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.     

An intersection of the cAMP/PKA and two-component signal transduction systems in Dictyostelium.

Terminal differentiation of both stalk and spore cells in Dictyostelium can be triggered by activation of cAMP-dependent protein kinase (PKA). A screen for mutants where stalk and spore cells mature in isolation produced three genes which may act as negative regulators of PKA: rdeC (encoding the PKA regulatory subunit), regA and rdeA. The biochemical properties of RegA were studied in detail. One domain is a cAMP phosphodiesterase (Km approximately 5 microM); the other is homologous to response regulators (RRs) of two-component signal transduction systems. It can accept phosphate from acetyl phosphate in a reaction typical of RRs, with transfer dependent on Asp212, the predicted phosphoacceptor. RegA phosphodiesterase activity is stimulated up to 8-fold by the phosphodonor phosphoramidate, with stimulation again dependent on Asp212. This indicates that phosphorylation of the RR domain activates the phosphodiesterase domain. Overexpression of the RR domain in wild-type cells phenocopies a regA null. We interpret this dominant-negative effect as due to a diversion of the normal flow of phosphates from RegA, thus preventing its activation. Mutation of rdeA is known to produce elevated cAMP levels. We propose that cAMP breakdown is controlled by a phosphorelay system which activates RegA, and may include RdeA. Cell maturation should be triggered when this system is inhibited.     

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 amebae alter motility differently in response to increasing versus decreasing temporal gradients of cAMP

Using a perfusion chamber, we examined the behavior of individual amebae in increasing and decreasing temporal gradients of cAMP. We demonstrated that amebae respond to increasing temporal gradients of cAMP with stimulated motility and to corresponding decreasing temporal gradients with depressed motility. Depressed motility observed in decreasing temporal gradients corresponded to the inhibited levels observed when cAMP was applied at constant concentrations. These results were consistent with a simple model for the motile behavior of amebae in an early aggregation territory in which nondissipating waves of cAMP originate at the aggregation center and travel outward periodically. We conclude that chemotactically responsive amebae can assess whether a temporal gradient of chemoattractant is increasing or decreasing in the absence of a spatial gradient, and can adjust their motility accordingly.     

Amebae of Dictyostelium discoideum respond to an increasing temporal gradient of the chemoattractant cAMP with a reduced frequency of turning: evidence for a temporal mechanism in ameboid chemotaxis

In an aggregation territory of Dictyostelium discoideum, outwardly moving, nondissipating waves of the chemoattractant cAMP sweep across each ameba. At the front of each wave, an ameba experiences an increasing temporal and a positive spatial gradient of cAMP. At the back of a wave, an ameba experiences a decreasing temporal and a negative spatial gradient of cAMP. Employing a perfusion chamber, we have mimicked the temporal dynamics of these waves in the absence of a spatial gradient and demonstrated that the frequency of lateral pseudopod formation and the frequency of turning are dramatically affected by the direction and dynamics of the temporal gradient. In addition, since an ameba will move in a directed fashion up a shallow, nonpulsatile gradient of cAMP, we also mimicked the increasing temporal gradient generated by an ameba moving up a shallow spatial gradient. The frequency of lateral pseudopod formation and the frequency of turning were depressed. Together, these results demonstrate that amebae can assess the direction of a temporal gradient of chemoattractant in the absence of a spatial gradient and alter both the frequency of pseudopod extension and turning, accordingly. Although these results do not rule out the involvement of a spatial mechanism in assessing a spatial gradient, they strongly suggest that the temporal dynamics of a cAMP wave or the temporal gradient generated by an ameba moving through a spatial gradient may play a major role in chemotaxis.     

RasC plays a role in transduction of temporal gradient information in the cyclic-AMP wave of Dictyostelium discoideum

To define the role that RasC plays in motility and chemotaxis, the behavior of a rasC null mutant, rasC-, in buffer and in response to the individual spatial, temporal, and concentration components of a natural cyclic AMP (cAMP) wave was analyzed by using computer-assisted two-dimensional and three-dimensional motion analysis systems. These quantitative studies revealed that rasC- cells translocate at the same velocity and exhibit chemotaxis up spatial gradients of cAMP with the same efficiency as control cells. However, rasC- cells exhibit defects in maintaining anterior-posterior polarity along the substratum and a single anterior pseudopod when translocating in buffer in the absence of an attractant. rasC- cells also exhibit defects in their responses to both the increasing and decreasing temporal gradients of cAMP in the front and the back of a wave. These defects result in the inability of rasC- cells to exhibit chemotaxis in a natural wave of cAMP. The inability to respond normally to temporal gradients of cAMP results in defects in the organization of the cytoskeleton, most notably in the failure of both F actin and myosin II to exit the cortex in response to the decreasing temporal gradient of cAMP in the back of the wave. While the behavioral defect in the front of the wave is similar to that of the myoA-/myoF- myosin I double mutant, the behavioral and cytoskeletal defects in the back of the wave are similar to those of the S13A myosin II regulatory light-chain phosphorylation mutant. Expression array data support the premise that the behavioral defects exhibited by the rasC- mutant are the immediate result of the absence of RasC function.     

The RdeA-RegA system, a eukaryotic phospho-relay controlling cAMP breakdown.

The regA and rdeA gene products of Dictyostelium are involved in the regulation of cAMP signaling. The response regulator, RegA, is composed of an N-terminal receiver domain linked to a C-terminal cAMP-phosphodiesterase domain. RdeA may be a phospho-transfer protein that supplies phosphates to RegA. We show genetically that phospho-RegA is the activated form of the enzyme in vivo, in that the predicted site of aspartate phosphorylation is required for full activity. We show biochemically that RdeA and RegA communicate, as evidenced by phospho-transfer between the two proteins in vitro. Phospho-transfer is dependent on the presumed phospho-accepting amino acids, histidine 65 of RdeA and aspartate 212 of RegA, and occurs in both directions. Phosphorylation of RegA by a heterologous phospho-donor protein activates RegA phosphodiesterase activity at least 20-fold. Our results suggest that the histidine phosphotransfer protein, RdeA, and the response regulator, RegA, constitute two essential elements in a eukaryotic His-Asp phospho-relay network that regulates Dictyostelium development and fruiting body maturation.     

Autogenous Regulation of the Rega Gene of Bacteriophage T4: Derepression of Translation

The regA gene of phage T4 encodes a translational repressor that inhibits utilization of its own mRNA as well as the translation of a number of other phage-induced mRNAs. In recombinant plasmids, autogenous translational repression limits production of the RegA protein when the cloned structural gene is expressed under control of a strong, plasmid-borne promoter (lambda PL). We have found that a genetic fusion which places the regA ribosome binding domain in proximity to active translation leads to partial derepression of wild-type RegA protein synthesis. The derepression is not due to increased synthesis of regA RNA, suggesting that it occurs at the translational level. Derepressed clones of the wild-type regA gene were used to overproduce and purify the repressor. In an in vitro assay the wild-type target was sensitive and a mutant target was resistant to inhibition by the added protein. The results suggest that the sensitivity of a regA-regulated cistron to translational repression may depend on the competition between ribosomes and RegA protein for overlapping recognition sequences in the translation initiation domain of the mRNA.     

Constitutively active protein kinase A disrupts motility and chemotaxis in Dictyostelium discoideum

The deletion of the gene for the regulatory subunit of protein kinase A (PKA) results in constitutively active PKA in the pkaR mutant. To investigate the role of PKA in the basic motile behavior and chemotaxis of Dictyostelium discoideum, pkaR mutant cells were subjected to computer-assisted two- and three-dimensional motion analysis. pkaR mutant cells crawled at only half the speed of wild-type cells in buffer, chemotaxed in spatial gradients of cyclic AMP (cAMP) but with reduced efficiency, were incapable of suppressing lateral pseudopods in the front of temporal waves of cAMP, a requirement for natural chemotaxis, did not exhibit the normal velocity surge in response to the front of a wave, and were incapable of chemotaxing toward an aggregation center in natural waves generated by wild-type cells that made up the majority of cells in mixed cultures. Many of the behavioral defects appeared to be the result of the constitutively ovoid shape of the pkaR mutant cells, which forced the dominant pseudopod off the substratum and to the top of the cell body. The behavioral abnormalities that pkaR mutant cells shared with regA mutant cells are discussed by considering the pathway ERK2 —| RegA —| [cAMP] → PKA, which emanates from the front of a wave. The results demonstrate that cells must suppress PKA activity in order to elongate along a substratum, suppress lateral-pseudopod formation, and crawl and chemotax efficiently. The results also implicate PKA activation in dismantling cell polarity at the peak and in the back of a natural cAMP wave.     

Translational repression: biological activity of plasmid-encoded bacteriophage T4 RegA protein

The RegA protein of bacteriophage T4 is a translational repressor that regulates expression of several phage early mRNAs. We have cloned wild-type and mutant alleles of the T4 regA gene under control of the heat-inducible, plasmid-borne leftward promoter (PL) of phage lambda. Expression of the cloned regA+ gene resulted in the synthesis of a protein that closely resembled phage-encoded RegA protein in biological properties. It repressed its own synthesis (autogenous translational control) as well as the synthesis of specific T4-encoded proteins that are known from other studies to be under RegA-mediated translational control. Cloned mutant alleles of regA exhibited derepressed synthesis of the mutant regA gene products and were ineffective in trans against RegA-sensitive mRNA targets. The effects of plasmid-encoded RegA proteins were also demonstrated in experiments using two compatible plasmids in uninfected Escherichia coli. The two-plasmid assays confirm the sensitivities of several cloned T4 genes to RegA-mediated translational repression and are well-suited for genetic analysis of RegA target sites. Repression specificity in this system was demonstrated by using wild-type and operator-constitutive translational initiation sites of T4 rIIB fused to lacZ. The results show that no additional T4 products are required for RegA-mediated translational repression. Additional evidence is provided for the proposal that uridine-rich mRNA sequences are preferred targets for the repressor. Surprisingly, plasmid-generated RegA protein represses the synthesis of some E. coli proteins and appears to enhance selectively the synthesis of others. The RegA protein may have multiple functions, and its binding sites are not restricted to phage mRNAs.     

Cell communication by periodic cyclic-AMP pulses.

At the surface of aggregating cells of the slime mould, Dictyostelium discoideum, two different sites interacting with extracellular cAMP are detectable: binding sites and cycl-nucleotide phosphodiesterase. Both sites are developmentally regulated. An adequate stimulus for the chemoreceptor system in D. discoideum is the change of cAMP concentration in time, rather than concentration per se: long-term binding of cAMP causes only short-term response. The system is, consequently, adapted to the recognition of pulses rather than to steady-state concentrations of cAMP. The ce,lls are, nevertheless, able to sense stationary spatial gradients and to respond to them by chemotactic orientation. The possibility is discussed that they do so by transforming spatial concentration changes into temporal ones, using extending pseudopods as sensors. The cAMP recognition system is part of a molecular network involved in the generation of spatio-temporal patterns of cellular activities. This system controls the periodic formation of chemotactic signals and their propagation from cell to cell. The phosphodiesterase limits the duration of the cAMP pulses and thus sharply separates the periods of signalling; the binding sites at the cell surface are supposed to be the chemoreceptors. The control of cellular activities via cAMP receptors can be studied with biochemical techniques with cell suspensions in which spatial inhomogeneities are suppressed by intense stirring, whereas the temporal aspect of the spatiotemporal pattern is preserved. Under these conditions it can be shown that the extracellular cAMP concentration changes periodically, and that the phase of the cellular oscillator can be shifted by external pulses of cAMP. It can also be shown that small cAMP pulses induce a high output of cAMP, which demonstrates signal amplification, a function necessary for a cellular relay system.     

Chemoattraction and chemotaxis in Dictyostelium discoideum: myxamoeba cannot read spatial gradients of cyclic adenosine monophosphate

Myxamoebae of the morphogenetic cellular slime mold Dictyostelium discoideum are thought to be able to accurately read and respond to directional information in spatial gradients of cyclic AMP. We examined the spatial and temporal mechanisms proposed for chemotaxis by comparing the behavior of spreading or evenly distributed cell populations after exposure to well-defined spatial gradients. The effects of gradient generation on cells were avoided by using predeveloped gradients. Qualitatively different responses were obtained using (a) isotropic, (b) static spatial, or (c) temporal (impulse) gradients in a simple chamber of penetrable micropore filters. We simulated models of chemotaxis and chemokinesis to aid our interpretations. The attractive and locomotory responses of populations were maximally stimulated by 0.05 microM cyclic AMP, provided that cellular phosphodiesterase was inhibited. But a single impulse of cyclic AMP during gradient development caused a greater and qualitatively different attraction. Attraction in spatial gradients was only transient, in that populations eventually developed a random distribution when confined to a narrow territory. Populations never accumulated nor lost their random distribution even in extremely steep spatial gradients. Attraction in spatial gradients was inducible only in spreading populations, not randomly distributed ones. Thus, spatial gradients effect biased-random locomotion: i.e., chemokinesis without adaptation. Cells cannot read gradients; the reaction of the cells is stochastic. Spatial gradients do not cause chemotaxis, which probably requires a sharp stimulant concentration increase (a temporal gradient) as a pulse or impulse. The results also bear on concepts of how embryonic cells might be able to decipher the positional information in a morphogen spatial gradient during development.     

Identification of RegA protein from Pseudomonas aeruginosa using anti-RegA antibody

The product of Pseudomonas aeruginosa regA gene acts as a positive regulator of exotoxin A expression. The protein, RegA, was overproduced in E. coli transformed with an expression vector containing the regA gene. The overproduced RegA accumulated in E. coli in the form of inclusion bodies. The latter were isolated and served as a source of antigen for raising polyclonal antibodies. The antibodies reacted specifically with a P. aeruginosa protein whose molecular weight corresponded to that predicted for RegA from its known DNA sequence, and whose response to modulating factors matched that expected for RegA. The immunodetectable RegA was localized in the membrane fraction of P. aeruginosa strain PA103.     

The cyclic AMP phosphodiesterase RegA critically regulates encystation in social and pathogenic amoebas

Amoebas survive environmental stress by differentiating into encapsulated cysts. As cysts, pathogenic amoebas resist antibiotics, which particularly counteracts treatment of vision-destroying Acanthamoeba keratitis. Limited genetic tractability of amoeba pathogens has left their encystation mechanisms unexplored. The social amoeba Dictyostelium discoideum forms spores in multicellular fruiting bodies to survive starvation, while other dictyostelids, such as Polysphondylium pallidum can additionally encyst as single cells. Sporulation is induced by cAMP acting on PKA, with the cAMP phosphodiesterase RegA critically regulating cAMP levels. We show here that RegA is deeply conserved in social and pathogenic amoebas and that deletion of the RegA gene in P. pallidum causes precocious encystation and prevents cyst germination. We heterologously expressed and characterized Acanthamoeba RegA and performed a compound screen to identify RegA inhibitors. Two effective inhibitors increased cAMP levels and triggered Acanthamoeba encystation. Our results show that RegA critically regulates Amoebozoan encystation and that components of the cAMP signalling pathway could be effective targets for therapeutic intervention with encystation.     

Genetic interactions of the E3 ubiquitin ligase component FbxA with cyclic AMP metabolism and a histidine kinase signaling pathway during Dictyostelium discoideum development

Dictyostelium discoideum amoebae with an altered fbxA gene, which is thought to encode a component of an SCF E3 ubiquitin ligase, have defective regulation of cell type proportionality. In chimeras with wild-type cells, the mutant amoebae form mainly spores, leaving the construction of stalks to wild-type cells. To examine the role of fbxA and regulated proteolysis, we have recovered the promoter of fbxA and shown that it is expressed in a pattern resembling that of a prestalk-specific gene until late in development, when it is also expressed in developing spore cells. Because fbxA cells are developmentally deficient in pure culture, we were able to select suppressor mutations that promote sporulation of the original mutant. One suppressor mutation resides within the gene regA, which encodes a cyclic AMP (cAMP) phosphodiesterase linked to an activating response regulator domain. In another suppressor, there has been a disruption of dhkA, a gene encoding a two-component histidine kinase known to influence Dictyostelium development. RegA appears precociously and in greater amounts in the fbxA mutant than in the wild type, but in an fbxA/dhkA double mutant, RegA is restored to wild-type levels. Because the basis of regA suppression might involve alterations in cAMP levels during development, the concentrations of cAMP in all strains were determined. The levels of cAMP are relatively constant during multicellular development in all strains except the dhkA mutant, in which it is reduced at least sixfold. The level of cAMP in the double mutant dhkA/fbxA is relatively normal. The levels of cAMP in the various mutants do not correlate with spore formation, as would be expected on the basis of our present understanding of the signaling pathway leading to the induction of spores. Altered amounts of RegA and cAMP early in the development of the mutants suggest that both fbxA and dhkA genes act earlier than previously thought.     

Sequence analysis of conserved regA and variable orf43.1 genes in T4-like bacteriophages.

Bacteriophage T4 RegA protein is a translational repressor of several phage mRNAs. In the T4-related phages examined, regA nucleotide sequences are highly conserved and the inferred amino acid sequences are identical. The exceptional phage, RB69, did not produce a RegA protein reproducibly identifiable by Western blots (immunoblots) nor did it produce mRNA that hybridized to T4 regA primers. Nucleotide sequences of either 223 or 250 base pairs were identified immediately 3' to regA in RB18 and RB51 that were absent in T-even phages. Open reading frames in these regions, designated orf43.1RB18 and orf43.1RB51, potentially encode related proteins of 8.5 and 9.2 kilodaltons, respectively. orf43.1 sequences, detected in 13 of 27 RB bacteriophage chromosomes analyzed by polymerase chain reaction, are either RB18- or RB51-like and have flanking repeat sequences that may promote orf43.1 deletion.     

Osmotic stress response in Dictyostelium is mediated by cAMP

DokA, a homolog of bacterial hybrid histidine kinases, is essential for hyperosmotic stress resistance in Dictyostelium: We show that a transient intracellular cAMP signal, dependent on the presence of DokA, is generated in response to an osmotic shock. This variation of cAMP levels contributes to survival under hypertonic conditions. In contrast to the low cAMP levels observed in dokA(-) strains, overexpression of the receiver domain of DokA causes an increase in cAMP levels, resulting in a rapidly developing phenotype. We present biochemical and cell biological data indicating that the DokA receiver domain is a dominant-negative regulator of a phosphorelay, which controls the intracellular cAMP phosphodiesterase RegA. The activity of the DokA receiver domain depends on a conserved aspartate, mutation of which reverses the developmental phenotype, as well as the deregulation of cAMP metabolism.