Adenosine receptor A2B mediates alcoholic hepatitis by regulating cAMP levels and the NF-KB pathway.

Alcoholic hepatitis is a serious form of liver damage. Inflammation is a key factor in alcoholic hepatitis and plays a key role in the progression of alcoholic liver disease. Adenosine receptor A2B (A2BAR) is a member of the adenosine receptor family and generally considered to be a negative regulator of the inflammatory response. We found that A2BAR was the most highly expressed adenosine receptor in ETOH-fed mouse liver tissue and was also highly expressed in primary Kupffer cells and ETOH-induced RAW264.7 cells. In addition, injection of BAY 60-6583 stimulated A2BAR, induced upregulation of the expression levels of cAMP, and reduced ETOH-induced steatosis and inflammation in mice. At the same time, knockdown of A2BAR in vitro increased the inflammatory response in RAW264.7 cells triggered by ETOH. After knockdown of A2BAR in vitro, the release of the inflammatory cytokines IL-6, IL-1ß and TNF-α was increased. After overexpression of A2BAR in vitro, the cAMP level was significantly increased, PKA expression was increased, the expression of phosphorylated proteins in the NF-kB signal transduction pathway was significantly affected, and the expression of the key phosphorylated protein p-P65 was decreased. However, after the simultaneous overexpression of A2BAR and inhibition of PKA, the expression of the key phosphorylated protein p-P65 was still significantly decreased. In addition, after the expression of A2BAR increased or decreased in RAW264.7 cells, AML-12 cells were cultured in the supernatant of RAW264.7 cells stimulated by ETOH, and the apoptosis rate was significantly changed by flow cytometry. These results suggest that A2BAR can reduce alcoholic steatohepatitis by upregulating cAMP levels and negatively regulating the NF-kB pathway. Overall, these findings suggest the significance of A2BAR-mediated inflammation in alcoholic liver disease.

Signal Transmission in Escherichia coli Cyclic AMP Receptor Protein for Survival in Extreme Acidic Conditions.

During the life cycle of enteric bacterium Escherichia coli, it encounters a wide spectrum of pH changes. The asymmetric dimer of the cAMP receptor protein, CRP, plays a key role in regulating the expressions of genes and the survival of E. coli. To elucidate the pH effects on the mechanism of signal transmission, we present a combination of results derived from ITC, crystallography, and computation. CRP responds to a pH change by inducing a differential effect on the affinity for the binding events to the two cAMP molecules, ensuing in a reversible conversion between positive and negative cooperativity at high and low pH, respectively. The structures of four crystals at pH ranging from 7.8 to 6.5 show that CRP responds by inducing a differential effect on the structures of the two subunits, particularly in the DNA binding domain. Employing the COREX/BEST algorithm, computational analysis shows the change in the stability of residues at each pH. The change in residue stability alters the connectivity between residues including those in cAMP and DNA binding sites. Consequently, the differential impact on the topology of the connectivity surface among residues in adjacent subunits is the main reason for differential change in affinity; that is, the pH-induced differential change in residue stability is the biothermodynamic basis for the change in allosteric behavior. Furthermore, the structural asymmetry of this homodimer amplifies the differential impact of any perturbations. Hence, these results demonstrate that the combination of these approaches can provide insights into the underlying mechanism of an apparent complex allostery signal and transmission in CRP.

Adrenomedullin 2 attenuates LPS-induced inflammation in microglia cells by receptor-mediated cAMP-PKA pathway.

Inflammation plays a critical role in the development of neurodegenerative diseases. Adrenomedullin 2 (AM2), a member of the calcitonin gene-related peptide family, has been known to have anti-inflammatory effects. Here, we evaluated the anti-inflammatory effects of AM2 in LPS-activated microglia and BV2 cells. The endogenous mRNA and protein expressions of AM2, calcitonin receptor-like receptor (CLR), receptor activity-modifying proteins (RAMPs) including RAMP1, RAMP2 and RAMP3 and the production of inflammatory mediators including tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) were detected by RT-PCR and Western blot. Our results revealed that LPS (1 µg/mL) significantly stimulated CLR, RAMP1, RAMP2 and RAMP3 protein expressions in BV2 microglia cells, but AM2 had a significant decrease. However, the mRNA levels of AM2, CLR, and RAMP1/2/3 were all markedly increased. LPS also induced obvious increases in mRNA and protein levels of the inflammatory mediators (TNF-α, IL-1ß, COX2 and iNOS). More importantly, AM2 (10 nM) administration effectively inhibited the mRNA and protein expressions of these mediators induced by LPS and increased the cAMP content in LPS-stimulated BV2 cells. Furthermore, the antagonism with AM2 receptor antagonist IMD17-47, adrenomedullin (AM) receptor antagonist by AM22-52 or the inhibition of protein kinase A (PKA) activation by P1195 effectively prevented the inhibitory role of AM2 in LPS-induced production of the above inflammatory mediators. In conclusion, AM2 inhibits LPS-induced inflammation in BV2 microglia cells that may be mainly through AM receptor-mediated cAMP-PKA pathway. Our results indicate AM2 plays an important protective role in microglia inflammation, suggesting therapeutic potential for AM2 in neuroinflammation diseases caused by activated microglia.

NaCl triggers the CRP-dependent increase of cAMP in Mycobacterium tuberculosis.

The second messenger 3',5'-cyclic adenosine monophosphate (3',5'-cAMP) has been shown to be involved in the regulation of many biological processes ranging from carbon catabolite repression in bacteria to cell signalling in eukaryotes. In mycobacteria, the role of cAMP and the mechanisms utilized by the bacterium to adapt to and resist immune and pharmacological sterilization remain poorly understood. Among the stresses encountered by bacteria, ionic and non-ionic osmotic stresses are among the best studied. However, in mycobacteria, the link between ionic osmotic stress, particularly sodium chloride, and cAMP has been relatively unexplored. Using a targeted metabolic analysis combined with stable isotope tracing, we show that the pathogenic Mycobacterium tuberculosis but not the opportunistic pathogen Mycobacterium marinum nor the non-pathogenic Mycobacterium smegmatis responds to NaCl stress via an increase in intracellular cAMP levels. We further showed that this increase in cAMP is dependent on the cAMP receptor protein and in part on the threonine/serine kinase PnkD, which has previously been associated with the NaCl stress response in mycobacteria.

Some chemotactic mutants can be progress through development in chimeric populations.

Cell migration in response to morphogen gradients affects morphogenesis. Chemotaxis towards adenosine 3', 5'-monophosphate (cAMP) is essential for the early stage of morphogenesis in the slime mold Dictyostelium discoideum. Here, we show that D. discoideum completes morphogenesis without cAMP-chemotaxis-dependent cell migration. The extracellular cAMP gradient is believed to cause cells to form a slug-shaped multicellular structure and fruiting body. The cAMP receptor, cAR1, was not expressed at the cell surface during these stages, correlating with reduced chemotactic activity. Gß-null cells expressing temperature sensitive Gß are unable to generate extracellular cAMP (Jin et al., 1998) and thus unable to aggregate and exhibit proper morphogenesis under restrictive temperature. However, when mixed with wild type cells ts-Gß expressing gß-null cells normally aggregated and exhibited normal morphogenesis under restrictive temperature. Furthermore, cells migrated after aggregation in a mixture containing wild-type cells. KI-5 cells, which do not show aggregation or morphogenesis, spontaneously migrated to a transplanted wild-type tip and underwent normal morphogenesis and cell differentiation; this was not observed in cells lacking tgrB1and tgrC1 cells adhesion molecules. Thus, cAMP gradient-dependent cell migration may not be required for multicellular pattern formation in late Dictyostelium development.

Sycrp2 Is Essential for Twitching Motility in the Cyanobacterium Synechocystis sp. Strain PCC 6803.

Two cAMP receptor proteins (CRPs), Sycrp1 (encoded by sll1371) and Sycrp2 (encoded by sll1924), exist in the cyanobacterium Synechocystis sp. strain PCC 6803. Previous studies have demonstrated that Sycrp1 has binding affinity for cAMP and is involved in motility by regulating the formation of pili. However, the function of Sycrp2 remains unknown. Here, we report that sycrp2 disruption results in the loss of motility of Synechocystis sp. PCC 6803, and that the phenotype can be recovered by reintroducing the sycrp2 gene into the genome of sycrp2-disrupted mutants. Electron microscopy showed that the sycrp2-disrupted mutant lost the pilus apparatus on the cell surface, resulting in a lack of cell motility. Furthermore, the transcript level of the pilA9-pilA11 operon (essential for cell motility and regulated by the cAMP receptor protein Sycrp1) was markedly decreased in sycrp2-disrupted mutants compared with the wild-type strain. Moreover, yeast two-hybrid analysis and a pulldown assay demonstrated that Sycrp2 interacted with Sycrp1 to form a heterodimer and that Sycrp1 and Sycrp2 interacted with themselves to form homodimers. Gel mobility shift assays revealed that Sycrp1 specifically binds to the upstream region of pilA9 Together, these findings indicate that in Synechocystis sp. PCC 6803, Sycrp2 regulates the formation of pili and cell motility by interacting with Sycrp1.IMPORTANCE cAMP receptor proteins (CRPs) are widely distributed in cyanobacteria and play important roles in regulating gene expression. Although many cyanobacterial species have two cAMP receptor-like proteins, the functional links between them are unknown. Here, we found that Sycrp2 in the cyanobacterium Synechocystis sp. strain PCC 6803 is essential for twitching motility and that it interacts with Sycrp1, a known cAMP receptor protein involved with twitching motility. Our findings indicate that the two cAMP receptor-like proteins in cyanobacteria do not have functional redundancy but rather work together.

Intracellular cAMP Sensor EPAC: Physiology, Pathophysiology, and Therapeutics Development.

This review focuses on one family of the known cAMP receptors, the exchange proteins directly activated by cAMP (EPACs), also known as the cAMP-regulated guanine nucleotide exchange factors (cAMP-GEFs). Although EPAC proteins are fairly new additions to the growing list of cAMP effectors, and relatively "young" in the cAMP discovery timeline, the significance of an EPAC presence in different cell systems is extraordinary. The study of EPACs has considerably expanded the diversity and adaptive nature of cAMP signaling associated with numerous physiological and pathophysiological responses. This review comprehensively covers EPAC protein functions at the molecular, cellular, physiological, and pathophysiological levels; and in turn, the applications of employing EPAC-based biosensors as detection tools for dissecting cAMP signaling and the implications for targeting EPAC proteins for therapeutic development are also discussed.

Functional dynamics in cyclic nucleotide signaling and amyloid inhibition.

It is now established that understanding the molecular basis of biological function requires atomic resolution maps of both structure and dynamics. Here, we review several illustrative examples of functional dynamics selected from our work on cyclic nucleotide signaling and amyloid inhibition. Although fundamentally diverse, a central aspect common to both fields is that function can only be rationalized by considering dynamic equilibria between distinct states of the accessible free energy landscape. The dynamic exchange between ground and excited states of signaling proteins is essential to explain auto-inhibition and allosteric activation. The dynamic exchange between non-toxic monomeric species and toxic oligomers of amyloidogenic proteins provides a foundation to understand amyloid inhibition. NMR ideally probes both types of dynamic exchange at atomic resolution. Specifically, we will show how NMR was utilized to reveal the dynamical basis of cyclic nucleotide affinity, selectivity, agonism and antagonism in multiple eukaryotic cAMP and cGMP receptors. We will also illustrate how NMR revealed the mechanism of action of plasma proteins that act as extracellular chaperones and inhibit the self-association of the prototypical amyloidogenic Aß peptide. The examples outlined in this review illustrate the widespread implications of functional dynamics and the power of NMR as an indispensable tool in molecular pharmacology and pathology.

Unusually Situated Binding Sites for Bacterial Transcription Factors Can Have Hidden Functionality.

A commonly accepted paradigm of molecular biology is that transcription factors control gene expression by binding sites at the 5' end of a gene. However, there is growing evidence that transcription factor targets can occur within genes or between convergent genes. In this work, we have investigated one such target for the cyclic AMP receptor protein (CRP) of enterotoxigenic Escherichia coli. We show that CRP binds between two convergent genes. When bound, CRP regulates transcription of a small open reading frame, which we term aatS, embedded within one of the adjacent genes. Our work demonstrates that non-canonical sites of transcription factor binding can have hidden functionality.

Rice TUTOU1 Encodes a Suppressor of cAMP Receptor-Like Protein That Is Important for Actin Organization and Panicle Development.

Panicle development, a key event in rice (Oryza sativa) reproduction and a critical determinant of grain yield, forms a branched structure containing multiple spikelets. Genetic and environmental factors can perturb panicle development, causing panicles to degenerate and producing characteristic whitish, small spikelets with severely reduced fertility and yield; however, little is known about the molecular basis of the formation of degenerating panicles in rice. Here, we report the identification and characterization of the rice panicle degenerative mutant tutou1 (tut1), which shows severe defects in panicle development. The tut1 also shows a pleiotropic phenotype, characterized by short roots, reduced plant height, and abnormal development of anthers and pollen grains. Molecular genetic studies revealed that TUT1 encodes a suppressor of cAMP receptor/Wiskott-Aldrich syndrome protein family verprolin-homologous (SCAR/WAVE)-like protein. We found that TUT1 contains conserved functional domains found in eukaryotic SCAR/WAVE proteins, and was able to activate Actin-related protein2/3 to promote actin nucleation and polymerization in vitro. Consistently, tut1 mutants show defects in the arrangement of actin filaments in trichome. These results indicate that TUT1 is a functional SCAR/WAVE protein and plays an important role in panicle development.

Parallel Allostery by cAMP and PDE Coordinates Activation and Termination Phases in cAMP Signaling.

The second messenger molecule cAMP regulates the activation phase of the cAMP signaling pathway through high-affinity interactions with the cytosolic cAMP receptor, the protein kinase A regulatory subunit (PKAR). Phosphodiesterases (PDEs) are enzymes responsible for catalyzing hydrolysis of cAMP to 5' AMP. It was recently shown that PDEs interact with PKAR to initiate the termination phase of the cAMP signaling pathway. While the steps in the activation phase are well understood, steps in the termination pathway are unknown. Specifically, the binding and allosteric networks that regulate the dynamic interplay between PKAR, PDE, and cAMP are unclear. In this study, PKAR and PDE from Dictyostelium discoideum (RD and RegA, respectively) were used as a model system to monitor complex formation in the presence and absence of cAMP. Amide hydrogen/deuterium exchange mass spectrometry was used to monitor slow conformational transitions in RD, using disordered regions as conformational probes. Our results reveal that RD regulates its interactions with cAMP and RegA at distinct loci by undergoing slow conformational transitions between two metastable states. In the presence of cAMP, RD and RegA form a stable ternary complex, while in the absence of cAMP they maintain transient interactions. RegA and cAMP each bind at orthogonal sites on RD with resultant contrasting effects on its dynamics through parallel allosteric relays at multiple important loci. RD thus serves as an integrative node in cAMP termination by coordinating multiple allosteric relays and governing the output signal response.

The thyroxine inactivating gene, type III deiodinase, suppresses multiple signaling centers in Dictyostelium discoideum.

Thyroxine deiodinases, the enzymes that regulate thyroxine metabolism, are essential for vertebrate growth and development. In the genome of Dictyostelium discoideum, a single intronless gene (dio3) encoding type III thyroxine 5' deiodinase is present. The amino acid sequence of D. discoideum Dio3 shares 37% identity with human T4 deiodinase and is a member of the thioredoxin reductase superfamily. dio3 is expressed throughout growth and development and by generating a knockout of dio3, we have examined the role of thyroxine 5' deiodinase in D. discoideum. dio3(-) had multiple defects that affected growth, timing of development, aggregate size, cell streaming, and cell-type differentiation. A prominent phenotype of dio3(-) was the breaking of late aggregates into small signaling centers, each forming a fruiting body of its own. cAMP levels, its relay, photo- and chemo-taxis were also defective in dio3(-). Quantitative RT-PCR analyses suggested that expression levels of genes encoding adenylyl cyclase A (acaA), cAMP-receptor A (carA) and cAMP-phosphodiesterases were reduced. There was a significant reduction in the expression of CadA and CsaA, which are involved in cell-cell adhesion. The dio3(-) slugs had prestalk identity, with pronounced prestalk marker ecmA expression. Thus, Dio3 seems to have roles in mediating cAMP synthesis/relay, cell-cell adhesion and slug patterning. The phenotype of dio3(-) suggests that Dio3 may prevent the formation of multiple signaling centers during D. discoideum development. This is the first report of a gene involved in thyroxine metabolism that is also involved in growth and development in a lower eukaryote.

Signaling in chemotactic amoebae remains spatially confined to stimulated membrane regions.

Recent work has demonstrated that the receptor-mediated signaling system in chemotactic amoeboid cells shows typical properties of an excitable system. Here, we delivered spatially confined stimuli of the chemoattractant cAMP to the membrane of differentiated Dictyostelium discoideum cells to investigate whether localized receptor stimuli can induce the spreading of excitable waves in the G-protein-dependent signal transduction system. By imaging the spatiotemporal dynamics of fluorescent markers for phosphatidylinositol (3,4,5)-trisphosphate (PIP3), PTEN and filamentous actin, we observed that the activity of the signaling pathway remained spatially confined to the stimulated membrane region. Neighboring parts of the membrane were not excited and no receptor-initiated spatial spreading of excitation waves was observed. To generate localized cAMP stimuli, either particles that carried covalently bound cAMP molecules on their surface were brought into contact with the cell or a patch of the cell membrane was aspirated into a glass micropipette to shield this patch against freely diffusing cAMP molecules in the surrounding medium. Additionally, the binding site of the cAMP receptor was probed with different surface-immobilized cAMP molecules, confirming results from earlier ligand-binding studies.

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.

Cyclic AMP receptor protein regulates cspD, a bacterial toxin gene, in Escherichia coli.

cspD, a member of cspA family of cold shock genes in Escherichia coli, is not induced during cold shock. Its expression is induced during stationary phase. CspD inhibits DNA replication, and a high level of the protein is toxic to cells. Recently, CspD was proposed to be associated with persister cell formation in E. coli. Here, we show that cyclic AMP receptor protein (CRP) upregulates cspD transcription. Sequence analysis of the cspD upstream region revealed two tandem CRP target sites, CRP site-I (the proximal site centered at -83.5 with respect to the transcription start) and CRP site-II (the distal site centered at -112.5). The results from electrophoretic mobility shift assays showed that CRP indeed binds to these two target sites in PcspD. The promoter-proximal CRP target site was found to play a major role in PcspD activation by CRP, as studied by transcriptional fusions carrying mutations in the target sites. The results from in vitro transcription assays demonstrated that CRP activates PcspD transcription in the absence of additional factors other than RNA polymerase. The requirement for activating region 1 of CRP in PcspD activation, along with the involvement of the 287, 265, and 261 determinants of the α-CTD, suggest that CRP activates by a class I-type mechanism. However, only moderate activation in vitro was observed compared to high activation in vivo, suggesting there might be additional activators of PcspD. Overall, our findings show that CRP, a global metabolic regulator in E. coli, activates a gene potentially related to persistence.

Alkaline pH Is a signal for optimal production and secretion of the heat labile toxin, LT in enterotoxigenic Escherichia coli (ETEC).

Enterotoxigenic Escherichia coli (ETEC) cause secretory diarrhea in children and travelers to endemic areas. ETEC spreads through the fecal-oral route. After ingestion, ETEC passes through the stomach and duodenum before it colonizes the lower part of the small intestine, exposing bacteria to a wide range of pH and environmental conditions. This study aimed to determine the impact of external pH and activity of the Cyclic AMP receptor protein (CRP) on the regulation of production and secretion of heat labile (LT) enterotoxin. ETEC strain E2863wt and its isogenic mutant E2863ΔCRP were grown in LBK media buffered to pH 5, 7 and 9. GM1 ELISA, cDNA and cAMP analyses were carried out on bacterial pellet and supernatant samples derived from 3 and 5 hours growth and from overnight cultures. We confirm that CRP is a repressor of LT transcription and production as has been shown before but we show for the first time that CRP is a positive regulator of LT secretion both in vitro and in vivo. LT secretion increased at neutral to alkaline pH compared to acidic pH 5 where secretion was completely inhibited. At pH 9 secretion of LT was optimal resulting in 600 percent increase of secreted LT compared to unbuffered LBK media. This effect was not due to membrane leakage since the bacteria were viable at pH 9. The results indicate that the transition to the alkaline duodenum and/or exposure to high pH close to the epithelium as well as activation of the global transcription factor CRP are signals that induce secretion of the LT toxin in ETEC.

Cyclic AMP receptor protein regulates pheromone-mediated bioluminescence at multiple levels in Vibrio fischeri ES114.

Bioluminescence in Vibrio fischeri ES114 is activated by autoinducer pheromones, and this regulation serves as a model for bacterial cell-cell signaling. As in other bacteria, pheromone concentration increases with cell density; however, pheromone synthesis and perception are also modulated in response to environmental stimuli. Previous studies suggested that expression of the pheromone-dependent bioluminescence activator LuxR is regulated in response to glucose by cyclic AMP (cAMP) receptor protein (CRP) (P. V. Dunlap and E. P. Greenberg, J. Bacteriol. 164:45-50, 1985; P. V. Dunlap and E. P. Greenberg, J. Bacteriol. 170:4040-4046, 1988; P. V. Dunlap, J. Bacteriol. 171:1199-1202, 1989; and W. F. Friedrich and E. P. Greenberg, Arch. Microbiol. 134:87-91, 1983). Consistent with this model, we found that bioluminescence in V. fischeri ES114 is modulated by glucose and stimulated by cAMP. In addition, a Δcrp mutant was ∼100-fold dimmer than ES114 and did not increase luminescence in response to added cAMP, even though cells lacking crp were still metabolically capable of producing luminescence. We further discovered that CRP regulates not only luxR but also the alternative pheromone synthase gene ainS. We found that His-tagged V. fischeri CRP could bind sequences upstream of both luxR and ainS, supporting bioinformatic predictions of direct regulation at both promoters. Luminescence increased in response to cAMP if either the ainS or luxR system was under native regulation, suggesting cAMP-CRP significantly increases luminescence through both systems. Finally, using transcriptional reporters in transgenic Escherichia coli, we elucidated two additional regulatory connections. First, LuxR-independent basal transcription of the luxI promoter was enhanced by CRP. Second, the effect of CRP on the ainS promoter depended on whether the V. fischeri regulatory gene litR was also introduced. These results suggest an integral role for CRP in pheromone signaling that goes beyond sensing cell density.

How do amoebae swim and crawl?

The surface behaviour of swimming amoebae was followed in cells bearing a cAR1-paGFP (cyclic AMP receptor fused to a photoactivatable-GFP) construct. Sensitized amoebae were placed in a buoyant medium where they could swim toward a chemoattractant cAMP source. paGFP, activated at the cell's front, remained fairly stationary in the cell's frame as the cell advanced; the label was not swept rearwards. Similar experiments with chemotaxing cells attached to a substratum gave the same result. Furthermore, if the region around a lateral projection near a crawling cell's front is marked, the projection and the labelled cAR1 behave differently. The label spreads by diffusion but otherwise remains stationary in the cell's frame; the lateral projection moves rearwards on the cell (remaining stationary with respect to the substrate), so that it ends up outside the labelled region. Furthermore, as cAR1-GFP cells move, they occasionally do so in a remarkably straight line; this suggests they do not need to snake to move on a substratum. Previously, we suggested that the surface membrane of a moving amoeba flows from front to rear as part of a polarised membrane trafficking cycle. This could explain how swimming amoebae are able to exert a force against the medium. Our present results indicate that, in amoebae, the suggested surface flow does not exist: this implies that they swim by shape changes.

Phosphorylation of chemoattractant receptors regulates chemotaxis, actin reorganization and signal relay.

Migratory cells, including mammalian leukocytes and Dictyostelium, use G-protein-coupled receptor (GPCR) signaling to regulate MAPK/ERK, PI3K, TORC2/AKT, adenylyl cyclase and actin polymerization, which collectively direct chemotaxis. Upon ligand binding, mammalian GPCRs are phosphorylated at cytoplasmic residues, uncoupling G-protein pathways, but activating other pathways. However, connections between GPCR phosphorylation and chemotaxis are unclear. In developing Dictyostelium, secreted cAMP serves as a chemoattractant, with extracellular cAMP propagated as oscillating waves to ensure directional migratory signals. cAMP oscillations derive from transient excitatory responses of adenylyl cyclase, which then rapidly adapts. We have studied chemotactic signaling in Dictyostelium that express non-phosphorylatable cAMP receptors and show through chemotaxis modeling, single-cell FRET imaging, pure and chimeric population wavelet quantification, biochemical analyses and TIRF microscopy, that receptor phosphorylation is required to regulate adenylyl cyclase adaptation, long-range oscillatory cAMP wave production and cytoskeletal actin response. Phosphorylation defects thus promote hyperactive actin polymerization at the cell periphery, misdirected pseudopodia and the loss of directional chemotaxis. Our data indicate that chemoattractant receptor phosphorylation is required to co-regulate essential pathways for migratory cell polarization and chemotaxis. Our results significantly extend the understanding of the function of GPCR phosphorylation, providing strong evidence that this evolutionarily conserved mechanism is required in a signal attenuation pathway that is necessary to maintain persistent directional movement of Dictyostelium, neutrophils and other migratory cells.

Bacterial cyclic AMP-phosphodiesterase activity coordinates biofilm formation.

Biofilm-related infections are a major contributor to human disease, and the capacity for surface attachment and biofilm formation are key attributes for the pathogenesis of microbes. Serratia marcescens type I fimbriae-dependent biofilms are coordinated by the adenylate cyclase, CyaA, and the cyclic 3',5'-adenosine monophosphate (cAMP)-cAMP receptor protein (CRP) complex. This study uses S. marcescens as a model system to test the role of cAMP-phosphodiesterase activity in controlling biofilm formation. Herein we describe the characterization of a putative S. marcescens cAMP-phosphodiesterase gene (SMA3506), designated as cpdS, and demonstrated to be a functional cAMP-phosphodiesterase both in vitro and in vivo. Deletion of cpdS resulted in defective biofilm formation and reduced type I fimbriae production, whereas multicopy expression of cpdS conferred a type I fimbriae-dependent hyper-biofilm. Together, these results support a model in which bacterial cAMP-phosphodiesterase activity modulates biofilm formation.