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.     

Becoming Multicellular by Aggregation; The Morphogenesis of the Social Amoebae Dicyostelium discoideum

The organisation and form of most organisms is generated during theirembryonic development and involves precise spatial and temporal controlof cell division, cell death, cell differentiation and cell movement.Differential cell movement is a particularly important mechanism in thegeneration of form. Arguably the best understood mechanism of directedmovement is chemotaxis. Chemotaxis plays a major role in the starvationinduced multicellular development of the social amoebae Dictyostelium.Upon starvation up to 10⁵ individual amoebae aggregate to form afruiting body. In this paper we review the evidence that the movement ofthe cells during all stages of Dictyostelium development is controlled bypropagating waves of cAMP which control the chemotactic movement ofthe cells. We analyse the complex interactions between cell-cell signallingresulting in cAMP waves of various geometries and cell movement whichresults in a redistribution of the signalling sources and therefore changes thegeometry of the waves. We proceed to show how the morphogenesis,including aggregation stream and mound formation, slug formation andmigration, of this relatively simple organism is beginning to be understoodat the level of rules for cell behaviour, which can be tested experimentallyand theoretically by model calculations.     

The control of chemotactic cell movement during Dictyostelium morphogenesis

Differential cell movement is an important mechanism in the development and morphogenesis of many organisms. In many cases there are indications that chemotaxis is a key mechanism controlling differential cell movement. This can be particularly well studied in the starvation-induced multicellular development of the social amoeba Dictyostelium discoideum. Upon starvation, up to 10(5) individual amoebae aggregate to form a fruiting body The cells aggregate by chemotaxis in response to propagating waves of cAMP, initiated by an aggregation centre. During their chemotactic aggregation the cells start to differentiate into prestalk and prespore cells, precursors to the stalk and spores that form the fruiting body. These cells enter the aggregate in a random order but then sort out to form a simple axial pattern in the slug. Our experiments strongly suggest that the multicellular aggregates (mounds) and slugs are also organized by propagating cAMP waves and, furthermore, that cell-type-specific differences in signalling and chemotaxis result in cell sorting, slug formation and movement.     

Signaling to cytoskeletal dynamics during chemotaxis

We review insights in signaling pathways controlling cell polarization and cytoskeletal organization during chemotactic movement in Dictyostelium amoebae and neutrophils. We compare and contrast these insights with our current understanding of pathways controlling chemotactic movements in more-complex multicellular developmental contexts.     

Dictyostelium morphogenesis

During starvation-induced Dictyostelium development, up to several hundred thousand amoeboid cells aggregate, differentiate and form a fruiting body. The chemotactic movement of the cells is guided by the rising phase of the outward propagating cAMP waves and results in directed periodic movement towards the aggregation centre. In the mound and slug stages of development, cAMP waves continue to play a major role in the coordination of cell movement, cell-type-specific gene expression and morphogenesis; however, in these stages where cells are tightly packed, cell-cell adhesion/contact-dependent signalling mechanisms also play important roles in these processes.     

Chemoresponsiveness to cAMP and Folic Acid during Growth, Development, and Dedifferentiation in Dictyostelium discoideum

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

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

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

Signal transduction, chemotaxis, and cell aggregation in Dictyostelium discoideum cells without myosin heavy chain

Dictyostelium discoideum cells have been generated that lack myosin heavy chain (MHC) due to antisense RNA inactivation of the endogenous mRNA or to insertional mutagenesis of the myosin gene. These cells retain chemotactic movement in gradients of the chemoattractant cAMP. Furthermore, cAMP does induce many biochemical and physiological responses in aggregative cells, including binding of cAMP to surface receptors, modification, and down-regulation of the receptor; activation of adenylate and guanylate cyclase, secretion of cAMP; and the association of actin to the Triton-insoluble cytoskeleton. Cells lacking MHC were found to have a requirement for bivalent cations in the medium for optimal chemotaxis and cell aggregation.     

Navigation of Chemotactic Cells by Parallel Signaling to Pseudopod Persistence and Orientation

The mechanism of chemotaxis is one of the most interesting issues in modern cell biology. Recent work shows that shallow chemoattractant gradients do not induce the generation of pseudopods, as has been predicted in many models. This poses the question of how else cells can steer towards chemoattractants. Here we use a new computational algorithm to analyze the extension of pseudopods by Dictyostelium cells. We show that a shallow gradient of cAMP induces a small bias in the direction of pseudopod extension, without significantly affecting parameters such as pseudopod frequency or size. Persistent movement, caused by alternating left/right splitting of existing pseudopodia, amplifies the effects of this bias by up to 5-fold. Known players in chemotactic pathways play contrasting parts in this mechanism; PLA2 and cGMP signal to the cytoskeleton to regulate the splitting process, while PI 3-kinase and soluble guanylyl cyclase mediate the directional bias. The coordinated regulation of pseudopod generation, orientation and persistence by multiple signaling pathways allows eukaryotic cells to detect extremely shallow gradients.     

Cyclic-AMP binding to the cell surface during development of Dictyostelium discoideum

Abstract. Cell aggregation in Dictyostelium discoideum is a chemotactic process mediated by cyclic adenosine monophosphate (CAMP), which is detected by cell surface receptors. The cAMP signal is degraded by cAMP phosphodiesterase. The possibility that cAMP signals are also used for cell communication in the multicellular stages was studied by determining whether the cAMP receptors, which are essential for signal transduction, continue to function in these stages. During slug migration, the number of binding sites per cell decreases to about 15% of the maximum level acquired during aggregation. At the onset of fruiting body formation, a three- to Four-Fold increase in cAMP binding activity occurs. This increase coincides with an increase in cAMP phosphodiesterase. Both phenomena suggest that cell-cell communication mediated by cAMP is used during culmination. During both slug migration and early culmination, the prestalk cells exhibit about twice as much binding activity as the prespore cells.     

Guanylate Cyclase Activity in Dictyostelium discoideum and Its Increase during Cell Development

Following consumption of the food supply, cells of the cellular slime mould Dictyostelium discoideum aggregate and form a multicellular organism. The mechanism for cell aggregation is chemotaxis. The chemotactic signal in D. discoideum is released periodically from aggregation centers and propagated from cell to cell. cAMP mediates cell aggregation by acting as chemotactic attractant and as propagator of the signal. cAMP signals are measured by cell-surface receptors. Recent evidence indicates a role for cGMP during cAMP-mediated cell aggregation in D. discoideum .
During cell differentiation to aggregation competence, cAMP binding sites appear at the cell surface, and the activity of the enzymes adenylate cyclase and phosphodiesterase increases several-fold. In the present work we investigate the synthesis of cGMP in D. discoideum . Conditions for the assay of guanylate cyclase in cell homogenates are described. Guanylate cyclase activity was followed during cell differentiation to aggregation competence and found to increase fourfold. These results indicate that cGMP is involved in cell differentiation of D. discoideum . In contrast to adenylate cyclase, which is activated by cAMP, guanylate cyclase was under our conditions activated neither by cAMP, nor by folic acid.     

Different molecular mechanisms for cAMP regulation of gene expression during Dictyostelium development

Alterations in cAMP concentrations have been implicated in developmentally regulated gene expression in Dictyostelium. Using a variety of culture conditions to control the metabolism of cAMP during cytodifferentiation, I have examined the role of the cyclic nucleotide in development. Conditions which allow intracellular synthesis of cAMP promote the normal developmental repression of gene M4-1 by a mechanism which is completely independent of the formation of multicellular aggregates. If, however, cells are inhibited in their ability to activate adenylate cyclase and, thus, intracellular cAMP signaling, they prove unable to repress M4-1, even in the presence of exogenous cAMP. In contrast, expression of genes which exhibit maximal activity after aggregate formation depends upon accumulation of extracellular cAMP. Inhibition of intracellular cAMP signaling does not prevent the expression of these genes if cultures are simultaneously exposed to high levels of exogenously added extracellular cAMP. These results indicate that there are at least two independent mechanisms involved in the developmental regulation of gene expression by cAMP in Dictyostelium. I discuss plausible molecular mechanisms through which cAMP might alter gene expression.     

Dictyostelium discoideum chemotaxis: threshold for directed motion

The chemotactic response of Dictyostelium discoideum cells to stationary, linear gradients of cyclic adenosine 3',5'-monophosphate (cAMP) was studied using microfluidic devices. In shallow gradients of less than 10(-3) nM/microm, the cells showed no directional response and exhibited a constant basal motility. In steeper gradients, cells moved up the gradient on average. The chemotactic speed and the motility increased with increasing steepness up to a plateau at around 10(-1) nM/microm. In very steep gradients, above 10 nM/microm, the cells lost directionality and the motility returned to the sub-threshold level. In the regime of optimal response the difference in receptor occupancy at the front and back of the cell is estimated to be only about 100 molecules.     

Stochastic model of chemoattractant receptor dynamics in leukocyte chemosensory movement

Mammalian white blood cells are known to bias the direction of their movement along concentration gradients of specific chemical stimuli, a phenomenon called chemotaxis. Chemotaxis of leukocyte cells is central to the acute inflammatory response in living organisms and other critical physiological functions. On a molecular level, these cells sense the stimuli termed chemotactic factor (CF) through specific cell surface receptors that bind CF molecules. This triggers a complex signal transduction process involving intracellular biochemical pathways and biophysical events, eventually leading to the observable chemotactic response. Several investigators have shown theoretically that statistical fluctuations in receptor binding lead to “noisy” intracellular signals, which may explain the observed imperfect chemotactic response to a CF gradient. The most recent dynamic model (Tranquillo and Lauffenburger,J. Math. Biol. 25, 229–262. 1987) couples a scheme for intracellular signal transduction and cell motility response with fluctuations in receptor binding. However, this model employs several assumptions regarding receptor dynamics that are now known to be oversimplifications. We extend the earlier model by accounting for several known and speculated chemotactic receptor dynamics, namely, transient G-protein signaling, cytoskeletal association, and receptor internalization and recycling, including statistical fluctuations in the numbers of receptors among the various states. Published studies are used to estimate associated constants and ensure the predicted receptor distribution is accurate. Model analysis indicates that directional persistence in uniform CF concentrations is enhanced by increasing rate constants for receptor cytoskeletal inactivation, ternary complex dissociation, and binary complex dissociation, and by decreasing rate constants for receptor internalization and recycling. For most rate constants, we have detected an optimal range that maximizes orientation bias in CF gradients. We have also examined different desensitization and receptor recycling mechanisms that yield experimentally documented orientation behavior. These yield novel insights into the relationship between receptor dynamics and leukocyte chemosensory movement behavior.     

The internal phosphodiesterase RegA is essential for the suppression of lateral pseudopods during Dictyostelium chemotaxis

Dictyostelium strains in which the gene encoding the cytoplasmic cAMP phosphodiesterase RegA is inactivated form small aggregates. This defect was corrected by introducing copies of the wild-type regA gene, indicating that the defect was solely the consequence of the loss of the phosphodiesterase. Using a computer-assisted motion analysis system, regA(-) mutant cells were found to show little sense of direction during aggregation. When labeled wild-type cells were followed in a field of aggregating regA(-) cells, they also failed to move in an orderly direction, indicating that signaling was impaired in mutant cell cultures. However, when labeled regA(-) cells were followed in a field of aggregating wild-type cells, they again failed to move in an orderly manner, primarily in the deduced fronts of waves, indicating that the chemotactic response was also impaired. Since wild-type cells must assess both the increasing spatial gradient and the increasing temporal gradient of cAMP in the front of a natural wave, the behavior of regA(-) cells was motion analyzed first in simulated temporal waves in the absence of spatial gradients and then was analyzed in spatial gradients in the absence of temporal waves. Our results demonstrate that RegA is involved neither in assessing the direction of a spatial gradient of cAMP nor in distinguishing between increasing and decreasing temporal gradients of cAMP. However, RegA is essential for specifically suppressing lateral pseudopod formation during the response to an increasing temporal gradient of cAMP, a necessary component of natural chemotaxis. We discuss the possibility that RegA functions in a network that regulates myosin phosphorylation by controlling internal cAMP levels, and, in support of that hypothesis, we demonstrate that myosin II does not localize in a normal manner to the cortex of regA(-) cells in an increasing temporal gradient of cAMP.     

Ammonia differentially suppresses the cAMP chemotaxis of anterior-like cells and prestalk cells indictyostelium discoideum

A drop assay for chemotaxis to cAMP confirms that both anterior-like cells (ALC) and prestalk cells (pst cells) respond to cAMP gradients. We present evidence that the chemotactic response of both ALC and pst cells is suppressed by ammonia, but a higher concentration of ammonia is required to suppress the response in pst cells. ALC show a chemotactic response to cAMP when moving on a substratum of prespore cells in isolated slug posteriors incubated under oxygen. ALC chemotaxis on a prespore cell substratum is suppressed by the same concentration of ammonia that suppresses ALC chemotaxis on the agar substratum in drop assays. Chemotaxis suppression is mediated by the unprotonated (NH₃) species of ammonia. The observed suppression, by ammonia, of ALC chemotaxis to cAMP supports our earlier hypothesis that ammonia is the tip-produced suppressor of such chemotaxis. We discuss implications of ammonia sensitivity of pst cells and ALC with regard to the movement and localization of ALC and pst cells in the slug and to the roles played by ALC in fruiting body formation. In addition, we suggest that a progressive decrease in sensitivity to ammonia is an important part of the maturation of ALC into pst cells.     

Sphingosine-1-phosphate plays a role in the suppression of lateral pseudopod formation during Dictyostelium discoideum cell migration and chemotaxis

Sphingosine-1-phosphate (S-1-P) is a bioactive lipid that plays a role in diverse biological processes. It functions both as an extracellular ligand through a family of high-affinity G-protein-coupled receptors, and intracellularly as a second messenger. A growing body of evidence has implicated S-1-P in controlling cell movement and chemotaxis in cultured mammalian cells. Mutant D. discoideum cells, in which the gene encoding the S-1-P lyase had been specifically disrupted by homologous recombination, previously were shown to be defective in pseudopod formation, suggesting that a resulting defect might exist in motility and/or chemotaxis. To test this prediction, we analyzed the behavior of mutant cells in buffer, and in both spatial and temporal gradients of the chemoattractant cAMP, using computer-assisted 2-D and 3-D motion analysis systems. Under all conditions, S-1-P lyase null mutants were unable to suppress lateral pseudopod formation like wild-type control cells. This resulted in a reduction in velocity in buffer and spatial gradients of cAMP. Mutant cells exhibited positive chemotaxis in spatial gradients of cAMP, but did so with lowered efficiency, again because of their inability to suppress lateral pseudopod formation. Mutant cells responded normally to simulated temporal waves of cAMP but mimicked the temporal dynamics of natural chemotactic waves. The effect must be intracellular since no homologs of the S-1-P receptors have been identified in the Dictyostelium genome. The defects in the S-1-P lyase null mutants were similar to those seen in mutants lacking the genes for myosin IA, myosin IB, and clathrin, indicating that S-1-P signaling may play a role in modulating the activity or organization of these cytoskeletal elements in the regulation of lateral pseudopod formation.     

Dictyostelium Erk2 is an atypical MAPK required for chemotaxis

The Dictyostelium genome encodes only two MAPKs, Erk1 and Erk2, and both are expressed during growth and development. Reduced levels of Erk2 expression have been shown previously to restrict cAMP production during development but still allow for chemotactic movement. In this study the erk2 gene was disrupted to eliminate Erk2 function. The absence of Erk2 resulted in a complete loss of folate and cAMP chemotaxis suggesting that this MAPK plays an integral role in the signaling mechanisms involved with this cellular response. However, folate stimulation of early chemotactic responses, such as Ras and PI3K activation and rapid actin filament formation, were not affected by the loss of Erk2 function. The erk2⁻ cells had a severe defect in growth on bacterial lawns but assays of bacterial cell engulfment displayed only subtle changes in the rate of bacterial engulfment. Only cells with no MAPK function, erk1⁻erk2⁻ double mutants, displayed a severe proliferation defect in axenic medium. Loss of Erk2 impaired the phosphorylation of Erk1 in secondary responses to folate stimulation indicating that Erk2 has a role in the regulation of Erk1 activation during chemotaxis. Loss of the only known Dictyostelium MAPK kinase, MekA, prevented the phosphorylation of Erk1 but not Erk2 in response to folate and cAMP confirming that Erk2 is not regulated by a conventional MAP2K. This lack of MAP2K phosphorylation of Erk2 and the sequence similarity of Erk2 to mammalian MAPK15 (Erk8) suggest that the Dictyostelium Erk2 belongs to a group of atypical MAPKs. MAPK activation has been observed in chemotactic responses in a wide range of organisms but this study demonstrates an essential role for MAPK function in chemotactic movement. This study also confirms that MAPKs provide critical contributions to cell proliferation.     

Locally controlled inhibitory mechanisms are involved in eukaryotic GPCR-mediated chemosensing

Gprotein-coupled receptor (GPCR) signaling mediates a balance of excitatory and inhibitory activities that regulate Dictyostelium chemosensing to cAMP. The molecular nature and kinetics of these inhibitors are unknown. We report that transient cAMP stimulations induce PIP3 responses without a refractory period, suggesting that GPCR-mediated inhibition accumulates and decays slowly. Moreover, exposure to cAMP gradients leads to asymmetric distribution of the inhibitory components. The gradients induce a stable accumulation of the PIP3 reporter PHCrac-GFP in the front of cells near the cAMP source. Rapid withdrawal of the gradient led to the reassociation of G protein subunits, and the return of the PIP3 phosphatase PTEN and PHCrac-GFP to their pre-stimulus distribution. Reapplication of cAMP stimulation produces a clear PHCrac-GFP translocation to the back but not to the front, indicating that a stronger inhibition is maintained in the front of a polarized cell. Our study demonstrates a novel spatiotemporal feature of currently unknown inhibitory mechanisms acting locally on the PI3K activation pathway.