The organisation of fruiting body formation in Dictyostelium minutum

The process of culmination was investigated in three strains of the species Dictyostelium minutum. After aggregates have been formed a pulsatile signalling mechanism arises; the centre of signal emission becomes the apex of the developing fruiting structure. In the late aggregate, all cells differentiate into prespore cells. Cells that have reached the apex of the culminating cells mass redifferentiate into stalk cells. In two of the three D. minutum strains, interruption of regular stalk formation, more or less random formation of stalk cells and the synthesis of stalk supporting material from cell debris often takes place. The formation of multiple apices on aggregates and early fruiting structures is characteristic for these two strains. Within the species D. minutum, the exhibition of a marked pulsatile signalling mechanism is correlated with a capacity to form a regularly shaped stalk and to organize relatively large cell masses. The possible function of pulsatile signalling in the culmination process is discussed.     

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 effect of the disruption of a gene encoding a PI4 kinase on the developmental defect exhibited by Dictyostelium rasC(-) cells

The disruption of the gene encoding the Dictyostelium Ras subfamily protein, RasC results in a strain that fails to aggregate with defects in both cAMP signal relay and chemotaxis. Restriction enzyme mediated integration disruption of a second gene in the rasC(-) strain resulted in cells that were capable of forming multicellular structures in plaques on bacterial lawns. The disrupted gene, designated pikD(1), encodes a member of the phosphatidyl-inositol-4-kinase beta subfamily. Although the rasC(-)/pikD(1) cells were capable of progressing through early development, when starved on a plastic surface under submerged conditions, they did not form aggregation streams or exhibit pulsatile motion. The rasC(-)/pikD(1) cells were extremely efficient in their ability to chemotax to cAMP in a spatial gradient, although the reduced phosphorylation of PKB in response to cAMP observed in rasC(-) cells, was unchanged. In addition, the activation of adenylyl cyclase, which was greatly reduced in the rasC(-) cells, was only minimally increased in the rasC(-)/pikD(1) strain. Thus, although the rasC(-)/pikD(-) cells were capable of associating to form multicellular structures, normal cell signaling was clearly not restored. The disruption of the pikD gene in a wild type background resulted in a strain that was delayed in aggregation and formed large aggregation streams, when starved on a plastic surface under submerged conditions. This strain also exhibited a slight defect in terminal development. In conclusion, disruption of the pikD gene in a rasC(-) strain resulted in cells that were capable of forming multicellular structures, but which did so in the absence of normal signaling and aggregation stream formation.     

TOR complex 2 integrates cell movement during chemotaxis and signal relay in Dictyostelium

Dictyostelium cells form a multicellular organism through the aggregation of independent cells. This process requires both chemotaxis and signal relay in which the chemoattractant cAMP activates adenylyl cyclase through the G protein-coupled cAMP receptor cAR1. cAMP is produced and secreted and it activates receptors on neighboring cells, thereby relaying the chemoattractant signal to distant cells. Using coimmunoprecipitation and mass spectrometric analyses, we have identified a TOR-containing complex in Dictyostelium that is related to the TORC2 complex of Saccharomyces cerevisiae and regulates both chemotaxis and signal relay. We demonstrate that mutations in Dictyostelium LST8, RIP3, and Pia, orthologues of the yeast TORC2 components LST8, AVO1, and AVO3, exhibit a common set of phenotypes including reduced cell polarity, chemotaxis speed and directionality, phosphorylation of Akt/PKB and the related PKBR1, and activation of adenylyl cyclase. Further, we provide evidence for a role of Ras in the regulation of TORC2. We propose that, through the regulation of chemotaxis and signal relay, TORC2 plays an essential role in controlling aggregation by coordinating the two essential arms of the developmental pathway that leads to multicellularity in Dictyostelium.     

Galpha-mediated inhibition of developmental signal response

BACKGROUND: Seven-transmembrane receptor (7-TMR)-G protein networks are molecular sensors of extracellular signals in all eukarya. These pathways cycle through activated (sensitized) and inhibited (desensitized) states, and, while many of the molecular components for signal activation have been described, inhibitory mechanisms are not well characterized. In Dictyostelium, 7-TM cAMP receptors direct chemotaxis and development but also regulate the periodic synthesis of their own ligand, the chemoattractant/morphogen cAMP. We now demonstrate through loss-of-function/gain-of-function studies that the novel heterotrimeric Galpha9 protein subunit regulates an inhibitory pathway during early Dictyostelium development for the cAMP signal response.RESULTS: galpha9 null cells form more cAMP signaling centers, are more resistant to compounds that inhibit cAMP signaling, and complete aggregation sooner and at lower cell densities than wild-type cells. These phentoypes are consistent with the loss of an inhibitory signaling pathway during development of galpha9 null cells. Cells expressing constitutively activated Galpha9 are defective in cAMP signaling center formation and development at low cell density and display an increased sensitivity to cAMP signal inhibition that is characteristic of enhanced suppression of the cAMP signal response. Finally, we demonstrate that galpha9 null cells, which have been codeveloped with a majority of wild-type cells, primarily establish cAMP signaling centers and are able to non-autonomously direct wild-type cells to adopt a galpha9 null-like phenotype.CONCLUSIONS: We suggest that Galpha9 functions in an inhibitory-feedback pathway that regulates cAMP signaling center formation and propagation. Galpha9 may be part of the mechanism that regulates lateral signal inhibition or that modulates receptor desensitization.     

The role of Src family kinases in egg activation

Solitary amoebae of Dictyostelium discoideum are frequently exposed to stressful conditions in nature, and their multicellular development is one response to environmental stress. Here we analyzed an aggregation stage abundant gene, krsA, homologous to human krs1 (kinase responsive to stress 1) to understand the mechanisms for the initiation of development and cell fate determination. The krsA- cells exhibited reduced viability under hyperosmotic conditions. They produced smaller aggregates on membrane filters and did not form aggregation streams on a plastic surface under submerged starvation conditions, but were normal in sexual development. During early asexual development, the expression of cAMP-related genes peaked earlier in the knockout mutants. Neither cAMP oscillation in starved cells nor an increase in the cAMP level following osmotic stress was observed in krsA-. The nuclear export signal, as well as the kinase domain, in KrsA was necessary for stream formation. These results strongly suggest that krsA is involved in cAMP relay, and that signaling pathways for multicellular development have evolved in unison with the stress response.     

Aggregation during sexual development in Dictyostelium discoideum.

A microcinematographic analysis of the behaviour and movements of cells and cell masses in mated cultures (NC4 X VI2) of Dictyostelium discoideum indicates that a chemotactic process directs cell aggregation during macrocyst development. Zygote giant cells form before aggregation begins and act as the aggregation centres. Young multicellular macrocyst stages are sources of cyclic AMP, and amoebae from macrocyst cultures orient chemotactically to cyclic AMP. The data, coupled with other characteristics such as pulsatile streaming, suggest that the aggregation process leading to macrycyst development is the same as that occurring during fruit construction. Other aspects of sexual development are also discussed. Based upon these data, we propose a model for the sequence of events leading to macrocyst development in D. discoideum.     

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.     

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.     

Phospholipase D controls Dictyostelium development by regulating G protein signaling

Dictyostelium discoideum cells normally exist as individual amoebae, but will enter a period of multicellular development upon starvation. The initial stages of development involve the aggregation of individual cells, using cAMP as a chemoattractant. Chemotaxis is initiated when cAMP binds to its receptor, cAR1, and activates the associated G protein, Gα2βγ. However, chemotaxis will not occur unless there is a high density of starving cells present, as measured by high levels of the secreted quorum sensing molecule, CMF. We previously demonstrated that cells lacking PldB bypass the need for CMF and can aggregate at low cell density, whereas cells overexpressing pldB do not aggregate even at high cell density. Here, we found that PldB controlled both cAMP chemotaxis and cell sorting. PldB was also required by CMF to regulate G protein signaling. Specifically, CMF used PldB, to regulate the dissociation of Gα2 from Gβγ. Using fluorescence resonance energy transfer (FRET), we found that along with cAMP, CMF increased the dissociation of the G protein. In fact, CMF augmented the dissociation induced by cAMP. This augmentation was lost in cells lacking PldB. PldB appears to mediate the CMF signal through the production of phosphatidic acid, as exogenously added phosphatidic acid phenocopies overexpression of pldB. These results suggest that phospholipase D activity is required for CMF to alter the kinetics of cAMP-induced G protein signaling.     

Aggregation of human dental pulp cells into 3D spheroids enhances their migration ability after reseeding

Multicellular three-dimensional (3D) spheroids allow intimate cell–cell communication and cell–extracellular matrix interaction. Thus, 3D cell spheroids better mimic microenvironment in vivo than two-dimensional (2D) monolayer cultures. The purpose of this study was to evaluate the behaviors of human dental pulp cells (DPCs) cultured on chitosan and polyvinyl alcohol (PVA) membranes. The protein expression of hypoxia-inducible factor 1-α (HIF-1α) and vascular endothelial growth factor (VEGF), and the migration ability of the DPCs from 2D versus 3D environments were investigated. The results showed that both chitosan and PVA membranes support DPCs aggregation to form multicellular spheroids. In comparison to 2D cultures on tissue culture polystyrene, DPC spheroids exhibited higher protein expression of HIF-1α and VEGF. The treatment with YC-1 (inhibitor to HIF-1α) blocked the upregulation of VEGF, indicating a downstream event to HIF-1α expression. When DPC spheroids were collected and subjected to the transwell assay, the cells growing outward from 3D spheroids showed greater migration ability than those from 2D cultures. Moreover, DPCs aggregation and spheroid formation on chitosan membrane were abolished by Y-27632 (inhibitor to Rho-associated kinases), whereas the inhibitory effect did not exist on PVA membrane. This suggests that the mechanism regulating DPCs aggregation and spheroid formation on chitosan membrane is involved with the Rho-associated kinase signaling pathway. In summary, the multicellular spheroid structure was beneficial to the protein expression of HIF-1α and VEGF in DPCs and enhanced the migration ability of the cells climbing from spheroids. This study showed a new perspective in exploring novel strategies for DPC-based research and application.     

Signaling molecules involved in the transition of growth to development of Dictyostelium discoideum

The social amoeba Dictyostelium discoideum, a powerful paradigm provides clear insights into the regulation of growth and development. In addition to possessing complex individual cellular functions like a unicellular eukaryote, D. discoideum cells face the challenge of multicellular development. D. discoideum undergoes a relatively simple differentiation process mainly by cAMP mediated pathway. Despite this relative simplicity, the regulatory signaling pathways are as complex as those seen in metazoan development. However, the introduction of restriction-enzyme-mediated integration (REMI) technique to produce developmental gene knockouts has provided novel insights into the discovery of signaling molecules and their role in D. discoideum development. Cell cycle phase is an important aspect for differentiation of D. discoideum, as cells must reach a specific stage to enter into developmental phase and specific cell cycle regulators are involved in arresting growth phase genes and inducing the developmental genes. In this review, we present an overview of the signaling molecules involved in the regulation of growth to differentiation transition (GDT), molecular mechanism of early developmental events leading to generation of cAMP signal and components of cAMP relay system that operate in this paradigm.     

The Regulation of Dictyostelium Development by Transmembrane Signalling

ABSTRACT. Dictyostelium discoideum has a well characterized life cycle where unicellular growth and multicellular development are separated events. Development is dependent upon signal transduction mediated by cell surface, cAMP receptor/G protein linkages. Secreted cAMP acts extracellularly as a primary signal and chemoattractant. There are 4 genes for the distinct cAMP receptor subtypes, CAR1, CAR2, CAR3 and CAR4. These subtypes are expressed with temporally and spatially specific patterns and cells carrying null mutations for each gene have distinct developmental phenotypes. These results indicate an essential role for cAMP signalling throughout Dictyostelium development to regulate such diverse pathways as cell motility, aggregation (multicellularity), cytodifferentiation, pattern formation and cell type-specific gene expression.     

A discrete cell model with adaptive signalling for aggregation of Dictyostelium discoideum.

Dictyostelium discoideum (Dd) is a widely studied model system from which fundamental insights into cell movement, chemotaxis, aggregation and pattern formation can be gained. In this system aggregation results from the chemotactic response by dispersed amoebae to a travelling wave of the chemoattractant cAMP. We have developed a model in which the cells are treated as discrete points in a continuum field of the chemoattractant, and transduction of the extracellular cAMP signal into the intracellular signal is based on the G protein model developed by Tang & Othmer. The model reproduces a number of experimental observations and gives further insight into the aggregation process. We investigate different rules for cell movement the factors that influence stream formation the effect on aggregation of noise in the choice of the direction of movement and when spiral waves of chemoattractant and cell density are likely to occur. Our results give new insight into the origin of spiral waves and suggest that streaming is due to a finite amplitude instability.     

Localization and levels of cyclic AMP during development of Dictyostelium discoideum

Cyclic AMP functions as the chemotactic signal during aggregation of amoebae of the cellular slime mold Dictyostelium discoideum. Evidence suggests that cAMP also acts as a regulatory molecule during Dictyostelium multicellular differentiation. We have used ultramicrotechniques and a sensitive radioimmunoassay to measure the levels of cAMP within the culmination stage individual. We show that there is a peak of cAMP at the culmination stage of development and that in the individual at this stage the molecule is localized in a gradient within the spore mass.     

An information-theoretic characterization of the optimal gradient sensing response of cells

Many cellular systems rely on the ability to interpret spatial heterogeneities in chemoattractant concentration to direct cell migration. The accuracy of this process is limited by stochastic fluctuations in the concentration of the external signal and in the internal signaling components. Here we use information theory to determine the optimal scheme to detect the location of an external chemoattractant source in the presence of noise. We compute the minimum amount of mutual information needed between the chemoattractant gradient and the internal signal to achieve a prespecified chemotactic accuracy. We show that more accurate chemotaxis requires greater mutual information. We also demonstrate that a priori information can improve chemotaxis efficiency. We compare the optimal signaling schemes with existing experimental measurements and models of eukaryotic gradient sensing. Remarkably, there is good quantitative agreement between the optimal response when no a priori assumption is made about the location of the existing source, and the observed experimental response of unpolarized Dictyostelium discoideum cells. In contrast, the measured response of polarized D. discoideum cells matches closely the optimal scheme, assuming prior knowledge of the external gradient-for example, through prolonged chemotaxis in a given direction. Our results demonstrate that different observed classes of responses in cells (polarized and unpolarized) are optimal under varying information assumptions.     

A computational model of chemotaxis-based cell aggregation

We present a computational model that successfully captures the cell behaviors that play important roles in 2-D cell aggregation. A virtual cell in our model is designed as an independent, discrete unit with a set of parameters and actions. Each cell is defined by its location, size, rates of chemoattractant emission and response, age, life cycle stage, proliferation rate and number of attached cells. All cells are capable of emitting and sensing a chemoattractant chemical, moving, attaching to other cells, dividing, aging and dying. We validated and fine-tuned our in silico model by comparing simulated 24-h aggregation experiments with data derived from in vitro experiments using PC12 pheochromocytoma cells. Quantitative comparisons of the aggregate size distributions from the two experiments are produced using the Earth Mover's Distance (EMD) metric. We compared the two size distributions produced after 24 h of in vitro cell aggregation and the corresponding computer simulated process. Iteratively modifying the model's parameter values and measuring the difference between the in silico and in vitro results allow us to determine the optimal values that produce simulated aggregation outcomes closely matched to the PC12 experiments. Simulation results demonstrate the ability of the model to recreate large-scale aggregation behaviors seen in live cell experiments.