an independent effect from those of the intrinsic agonist (cAMP) and phosphodiesterase in activation of adenylate cyclase. Since BAL could induce activation just after the activation induced by a supersaturating concentration of the intrinsic agonist had ceased, the independent effect of BAL is not a simple enhancement of the cAMP-induced activation. Our result also suggests that the cAMP-induced adaptation (but not down regulation) suppresses the BAL-induced activation while BAL itself does not induce adaptation to cAMP or BAL. We propose that the thiol-reducing reagent induces or modifies the transmembrane activation signal for adenylate cyclase.     

The specificity of the cAMP receptor mediating activation of adenylate cyclase in Dictyostelium discoideum

In Dictyostelium discoideum amoebae, binding of cyclic AMP (cAMP) to surface receptors elicits numerous responses including chemotaxis, cyclic GMP (cGMP) accumulation, and activation of adenylate cyclase. The specificity of the surface cAMP receptor which mediates activation of adenylate cyclase and cAMP secretion was determined by testing the relative effectiveness of a series of 10 cAMP analogs. Each of the 10 analogs elicited cAMP secretion, chemotaxis, and cGMP accumulation in the same dose range. The order of potency for eliciting these responses (cAMP greater than 2'-H-cAMP greater than N1-O-cAMP greater than cAMPS(Sp) greater than 6-Cl-cAMP greater than cAMPN(CH3)2(Sp) greater than 3'-NH-cAMP greater than 8-Br-cAMP greater than cAMPS(Rp) greater than cAMPN(CH3)2(Rp] matches that for binding to the major cell surface cAMP binding sites and differs from that of the cell surface phosphodiesterase and the major intracellular cAMP binding protein.     

Defect in peroxisomal multifunctional enzyme MFE1 affects cAMP relay in Dictyostelium

We have previously reported that cells of Dictyostelium discoideum lacking the fatty acid oxidation enzyme MFE1 accumulate excess cyclopropane fatty acids from ingested bacteria. Cells in which mfeA(-) is disrupted fail to develop when grown in association with bacteria but form normal fruiting bodies when grown in axenic media. Bacterially grown mfeA(-) cells express the genes for the cyclic AMP (cAMP) receptor (carA) and adenylyl cyclase (acaA) but fail to respond to a cAMP pulse by synthesis of additional cAMP which normally relays the signal. Moreover, they do not accumulate the adhesion protein, gp80, which is encoded by the cAMP-induced gene, csaA. As a consequence, they do not acquire developmentally regulated EDTA-resistant cell-cell adhesion. When mutant cells are mixed with wild-type cells and allowed to develop together, they co-aggregate and differentiate into both spores and stalk cells. Thus, most of the developmental consequences of excess cyclopropane fatty acids appear to result from impaired cAMP relay.     

Expression of Y53A-Actin in Dictyostelium Disrupts the Cytoskeleton and Inhibits Intracellular and Intercellular Chemotactic Signaling

We showed previously that phosphorylation of Tyr⁵³, or its mutation to Ala, inhibits actin polymerization in vitro with formation of aggregates of short filaments, and that expression of Y53A-actin in Dictyostelium blocks differentiation and development at the mound stage (Liu, X., Shu, S., Hong, M. S., Levine, R. L., and Korn, E. D. (2006) Proc. Natl. Acad. Sci. U.S.A. 103, 13694–13699; Liu, X., Shu, S., Hong, M. S., Yu, B., and Korn, E. D. (2010) J. Biol. Chem. 285, 9729–9739). We now show that expression of Y53A-actin, which does not affect cell growth, phagocytosis, or pinocytosis, inhibits the formation of head-to-tail cell streams during cAMP-induced aggregation, although individual amoebae chemotax normally. We show that expression of Y53A-actin causes a 50% reduction of cell surface cAMP receptors, and inhibits cAMP-induced increases in adenylyl cyclase A activity, phosphorylation of ERK2, and actin polymerization. Trafficking of vesicles containing adenylyl cyclase A to the rear of the cell and secretion of the ACA vesicles are also inhibited. The actin cytoskeleton of cells expressing Y53A-actin is characterized by numerous short filaments, and bundled and aggregated filaments similar to the structures formed by copolymerization of purified Y53A-actin and wild-type actin in vitro. This disorganized actin cytoskeleton may be responsible for the inhibition of intracellular and intercellular cAMP signaling in cells expressing F-Y53A-actin.     

Adenosine and its derivatives inhibit the cAMP signaling response in Dictyostelium discoideum

In developmentally competent Dictyostelium discoideum amoebae, binding of cAMP to high-affinity surface receptors produces a rapid activation of adenylate cyclase which adapts within minutes. The result is a transient increase in intracellular cAMP which is rapidly secreted. Adenosine inhibited this cAMP signaling response with an apparent Ki of 300 microM. The apparent Ki's for 2'-O-methyladenosine and 2-chloroadenosine were approximately 30 and 100 microM, respectively. Inhibition by adenosine was rapid, reversible, and depended on the cAMP stimulus concentration. In addition, the adaptation of the cAMP signaling response was blocked by adenosine. As has been previously reported, adenosine inhibits cAMP binding to intact cells. Under the same developmental conditions as in the perfusion studies, we find the binding inhibition depends on both the cAMP and adenosine concentrations, with an apparent Ki of 100 microM. The apparent Ki's for 2'-O-methyl- and 2-chloroadenosine were approximately 8 and 35 microM, respectively. However, with cells developed for short times and with an axenic strain, inhibition was independent of cAMP concentration or cells showed mixed-type binding inhibition. The effect of adenosine on the cAMP signaling response is consistent with the reported effects of adenosine on other cAMP-mediated processes such as chemotaxis and the increase in intracellular cGMP, and may provide an explanation for the reported inhibition of center formation.     

Nitric oxide regulates adenylyl cyclase activity in rat striatal membranes

The regulation of adenylyl cyclase activity by nitric oxide (NO) was studied in rat (Sprague-Dawley) striatal membranes. Three chemically distinct NO donors attenuated forskolin-stimulated activity but did not alter basal activity. Maximum inhibition resulted in a 50% decrease in forskolin-stimulated activity, consistent with the presence of multiple isoforms of adenylyl cyclase and our previous findings that only the forskolin-stimulated activity of the type-5 and -6 isoform family of enzymes is inhibited by NO. To monitor primarily the type-5 isoform, we examined the ability of NO donors to attenuate D(1)-agonist-stimulated adenylyl cyclase activity. Under those conditions, complete inhibition was observed. The data indicate that NO attenuates neuromodulator-stimulated cAMP signaling in the striatum.     

A MAP kinase necessary for receptor-mediated activation of adenylyl cyclase in Dictyostelium

Analysis of a developmental mutant in Dictyostelium discoideum which is unable to initiate morphogenesis has shown that a protein kinase of the MAP kinase/ERK family affects relay of the cAMP chemotactic signal and cell differentiation. Strains in which the locus encoding ERK2 is disrupted respond to a pulse of cAMP by synthesizing cGMP normally but show little synthesis of cAMP. Since mutant cells lacking ERK2 contain normal levels of both the cytosolic regulator of adenylyl cyclase (CRAC) and manganese-activatable adenylyl cyclase, it appears that this kinase is important for receptor-mediated activation of adenylyl cyclase.     

Cyclic AMP relay and cyclic AMP-induced cyclic GMP accumulation during development of Dictyostelium discoideum

Abstract Cyclic AMP-induced cAMP and cGMP responses during development of Dictyostelium discoideum were investigated. The cAMP-induced cGMP response is maximal when aggregation is in full progress, and then decreases to about 10% of the maximal level during further multicellular development. The cAMP response increases upon starvation, reaches its maximum at the onset of aggregation, and then decreases to about 8% of the maximum level. The dynamics of the post-aggregative cAMP response are in qualitative agreement with the dynamics of the cAMP relay response in aggregation-competent cells.     

Involvement of cyclic AMP cell surface receptors and G-proteins in signal transduction during slug migration of Dictyostelium discoideum.

The presence of G-proteins, interacting with cAMP surface receptors, was investigated in vegetative cells, aggregation-competent cells, and migrating slugs of Dictyostelium discoideum. Our results indicate that G-proteins are present in all stages. In vegetative cells there is a limited number of cAMP receptors but no effect of GTP tau S on cAMP binding could be detected; in addition, no effect of cAMP on GTP tau S binding or GTPase activity was observed. In both aggregation-competent cells and slugs GTP tau S inhibits cAMP binding, while cAMP stimulates GTP tau S binding and high-affinity GTPase. Since the presence of G-proteins coupled to cAMP receptors could be demonstrated in slugs, the involvement of the effector enzymes adenylate cyclase and phospholipase C was investigated. The results show that adenylate cyclase activity is stimulated by GTP tau S in both stages and that in cells from migrating slugs the Ins(1,4,5)P3 production is increased upon stimulation with cAMP. The possible involvement of G-proteins in signal transduction during the slug stage of D. discoideum is discussed.     

Activation of Soluble Adenylyl Cyclase Protects against Secretagogue Stimulated Zymogen Activation in Rat Pancreaic Acinar Cells

An early feature of acute pancreatitis is activation of zymogens, such as trypsinogen, within the pancreatic acinar cell. Supraphysiologic concentrations of the hormone cholecystokinin (CCK; 100 nM), or its orthologue cerulein (CER), induce zymogen activation and elevate levels of cAMP in pancreatic acinar cells. The two classes of adenylyl cyclase, trans-membrane (tmAC) and soluble (sAC), are activated by distinct mechanisms, localize to specific subcellular domains, and can produce locally high concentrations of cAMP. We hypothesized that sAC activity might selectively modulate acinar cell zymogen activation. sAC was identified in acinar cells by PCR and immunoblot. It localized to the apical region of the cell under resting conditions and redistributed intracellularly after treatment with supraphysiologic concentrations of cerulein. In cerulein-treated cells, pre-incubation with a trans-membrane adenylyl cyclase inhibitor did not affect zymogen activation or amylase secretion. However, treatment with a sAC inhibitor (KH7), or inhibition of a downstream target of cAMP, protein kinase A (PKA), significantly enhanced secretagogue-stimulated zymogen activation and amylase secretion. Activation of sAC with bicarbonate significantly inhibited secretagogue-stimulated zymogen activation; this response was decreased by inhibition of sAC or PKA. Bicarbonate also enhanced secretagogue-stimulated cAMP accumulation; this effect was inhibited by KH7. Bicarbonate treatment reduced secretagogue-stimulated acinar cell vacuolization, an early marker of pancreatitis. These data suggest that activation of sAC in the pancreatic acinar cell has a protective effect and reduces the pathologic activation of proteases during pancreatitis.