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)     

Dictyostelium discoideum lipids modulate cell-cell cohesion and cyclic AMP signaling.

During Dictyostelium discoideum development, cell-cell communication is mediated through cyclic AMP (cAMP)-induced cAMP synthesis and secretion (cAMP signaling) and cell-cell contact. Cell-cell contact elicits cAMP secretion and modulates the magnitude of a subsequent cAMP signaling response (D. R. Fontana and P. L. Price, Differentiation 41:184-192, 1989), demonstrating that cell-cell contact and cAMP signaling are not independent events. To identify components involved in the contact-mediated modulation of cAMP signaling, amoebal membranes were added to aggregation-competent amoebae in suspension. The membranes from aggregation-competent amoebae inhibited cAMP signaling at all concentrations tested, while the membranes from vegetative amoebae exhibited a concentration-dependent enhancement or inhibition of cAMP signaling. Membrane lipids inhibited cAMP signaling at all concentrations tested. The lipids abolished cAMP signaling by blocking cAMP-induced adenylyl cyclase activation. The membrane lipids also inhibited amoeba-amoeba cohesion at concentrations comparable to those which inhibited cAMP signaling. The phospholipids and neutral lipids decreased cohesion and inhibited the cAMP signaling response. The glycolipid/sulfolipid fraction enhanced cohesion and cAMP signaling. Caffeine, a known inhibitor of cAMP-induced adenylyl cyclase activation, inhibited amoeba-amoeba cohesion. These studies demonstrate that endogenous lipids are capable of modulating amoeba-amoeba cohesion and cAMP-induced activation of the adenylyl cyclase. These results suggest that cohesion may modulate cAMP-induced adenylyl cyclase activation. Because the complete elimination of cohesion is accompanied by the complete elimination of cAMP signaling, these results further suggest that cohesion may be necessary for cAMP-induced adenylyl cyclase activation in D. discoideum.     

Cyclic 3'',5'' AMP relay in Dictyostelium discoideum. I. A technique to monitor responses to controlled stimuli

A perfusion technique was developed to deliver [14C]adenosine 3',5'-cyclic monophosphate (cAMP) stimuli of well-defined magnitude and duration to tritium-labeled Dictyostelium discoideum amoebae and simultaneously monitor the elicited secretion of [3H]cAMP (i.e., the relay response). The tritiated compounds secreted in response to [14C]cAMP stimuli were highly enriched in [3H]cAMP and reflected an increase in intracellular cAMP accompanying stimulation rather than the release of a preexisting store or bulk cellular contents. The secretory response (per 10(6) cells) to 2-min stimuli increased during differentiation from about 0.2 pmol at 0.5 h to approximately 5 pmol of cAMP at 7 h. Without adequate perfusion, amoebae altered the level of cAMP in their environment in two ways: phosphodiesterases destroyed cAMP stimuli under some conditions so as to attenuate the relay response; under other circumstances, secreted cAMP magnified minimal exogenous stimuli into maximal responses. Amoebae, furthermore, would respond to their basal secretion of cAMP autocatalytically if its removal or destruction were interrupted. The perfusion system minimized these cell-induced modifications, allowing control of the level of the stimulus and response in quantitative studies.     

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.     

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.     

Unsaturated free fatty acids regulate cAMP relay and cell-cell adhesion duringDictyostelium discoideum aggregation

Summary Cell-cell adhesion and cAMP-stimulated cAMP production (cAMP relay) regulate the aggregation that occurs early in theDictyostelium discoideum developmental cycle. Increasing the concentration of neutral lipids inD. discoideum membranes inhibits both cell-cell adhesion and cAMP relay. Fractionation experiments revealed that it was the free fatty acids, one group of the neutral lipids, that inhibited both cAMP relay and cell-cell adhesion. Work with commercially-available free fatty acids demonstrated that the addition of saturated free fatty acids, palmitic acid and stearic acid, did not alter cell-cell adhesion or cAMP relay. The addition of unsaturated free fatty acids inhibited both cAMP relay and cell-cell adhesion in a dose-dependent, saturable manner. To test the physiological significance of these observations, the concentrations of endogenous unsaturated fatty acids were modified by altering theD. discoideum diet. Decreasing the amount of fatty acids 18 carbons long with 2 double bonds enhanced cAMP relay and cell-cell adhesion. These results suggest that fatty acids may be important regulators of cAMP responsiveness and cell-cell adhesion duringD. discoideum aggregation, and therefore may play important roles in development.     

cAMP relay during early culmination of Dictyostelium minutum

Abstract. It has recently been found that the culmination process of Dictyostelium minutum is accompanied by the appearance of oscillatory cell movement, cell-surface cAMP receptors, and cAMP phosphodiesterase. In the present study, it is demonstrated that the cAMP analog 2'deoxyc-AMP induces a transient accumulation and secretion of cAMP in culminating structures. At least 50-fold-less cAMP is accumulated during relay of D. minutum than during relay of Dictyostelium discoideum aggregative cells. No cAMP relay could be induced in vegetative or aggregative cells of D. minutum . These combined results yield evidence that oscillatory cAMP secretion and relay are involved in the organization of cell movement during the culmination of D. minutum .     

Cyclic 3'',5'' AMP relay in Dictyostelium discoideum. II. Requirements for the initiation and termination of the response

The secretion of 3H-cyclic adenosine 3',5'-monophosphate (cAMP) by prelabeled and suitably differentiated Dictyostelium discoideum amoebae was elicited in a perfusion apparatus by 10(-10) to 10(-5) M [14C]cAMP stimuli of defined magnitude and duration. Exogenous stimuli evoked an immediate increase in the rate of [3H]cAMP secretion which accelerated continuously to reach a peak of up to 100 times the unstimulated rate after 2--3 min of stimulation. Withdrawal of the stimulus at any time during the response led to a rapid decline to basal levels. Furthermore, a spontaneous decline in secretion rate was observed during prolonged cAMP stimulation, with a return to basal levels after 3--8 min of stimulation. After the initial secretory event, cells did not respond further to the continued presence of external [14C]cAMP unless (a) it was interrupted by a brief recovery period or (b) the level of the stimulus was increased sufficiently. Since the second increment could follow the first at any time, continuous secretion of [3H]cAMP could be sustained for up to 30 min by progressively increasing the stimulus between 10(-10) and 10(-5) M cAMP. The total magnitude of spontaneously terminated responses depended on the size of the increment in applied cAMP, larger stimuli evoking both a more rapid acceleration and a slower deceleration in [3H]cAMP secretion rate. The integrated response to a given increment in stimulus level was apparently independent of its "shape" - i.e., the duration, magnitude, and number of sub-steps in the increment. These data support two mechanistic inferences: that amoebae respond in proportion to relative increases in extracellular cAMP concentration, but adapt to the concentration of cAMP itself. The data further suggest that the initiation and termination of the response are mediated by cellular component(s) beyond cAMP-occupied receptors.     

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.     

Follow the leader

Upon starvation, individual Dictyostelium amoebae chemotax toward aggregation centers in tightly packed streams in which cells are organized in head-to-tail chains. A recent report in Cell shows that this behavior requires localization of adenylyl cyclase and the production and secretion of the chemoattractant cAMP at the posterior of individual cells. These findings suggest a relay and communication system to regulate the long-range coordinated movement of cells.     

Multiple effects of differentiation-inducing factor on prespore differentiation and cyclic-AMP signal transduction in Dictyostelium

Abstract. The effects of the differentiation inducing factor (DIF) on several CAMP-induced responses in Dictyostelium were investigated. It was found that DIF reduces the apparent affinity of cell-surface cAMP receptors. DIF does not affect the CAMP-induced cGMP response, but it is a potent inhibitor of the CAMP-relay response. DIF also inhibits the induction of prespore differentiation by cAMP in aggregation-competent cells. We also compared the effects of DIF on CAMP-induced responses with those of the relay inhibitor, caffeine, and the morphogen, adenosine.     

Multiple effects of differentiation-inducing factor on prespore differentiation and cyclic-AMP signal transduction in Dictyostelium

Abstract. The effects of the differentiation inducing factor (DIF) on several cAMP-induced responses in Dictyostelium were investigated. It was found that DIF reduces the apparent affinity of cell-surface cAMP receptors. DIF does not affect the cAMP-induced cGMP response, but it is a potent inhibitor of the cAMP-relay response. DIF also inhibits the induction of prespore differentiation by cAMP in aggregation-competent cells. We also compared the effects of DIF on cAMP-induced responses with those of the relay inhibitor, caffeine, and the morphogen, adenosine.     

Differences in accumulation and phosphorylation of proteins in vegetatively growing and aggregation-competent cells of Dictyostelium discoideum.

A comparison of proteins from whole cell lysates of vegetative amoebae and aggregation-competent cells by high-resolution two-dimensional gel electrophoresis coupled with a sensitive silver staining method revealed distinct differences. In aggregation-competent cells, 16 proteins present in the vegetative amoebae disappeared, and 25 new proteins appeared. A few other proteins showed quantitative variation during the transition of vegetative amoebae to aggregation competence. Identification of phosphoproteins by in vivo labeling with [32P]orthophosphate showed that none of the developmentally regulated cellular proteins were modified. Phosphorylation was observed in four proteins. One protein was phosphorylated exclusively in aggregation-competent cells. The phosphorylation level of two other proteins was higher in aggregation-competent cells compared with vegetative amoebae. The data suggest that phosphorylation of cellular and certain ribosomal proteins may be regulated coordinately in Dictyostelium discoideum.     

NH3 and propionate modulate the morphological response of aggregation-competent Dictyostelium discoideum to cAMP

Two metabolites, NH3 and propionic acid, are known to act as morphogens during the development of Dictyostelium discoideum, specifically altering the course of morphogenesis and cytodifferentiation. They have also been shown to modulate the cAMP relay in this organism: NH3 by restricting intracellular accumulation, and propionate by inhibiting extracellular release. In the present study, we utilized the light-scattering properties of aggregation-competent cells in agitated suspension to demonstrate that the morphological responses of such cells to exogenous cAMP are also modulated by NH3 and propionate in a manner that has interesting implications for the overall control of morphogenetic movements in D. discoideum. Our experiments were conducted using a newly designed continuous-flow apparatus that represents a significant improvement in the technique. The apparatus is described in detail.     

Frequency encoding of pulsatile signals of cAMP based on receptor desensitization in Dictyostelium cells

Dictyostelium discoideum amoebae represent a prototype for the study of periodic signaling in intercellular communication. These cells synthesize cAMP in response to cAMP pulses. Cell responsiveness in Dictyostelium can be characterized by the capability to generate a large number of significant responses to cAMP signals in a given amount of time. The existence of a frequency of pulsatile cAMP signals yielding maximum responsiveness is demonstrated by analysis of a realistic model for cAMP synthesis, based on receptor desensitization. The optimal frequency of stimulation closely depends on the kinetics of receptor desensitization and resensitization in target cells. Synthesis of cAMP is determined both in conditions where cells are not excitable and in conditions where they relay suprathreshold pulses of cAMP. Moreover, the effect of the stimulus waveform is investigated, and several measures of cell responsiveness are compared. The results provide an explanation for the effectiveness of cAMP pulses delivered at 5 min intervals, and for the failure of pulses delivered at 2 min intervals, in inducing slime mold development. Besides applying to intercellular communication in Dictyostelium, the present analysis bears on patterns of pulsatile signaling observed for hormones and growth factors. In all these cases, it appears that pulsatile signals can be encoded in terms of their frequency on the basis of desensitization in target cells.     

A characterization of the preaggregative period of Dictyostelium discoideum

The preaggregative period of Dictyostelium discoideum has been characterized by measuring the reduction in time for the onset of aggregation under conditions which hinder close cell-cell associations, inhibit protein synthesis, and/or include continuous high concentrations or pulsed low concentrations of exogenous cAMP. The results demonstrate that: the preaggregative period (normally 7 hr for cells from log phase cultures) can be dissected into two distinct components: an initial component which includes the first 4.5 hr, and a second component which includes the last 2.5 hr; the first component will progress at normal rate in the continuous absence of close cell-cell associations (as single amoebae in suspension) or in the continuous absence of de novo protein synthesis; the second component will not progress in the continuous absence of close cell-cell associations or de novo protein synthesis; high concentrations of cAMP continuously present in suspension cultures do not affect progress through the first component, nor do they support progress through the second component; however, if cells are allowed to form close cell-cell associations during progress through the first component, high concentrations of cAMP will support progress through the second component in the absence of close cell-cell associations; these associations, which render cells sensitive to cAMP, will occur in the absence of de novo protein synthesis and before the acquisition of contact sites A; these associations may be completely bypassed if suspended cells are continuously pulsed with low concentrations of cAMP; in this case, pulses of cAMP will support progress through the final component in continuous suspension cultures; and the acquisition of contact sites A will not occur in the absence of progress through the second component; in contrast, the acquisition of cAMP binding sites on the cell's surface will occur. These results are considered in terms of the complexity and regulation of the preaggregative period of Dictyostelium.     

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.     

Dual chemotaxis signalling regulates Dictyostelium development: intercellular cyclic AMP pulses and intracellular F-actin disassembly waves induce each other

Aggregating Dictyostelium discoideum amoebae periodically emit and relay cAMP, which regulates their chemotaxis and morphogenesis into a multicellular, differentiated organism. Cyclic AMP also stimulates F-actin assembly and chemotactic pseudopodium extension. We used actin-GFP expression to visualise for the first time intracellular F-actin assembly as a spatio-temporal indicator of cell reactions to cAMP, and thus the kinematics of cell communication, in aggregating streams. Every natural cAMP signal pulse induces an autowave of F-actin disassembly, which propagates from each cell's leading end to its trailing end at a linear rate, much slower than the calculated and measured velocities of cAMP diffusion in aggregating Dictyostelium. A sequence of transient reactions follows behind the wave, including anterior F-actin assembly, chemotactic pseudopodium extension and cell advance at the cell front and, at the back, F-actin assembly, extension of a small retrograde pseudopodium (forcing a brief cell retreat) and chemotactic stimulation of the following cell, yielding a 20s cAMP relay delay. These dynamics indicate that stream cell behaviour is mediated by a dual signalling system: a short-range cAMP pulse directed from one cell tail to an immediately following cell front and a slower, long-range wave of intracellular F-actin disassembly, each inducing the other.     

Phenotypic suppression of morphogenetic mutants of Dictyostelium discoideum.

The effects of cAMP pulses on the capacity of 15 aggregateless mutants to differentiate and construct fruiting bodies are compared to those obtained when mutant cells are starved with wild-type amoebae. Mutant strains are classified into three main groups depending upon the degree to which their phenotypic defects can be corrected. These data extend studies published earlier [Darmon, M., Brachet, P., and Pereira da Silva, L. (1975). Chemotactic signals induce cell differentiation in Dictyostelium discoideum. Proc. Nat. Acad. Sci. USA72, 3163–3166; Pereira da Silva, L., Darmon, M., Brachet, P., Klein, C., and Barrand, P. (1975). Induction of cell differentiation by the chemotactic signal in Dictyostelium discoideum. In “Proceedings of the Tenth FEBS Meeting,” pp. 269–276]. (1) Only one mutant was unresponsive both to cAMP pulses and to the presence of wild-type amoebae and did not display any of the properties of differentiated cells. (2) Following treatment with cAMP pulses, 11 mutants developed certain properties of aggregation-competent amoebae. They increased their levels of cellular phosphodiesterase, showed an enhanced chemotactic sensitivity to cAMP, and established specific cell contacts. None of these amoebae could differentiate further. They did co-aggregate to some extent with wild-type cells, but failed to differentiate into spores. Rather, mutant cells were excluded from the pseudoplasmodium during the process of morphogenesis of the fruiting body. (3) In contrast, the aggregateless phenotype of three mutants was fully corrected by both cAMP pulses and the presence of wild-type cells. These findings are discussed on the basis of a relationship between the chemotactic signal and cell differentiation.