Actin cross-linking proteins cortexillin I and II are required for cAMP signaling during Dictyostelium chemotaxis and development.

Starvation induces Dictyostelium amoebae to secrete cAMP, toward which other amoebae stream, forming multicellular mounds that differentiate and develop into fruiting bodies containing spores. We find that the double deletion of cortexillin (ctx) I and II alters the actin cytoskeleton and substantially inhibits all molecular responses to extracellular cAMP. Synthesis of cAMP receptor and adenylyl cyclase A (ACA) is inhibited, and activation of ACA, RasC, and RasG, phosphorylation of extracellular signal regulated kinase 2, activation of TORC2, and stimulation of actin polymerization and myosin assembly are greatly reduced. As a consequence, cell streaming and development are completely blocked. Expression of ACA-yellow fluorescent protein in the ctxI/ctxII-null cells significantly rescues the wild-type phenotype, indicating that the primary chemotaxis and development defect is the inhibition of ACA synthesis and cAMP production. These results demonstrate the critical importance of a properly organized actin cytoskeleton for cAMP-signaling pathways, chemotaxis, and development in Dictyostelium.

Involvement of the glucose-regulated protein 94 (Dd-GRP94) in starvation response of Dictyostelium discoideum cells.

Upon deprivation of nutrients, Dictyostelium discoideum Ax-2 cells arrest proliferation and initiate a metamorphosed developmental program including induction of altered gene expressions which are necessary for differentiation. In Ax-2 cells, we found out a member of Hsp90 family usually contained in the endoplasmic reticulum (ER), Dd-GRP94 (Dictyostelium discoideum glucose-regulated protein 94). In general, GRP94 are induced either by glucose-depletion or by depletion of Ca(2+) in intracellular Ca(2+) stores. Unexpectedly, however, the expression of Dd-grp94 was greatly reduced within 60 min of starvation. Dd-grp94-overexpressing cells (GRP94(OE) cells) collected without forming distinct aggregation streams, and never formed normal fruiting bodies. Also, prespore differentiation as well as maturation into spores and stalk cells were particularly impaired in the GRP94(OE) cells. Thus Dd-GRP94 seems to be crucial in late differentiation as well as in starvation response.

Aberrant folate response and premature development in a mutant of Dictyostelium discoideum.

Growth and development are mutually exclusive in Dictyostelium discoideum. The transition between the two stages of the life cycle is regulated by the relative abundance of nutrients and proteins secreted by the cells which reflect population density. At the transition from growth to development, the discoidin genes--developmental markers--are induced by the "quorum" protein PSF. The effect of PSF is counteracted by food bacteria and by folate [8]. We show that folate treatment during growth delays morphologic development. Furthermore, we demonstrate that in a mutant of Dictyostelium discoideum (V188, renamed HBW3), which expresses discoidinI during growth and which develops rapidly [46], discoidinI expression is less sensitive to folate than in wild type cells. Finally, we present evidence that fragments of the discoidinI gamma promoter which are unresponsive to PSF and CM are sufficient for misregulation in the mutant. The only known regulator of these promoter elements is folate. Changes in the expression of other early developmental genes are also shown. Taken together, these data suggest that the reduced sensitivity to folate might be the cause for the "rapid development" phenotype of the mutant and that folate regulates developmental timing.

Sequence-specific protein interaction with a transcriptional enhancer involved in the autoregulated expression of cAMP receptor 1 in Dictyostelium.

Major stages of Dictyostelium development are regulated by secreted, extracellular cAMP through activation of a serpentine receptor family. During early development, oscillations of extracellular cAMP mobilize cells for aggregation; later, continuous exposure to higher extracellular cAMP concentrations downregulates early gene expression and promotes cytodifferentiation and cell-specific gene expression. The cAMP receptor 1 gene CAR1 has two promoters that are differentially responsive to these extracellular cAMP stimuli. The early CAR1 promoter is induced by nM pulses of cAMP, which in turn are generated by CAR1-dependent activation of adenylyl cyclase (AC). Higher, non-fluctuating concentrations of cAMP will adapt this AC stimulus-response, repress the activated early promoter and induce the dormant late promoter. We now identify a critical element of the pulse-induced CAR1 promoter and a nuclear factor with sequence-specific interaction. Mutation of four nucleotides within the element prevents both in vitro protein binding and in vivo expression of an otherwise fully active early CAR1 promoter and multimerization of the wild-type, but not mutant, sequence will confer cAMP regulation to a quiescent heterologous promoter. These cis and trans elements, thus, constitute a part of the molecular response to the cAMP transmembrane signal cascade that regulates early development of Dictyostelium.

Switching of chemoattractant receptors programs development and morphogenesis in Dictyostelium: receptor subtypes activate common responses at different agonist concentrations.

One of the common functional features among G-protein coupled receptors is the occurrence of multiple subtypes involved in similar signal transduction events. The cAMP chemoattractant receptor family of Dictyostelium discoideum is composed of four receptors (cAR1-cAR4), which are expressed sequentially throughout the developmental transition from a unicellular to a multicellular organism. The receptors differ in affinity for cAMP and in the sequences of their C-terminal domains. In this study, we constitutively expressed cAR1, cAR2, and cAR3 as well as a series of chimeric and mutant receptors and assessed the capacity of each to mediate chemotaxis, activation of adenylyl cyclase and actin polymerization, and rescue the developmental defect of car1-/car3- cells. We found that various receptors and mutants sense different concentration ranges of cAMP but all can mediate identical responses during the aggregation stage of development. The responses displayed very similar kinetics, suggesting no major differences in regulatory properties attributable to the C-terminal domains. We speculate that switching of receptor subtypes during development enables the organism to respond to the changing concentrations of the chemoattractant and thereby program morphogenesis appropriately.

Phosphorylation of chemoattractant receptors is not essential for chemotaxis or termination of G-protein-mediated responses.

In several G-protein-coupled signaling systems, ligand-induced receptor phosphorylation by specific kinases is suggested to lead to desensitization via mechanisms including receptor/G-protein uncoupling, receptor internalization, and receptor down-regulation. We report here that elimination of phosphorylation of a chemoattractant receptor of Dictyostelium, either by site-directed substitution of the serines or by truncation of the C-terminal cytoplasmic domain, completely prevented agonist-induced loss of ligand binding but did not impair the adaptation of several receptor-mediated responses including the activation of adenylyl and guanylyl cyclases and actin polymerization. In addition, the phosphorylation-deficient receptors were capable of mediating chemotaxis, aggregation, and differentiation. We propose that for chemoattractant receptors agonist-induced phosphorylation regulates surface binding activity but other phosphorylation-independent mechanisms mediate response adaptation.

G alpha 3 regulates the cAMP signaling system in Dictyostelium.

The Dictyostelium discoideum developmental program is initiated by starvation and its progress depends on G-protein-regulated transmembrane signaling. Disruption of the Dictyostelium G-protein alpha-subunit G alpha 3 (g alpha 3-) blocks development unless the mutant is starved in the presence of artificial cAMP pulses. The function of G alpha 3 was investigated by examining the expression of several components of the cAMP transmembrane signaling system in the g alpha 3- mutant. cAMP receptor 1 protein, cyclic nucleotide phosphodiesterase, phosphodiesterase inhibitor, and aggregation-stage adenylyl cyclase mRNA expression were absent or greatly reduced when cells were starved without exogenously applied pulses of cAMP. However, cAMP receptor 1 protein and aggregation-stage adenylyl cyclase mRNA expression were restored by starving the g alpha 3- cells in the presence of exogenous cAMP pulses. Adenylyl cyclase activity was also reduced in g alpha 3- cells starved without exogenous cAMP pulses compared with similarly treated wild-type cells but was elevated to a level twofold greater than wild-type cells in g alpha 3- cells starved in the presence of exogenous cAMP pulses. These results suggest that G alpha 3 is essential in early development because it controls the expression of components of the transmembrane signaling system.

Dynamic distribution of chemoattractant receptors in living cells during chemotaxis and persistent stimulation.

While the localization of chemoattractant receptors on randomly oriented cells has been previously studied by immunohistochemistry, the instantaneous distribution of receptors on living cells undergoing directed migration has not been determined. To do this, we replaced cAR1, the primary cAMP receptor of Dictyostelium, with a cAR1-green fluorescence protein fusion construct. We found that this chimeric protein is functionally indistinguishable from wild-type cAR1. By time-lapse imaging of single cells, we observed that the receptors remained evenly distributed on the cell surface and all of its projections during chemotaxis involving turns and reversals of polarity directed by repositioning of a chemoattractant-filled micropipet. Thus, cell polarization cannot result from a gradient-induced asymmetric distribution of chemoattractant receptors. Some newly extended pseudopods at migration fronts showed a transient drop in fluorescence signals, suggesting that the flow of receptors into these zones may slightly lag behind the protrusion process. Challenge with a uniform increase in chemoattractant, sufficient to cause a dramatic decrease in the affinity of surface binding sites and cell desensitization, also did not significantly alter the distribution profile. Hence, the induced reduction in binding activity and cellular sensitivity cannot be due to receptor relocalization. The chimeric receptors were able to "cap" rapidly during treatment with Con A, suggesting that they are mobile in the plane of the cell membrane. This capping was not influenced by pretreatment with chemoattractant.

Clathrin heavy chain is required for spore cell but not stalk cell differentiation in Dictyostelium discoideum.

Previous studies of a clathrin-minus Dictyostelium cell line revealed important roles for clathrin heavy chain (clathrin) in endocytosis, secretion of lysosomal hydrolases and osmoregulation. In this paper, we examine the contribution of clathrin-mediated membrane traffic to development in Dictyostelium discoideum. Clathrin-minus cells were delayed in early development. When exposed to starvation conditions, clathrin-minus cells streamed and aggregated more slowly than wild-type cells. Although clathrin-minus cells displayed only 40% the level of extracellular cyclic AMP binding normally found in wild-type cells, they responded chemotactically to extracellular cyclic AMP. Clathrin-minus cells down-regulated cyclic AMP receptors, but only to half the extent of wild-type cells. We found that the extent of development of clathrin-minus cells was variable and influenced by environmental conditions. Although the mutant cells always progressed beyond the tipped mound stage, the final structure varied from a finger-like projection to a short, irregular fruiting body. Microscopic examination of these terminal structures revealed the presence of intact stalks but a complete absence of spores. Clathrin-minus cells expressed prestalk (ecmA and ecmB) and prespore (psA and cotB) genes normally, but were blocked in expression of the sporulation gene spiA. Using clathrin-minus cells that had been transformed with various promoter-lacZ reporter constructs, we saw only partial sorting of clathrin-minus prestalk and prespore cells. Even when mixed with wild-type cells, clathrin-minus cells failed to sort correctly and never constructed functional spores. These results suggest three roles for clathrin during Dictyostelium development. First, clathrin increases the efficiency of early development. Second, clathrin enables proper and efficient patterning of prestalk and prespore cells during culmination. Third, clathrin is essential for differentiation of mature spore cells.

The regulation of Dictyostelium development by transmembrane signalling.

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.

Seven helix cAMP receptors stimulate Ca2+ entry in the absence of functional G proteins in Dictyostelium.

Surface cAMP receptors (cARs) in Dictyostelium transmit a variety of signals across the plasma membrane. The best characterized cAR, cAR1, couples to the heterotrimeric guanine nucleotide-binding protein (G protein) alpha-subunit G alpha 2 to mediate activation of adenylyl and guanylyl cyclases and cell aggregation. cAR1 also elicits other cAMP-dependent responses including receptor phosphorylation, loss of ligand binding (LLB), and Ca2+ influx through a G alpha 2-independent pathway that may not involve G proteins. Here, we have expressed cAR1 and a related receptor, cAR3, in a g beta- strain (Lilly, P., Wu. L., Welker, D. L., and Devreotes, P. N. (1993) Genes & Dev. 7,986-995), which lacks G protein activity. Both cell lines failed to aggregate, a process requiring the G alpha 2 and G beta- subunits. In contrast, cAR1 phosphorylation in cAR1/g beta- cells showed a time course and cAMP dose dependence indistinguishable from those of cAR1/G beta+ controls. cAMP-induced LLB was also normal in the cAR1/g beta- cells. Finally, cAR1/g beta- cells and cAR3/g beta- cells showed a Ca2+ response with kinetics, agonist dependence, ion specificity, and sensitivity to depolarization agents that were like those of G beta+ controls, although they accumulated fewer Ca2+ ions per cAMP receptor than the control strains. Together, these results suggest that the G beta-subunit is not required for the activation or attenuation of cAR1 phosphorylation, LLB, or Ca2+ influx. It may, however, serve to amplify the Ca2+ response, possibly by modulating other intracellular Ca2+ signal transduction pathways.

Localization of ligand-induced phosphorylation sites to serine clusters in the C-terminal domain of the Dictyostelium cAMP receptor, cAR1.

When Dictyostelium cells are stimulated with cyclic adenosine 3',5'-monophosphate (cAMP), the major surface cAMP receptor expressed in early development, cAR1, undergoes a rapid phosphorylation and parallel decrease in electrophoretic mobility which may serve to regulate the activity of this G protein-coupled receptor. Biochemical analyses indicate the electrophoretic mobility shift is caused by phosphorylation of serine residues within the C-terminal cytoplasmic domain. The 18 serines of this domain are grouped in four clusters, designated 1 to 4 (in N- to C-terminal order). Two approaches were taken to determine the distribution of phosphorylation sites among the serine clusters. First, a proteolytic analysis of the C-terminal domain was performed. Second, mutants lacking various combinations of the serine clusters were created by site-directed mutagenesis and their abilities to undergo ligand-induced modification were determined. Both approaches yielded corroborative results consistent with the following model: the stimulus induces the addition of approximately two phosphates to cluster 1 and one to cluster 2; basal phosphorylation occurs predominantly in cluster 3 and to a lesser extent in cluster 2; and cluster 4 is not phosphorylated. The phosphorylation-deficient receptor mutants should be useful for establishing the role of ligand-induced phosphorylation of cAR1 in chemotaxis, cell-cell signaling, and gene expression.

Overproduction of the cyclic AMP receptor protein of Escherichia coli and expression of the engineered C-terminal DNA-binding domain.

Overproduction of the cyclic AMP receptor protein (CRP) from Escherichia coli, up to 25% of the soluble cell protein, has been achieved in an inducible host-vector system under transcriptional control of the lambda promoter PL. This system is ideally suited for large scale production and purification of CRP. In addition, a structural gene for the DNA-binding domain of CRP has been constructed. To this end the nucleotide sequence coding for the C-terminus was fused to the sequence coding for the first 10 N-terminal amino acids and cloned into suitable vectors. Good expression was achieved using the lambda PL promoter. The gene product, beta CRP, is recognized by anti-CRP antibodies.

Homology between CAP and Fnr, a regulator of anaerobic respiration in Escherichia coli.

The fnr gene is essential for the expression of anaerobic respiratory metabolism in Escherichia coli. Genetic and biochemical studies support the view that its product. Fnr, is a transcriptional regulatory protein specific for genes encoding anaerobic respiratory functions (fumarate, nitrate and nitrite reductases, hydrogenase, etc.). In this respect Fnr may be considered analogous to the well-characterized catabolite gene activator protein (CAP), which mediates the control of catabolite-sensitive gene transcription. With a view to identifying its function, the fnr gene has recently been cloned and the primary structure of the Fnr protein deduced from the nucleotide sequence. This has revealed the presence of three regions of sequence homology with CAP. One corresponds to the DNA-binding site, a region of about 20 highly conserved amino acids that is believed to form a characteristic three-dimensional structure in several transcriptional regulators. The other regions of homology are in the nucleotide binding domain of CAP but the residues that interact with cAMP are not identical in Fnr. These homologies suggest that Fnr and CAP may have similar three-dimensional structures and that the regulation of anaerobic energy metabolism may involve interaction between Fnr and an unidentified effector molecule.