CbfA, the C-module DNA-binding factor, plays an essential role in the initiation of Dictyostelium discoideum development

We recently isolated from Dictyostelium discoideum cells a DNA-binding protein, CbfA, that interacts in vitro with a regulatory element in retrotransposon TRE5-A. We have generated a mutant strain that expresses CbfA at <5% of the wild-type level to characterize the consequences for D. discoideum cell physiology. We found that the multicellular development program leading to fruiting body formation is highly compromised in the mutant. The cells cannot aggregate and stay as a monolayer almost indefinitely. The cells respond properly to prestarvation conditions by expressing discoidin in a cell density-dependent manner. A genomewide microarray-assisted expression analysis combined with Northern blot analyses revealed a failure of CbfA-depleted cells to induce the gene encoding aggregation-specific adenylyl cyclase ACA and other genes required for cyclic AMP (cAMP) signal relay, which is necessary for aggregation and subsequent multicellular development. However, the cbfA mutant aggregated efficiently when mixed with as few as 5% wild-type cells. Moreover, pulsing cbfA mutant cells developing in suspension with nanomolar levels of cAMP resulted in induction of acaA and other early developmental genes. Although the response was less efficient and slower than in wild-type cells, it showed that cells depleted of CbfA are able to initiate development if given exogenous cAMP signals. Ectopic expression of the gene encoding the catalytic subunit of protein kinase A restored multicellular development of the mutant. We conclude that sensing of cell density and starvation are independent of CbfA, whereas CbfA is essential for the pattern of gene expression which establishes the genetic network leading to aggregation and multicellular development of D. discoideum.     

An adenylyl cyclase that functions during late development of Dictyostelium.

A variety of extracellular signals lead to the accumulation of cAMP which can act as a second message within cells by activating protein kinase A (PKA). Expression of many of the essential developmental genes in Dictyostelium discoideum are known to depend on PKA activity. Cells in which the receptor-coupled adenylyl cyclase gene, acaA, is genetically inactivated grow well but are unable to develop. Surprisingly, acaA(-) mutant cells can be rescued by developing them in mixtures with wild-type cells, suggesting that another adenylyl cyclase is present in developing cells that can provide the internal cAMP necessary to activate PKA. However, the only other known adenylyl cyclase gene in Dictyostelium, acgA, is only expressed during germination of spores and plays no role in the formation of fruiting bodies. By screening morphological mutants generated by Restriction Enzyme Mediated Integration (REMI) we discovered a novel adenylyl cyclase gene, acrA, that is expressed at low levels in growing cells and at more than 25-fold higher levels during development. Growth and development up to the slug stage are unaffected in acrA(-) mutant strains but the cells make almost no viable spores and produce unnaturally long stalks. Adenylyl cyclase activity increases during aggregation, plateaus during the slug stage and then increases considerably during terminal differentiation. The increase in activity following aggregation fails to occur in acrA(-) cells. As long as ACA is fully active, ACR is not required until culmination but then plays a critical role in sporulation and construction of the stalk.     

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.     

Fingerprinting of adenylyl cyclase activities during Dictyostelium development indicates a dominant role for adenylyl cyclase B in terminal differentiation.

Activation of cAMP-dependent protein kinase (PKA) triggers terminal differentiation in Dictyostelium, without an obvious requirement for the G-protein-coupled adenylyl cyclase, ACA, or the osmosensory adenylyl cyclase, ACG. A third adenylyl cyclase, ACB, was recently detected in rapidly developing mutants. The specific characteristics of ACA, ACG, and ACB were used to determine their respective activities during development of wild-type cells. ACA was highly active during aggregation, with negligible activity in the slug stage. ACG activity was not present at significant levels until mature spores had formed. ACB activity increased strongly after slugs had formed with optimal activity at early fruiting body formation. The same high activity was observed in slugs of ACG null mutants and ACA null mutants that overexpress PKA (acaA/PKA), indicating that it was not due to either ACA or ACG. The detection of high adenylyl cyclase activity in acaA/PKA null mutants contradicts earlier conclusions (B. Wang and A. Kuspa, Science 277, 251-254, 1997) that these mutants can develop into fruiting bodies in the complete absence of cAMP. In contrast to slugs of null mutants for the intracellular cAMP-phosphodiesterase REGA, where both intact cells and lysates show ACB activity, wild-type slugs only show activity in lysates. This indicates that cAMP accumulation by ACB in living cells is controlled by REGA. Both REGA inhibition and PKA overexpression cause precocious terminal differentiation. The developmental regulation of ACB and its relationship to REGA suggest that ACB activates PKA and induces terminal differentiation.     

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.     

Delineation of the roles played by RasG and RasC in cAMP-dependent signal transduction during the early development of Dictyostelium discoideum

On starvation, the cellular slime mold Dictyostelium discoideum initiates a program of development leading to formation of multicellular structures. The initial cell aggregation requires chemotaxis to cyclic AMP (cAMP) and relay of the cAMP signal by the activation of adenylyl cyclase (ACA), and it has been shown previously that the Ras protein RasC is involved in both processes. Insertional inactivation of the rasG gene resulted in delayed aggregation and a partial inhibition of early gene expression, suggesting that RasG also has a role in early development. Both chemotaxis and ACA activation were reduced in the rasG- cells, but the effect on chemotaxis was more pronounced. When the responses of rasG- cells to cAMP were compared with the responses of rasC- and rasC- rasG- strains, generated in otherwise isogenic backgrounds, these studies revealed that signal transduction through RasG is more important in chemotaxis and early gene expression, but that signal transduction through RasC is more important in ACA activation. Because the loss of either of the two Ras proteins alone did not result in a total loss of signal output down either of the branches of the cAMP signal-response pathway, there appears to be some overlap of function.     

Dual role of cAMP and involvement of both G-proteins and ras in regulation of ERK2 in Dictyostelium discoideum.

Dictyostelium discoideum expresses two Extracellular signal Regulated Kinases, ERK1 and ERK2, which are involved in growth, multicellular development and regulation of adenylyl cyclase. Binding of extracellular cAMP to cAMP receptor 1, a G-protein coupled cell surface receptor, transiently stimulates phosphorylation, activation and nuclear translocation of ERK2. Activation of ERK2 by cAMP is dependent on heterotrimeric G-proteins, since activation of ERK2 is absent in cells lacking the Galpha4 subunit. The small G-protein rasD also activates ERK2. In cells overexpressing a mutated, constitutively active rasD, ERK2 activity is elevated prior to cAMP stimulation. Intracellular cAMP and cAMP-dependent protein kinase (PKA) are essential for adaptation of the ERK2 response. This report shows that multiple signalling pathways are involved in regulation of ERK2 activity in D.discoideum.     

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.     

Intracellular role of adenylyl cyclase in regulation of lateral pseudopod formation during Dictyostelium chemotaxis

Cyclic AMP (cAMP) functions as the extracellular chemoattractant in the aggregation phase of Dictyostelium development. There is some question, however, concerning what role, if any, it plays intracellularly in motility and chemotaxis. To test for such a role, the behavior of null mutants of acaA, the adenylyl cyclase gene that encodes the enzyme responsible for cAMP synthesis during aggregation, was analyzed in buffer and in response to experimentally generated spatial and temporal gradients of extracellular cAMP. acaA- cells were defective in suppressing lateral pseudopods in response to a spatial gradient of cAMP and to an increasing temporal gradient of cAMP. acaA- cells were incapable of chemotaxis in natural waves of cAMP generated by majority control cells in mixed cultures. These results indicate that intracellular cAMP and, hence, adenylyl cyclase play an intracellular role in the chemotactic response. The behavioral defects of acaA- cells were surprisingly similar to those of cells of null mutants of regA, which encodes the intracellular phosphodiesterase that hydrolyzes cAMP and, hence, functions opposite adenylyl cyclase A (ACA). This result is consistent with the hypothesis that ACA and RegA are components of a receptor-regulated intracellular circuit that controls protein kinase A activity. In this model, the suppression of lateral pseudopods in the front of a natural wave depends on a complete circuit. Hence, deletion of any component of the circuit (i.e., RegA or ACA) would result in the same chemotactic defect.     

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.     

Requirements for the adenylyl cyclases in the development of Dictyostelium

It has been suggested that all intracellular signaling by cAMP during development of Dictyostelium is mediated by the cAMP-dependent protein kinase, PKA, since cells carrying null mutations in the acaA gene that encodes adenylyl cyclase can develop so as to form fruiting bodies under some conditions if PKA is made constitutive by overexpressing the catalytic subunit. However, a second adenylyl cyclase encoded by acrA has recently been found that functions in a cell autonomous fashion during late development. We have found that expression of a modified acaA gene rescues acrA- mutant cells indicating that the only role played by ACR is to produce cAMP. To determine whether cells lacking both adenylyl cyclase genes can develop when PKA is constitutive we disrupted acrA in a acaA- PKA-C(over) strain. When developed at high cell densities, acrA- acaA- PKA-C(over) cells form mounds, express cell type-specific genes at reduced levels and secrete cellulose coats but do not form fruiting bodies or significant numbers of viable spores. Thus, it appears that synthesis of cAMP is required for spore differentiation in Dictyostelium even if PKA activity is high.     

Two cell-counting factors regulate the aggregate size of the cellular slime mold Dictyostelium discoideum

Countin, a cell-counting factor in Dictyostelium discoideum, is considered to limit the maximum size of the multicellular structure, because a countin null strain forms a huge fruiting body compared to that of the wild-type. A novel gene, countin2, that is highly homologous to countin (40% identity in amino acid sequence) was identified in the D. discoideum genome. The countin2 null strain formed a 1.7-fold higher number of the aggregates, resulting in smaller fruiting bodies compared with those of wild-type cells. Thus, the Countin2 protein is thought to limit the minimum size of the multicellular structure. The size and number of aggregates formed by a mixture of countin null and countin2 null strains were the same as those of the wild-type. These findings demonstrate that a combination of Countin and Countin2 proteins determines the appropriate size of the multicellular structure of D. discoideum.