Hidekazu Kuwayama

Periodic Signaling Controlled by an Oscillatory Circuit That Includes Protein Kinases ERK2 and PKA

Self-regulating systems often use robust oscillatory circuits. One such system controls the chemotactic signaling mechanism of Dictyostelium, where pulses of adenosine 3′,5′-monophosphate (cAMP) are generated with a periodicity of 7 minutes. We have observed spontaneous oscillations in activation of the mitogen-activated protein (MAP) kinase ERK2 that occur in phase with peaks of cAMP, and we show that ERK2 modulates cAMP levels through the phosphodiesterase RegA. Computer modeling and simulations of the underlying circuit faithfully account for the ability of the cells to spontaneously generate periodic pulses during specific stages of development. Similar oscillatory processes may occur in cells of many different species.

Protection against osmotic stress by cGMP-mediated myosin phosphorylation

Conventional myosin functions universally as a generator of motive force in eukaryotic cells. Analysis of mutants of the microorganism Dictyostelium discoideum revealed that myosin also provides resistance against high external osmolarities. An osmo-induced increase of intracellular guanosine 3′,5′-monophosphate was shown to mediate phosphorylation of three threonine residues on the myosin tail, which caused a relocalization of myosin required to resist osmotic stress. This redistribution of myosin allowed cells to adopt a spherical shape and may provide physical strength to withstand extensive cell shrinkage in high osmolarities.

Evolutionary linkage between eukaryotic cytokinesis and chloroplast division by dynamin proteins

Chloroplasts have evolved from a cyanobacterial endosymbiont and been retained for more than 1 billion years by coordinated chloroplast division in multiplying eukaryotic cells. Chloroplast division is performed by ring structures at the division site, encompassing both the inside and the outside of the two envelopes. A part of the division machinery is derived from the cyanobacterial cytokinetic activity based on the FtsZ protein. In contrast, other parts of the division machinery involve proteins specific to eukaryotes, including a member of the dynamin family. Each member of the dynamin family is involved in the division or fusion of a distinct eukaryotic membrane system. To gain insight into the kind of ancestral dynamin protein and eukaryotic membrane activity that evolved to regulate chloroplast division, we investigated the functions of the dynamin proteins that are most closely related to chloroplast division proteins. These proteins in the amoeba Dictyostelium discoideum and Arabidopsis thaliana localize at the sites of cell division, where they are involved in cytokinesis. Our results suggest that the dynamin for chloroplast division is derived from that involved in eukaryotic cytokinesis. Therefore, the chloroplast division machinery is a mixture of bacterial and eukaryotic cytokinesis components, with the latter a key factor in the synchronization of endosymbiotic cell division with host cell division, thus helping to establish the permanent endosymbiotic relationship.

Switching direction in electric-signal-induced cell migration by cyclic guanosine monophosphate and phosphatidylinositol signaling.

Switching between attractive and repulsive migration in cell movement in response to extracellular guidance cues has been found in various cell types and is an important cellular function for translocation during cellular and developmental processes. Here we show that the preferential direction of migration during electrotaxis in Dictyostelium cells can be reversed by genetically modulating both guanylyl cyclases (GCases) and the cyclic guanosine monophosphate (cGMP)-binding protein C (GbpC) in combination with the inhibition of phosphatidylinositol-3-OH kinases (PI3Ks). The PI3K-dependent pathway is involved in cathode-directed migration under a direct-current electric field. The catalytic domains of soluble GCase (sGC) and GbpC also mediate cathode-directed signaling via cGMP, whereas the N-terminal domain of sGC mediates anode-directed signaling in conjunction with both the inhibition of PI3Ks and cGMP production. These observations provide an identification of the genes required for directional switching in electrotaxis and suggest that a parallel processing of electric signals, in which multiple-signaling pathways act to bias cell movement toward the cathode or anode, is used to determine the direction of migration.

Single-molecule analysis of chemoattractant-stimulated membrane recruitment of a PH-domain-containing protein

Molecular mechanisms of chemotactic response are highly conserved among many eukaryotic cells including human leukocytes and Dictyostelium discoideum cells. The cells can sense the differences in chemoattractant concentration across the cell body and respond by extending pseudopods from the cell side facing to a higher concentration. Pseudopod formation is regulated by binding of pleckstrin homology (PH)-domain-containing proteins to phosphatidylinositol 3,4,5-trisphosphates [PtdIns(3,4,5)P3] localized at the leading edge of chemotaxing cells. However, molecular mechanisms underlying dynamic features of a pseudopod have not been fully explained by the known properties of PH-domain-containing proteins. To investigate the mechanisms, we visualized single molecules of green fluorescent protein tagged to Crac (Crac-GFP), a PH-domain-containing protein in D. discoideum cells. Whereas populations of Crac molecules exhibited a stable steady-state localization at pseudopods, individual molecules bound transiently to PtdIns(3,4,5)P3 for ∼120 milliseconds, indicating dynamic properties of the PH-domain-containing protein. Receptor stimulation did not alter the binding stability but regulated the number of bound PH-domain molecules by metabolism of PtdIns(3,4,5)P3. These results demonstrate that the steady-state localization of PH-domain-containing proteins at the leading edge of chemotaxing cells is dynamically maintained by rapid recycling of individual PH-domain-containing proteins. The short interaction between PH domains and PtdIns(3,4,5)P3 contributes to accurate and sensitive chemotactic movements through the dynamic redistributions. These dynamic properties might be a common feature of signaling components involved in chemotaxis.

GMF is an evolutionarily developed Adf/cofilin-super family protein involved in the Arp2/3 complex-mediated organization of the actin cytoskeleton.

Actin‐depolymerizing factor (ADF)/cofilin is widely expressed in eukaryotes and plays a central role in reorganizing the actin cytoskeleton by disassembling actin filaments. The ADF‐homologous domain (ADF‐H) is conserved in several other actin‐modulating proteins such as twinfilin, Abp1/drebrin, and coactosin. Although these proteins interact with actin via ADF‐H, their effects on actin are not identical to each other. Here, we report a novel ADF/cofilin‐super family protein, Gmf1 (Glia maturation factor‐like protein 1), from the fission yeast Schizosaccharomyces pombe. Gmf1 is a component of actin patches, which are located on the cell cortex and required for endocytosis, and may be involved in the control of the disassembly of actin patches since its overexpression diminishes them. We provide evidence that Gmf1 binds weakly if at all to actin, but it associates with actin‐related protein (Arp) 2/3 complex and suppresses its functions such as the promotion of actin polymerization and branching filaments. Importantly, Arp2/3 complex‐suppressing activity is conserved among GMF‐family proteins from other organisms. Given the functional plasticity of ADF‐H, GMF‐family proteins possibly have changed their target from conventional actin to Arps through molecular evolution.

Identification and characterization of DdPDE3, a cGMP-selective phosphodiesterase from Dictyostelium

In Dictyostelium cAMP and cGMP have important functions as first and second messengers in chemotaxis and development. Two cyclic-nucleotide phosphodiesterases (DdPDE 1 and 2) have been identified previously, an extracellular dual-specificity enzyme and an intracellular cAMP-specific enzyme (encoded by the psdA and regA genes respectively). Biochemical data suggest the presence of at least one cGMP-specific phosphodiesterase (PDE) that is activated by cGMP. Using bioinformatics we identified a partial sequence in the Dictyostelium expressed sequence tag database that shows a high degree of amino acid sequence identity with mammalian PDE catalytic domains (DdPDE3). The deduced amino acid sequence of a full-length DdPDE3 cDNA isolated in this study predicts a 60 kDa protein with a 300-residue C-terminal PDE catalytic domain, which is preceded by approx. 200 residues rich in asparagine and glutamine residues. Expression of the DdPDE3 catalytic domain in Escherichia coli shows that the enzyme has Michaelis-Menten kinetics and a higher affinity for cGMP (K(m)=0.22 microM) than for cAMP (K(m)=145 microM); cGMP does not stimulate enzyme activity. The enzyme requires bivalent cations for activity; Mn(2+) is preferred to Mg(2+), whereas Ca(2+) yields no activity. DdPDE3 is inhibited by 3-isobutyl-1-methylxanthine with an IC(50) of approx. 60 microM. Overexpression of the DdPDE3 catalytic domain in Dictyostelium confirms these kinetic properties without indications of its activation by cGMP. The properties of DdPDE3 resemble those of mammalian PDE9, which also shows the highest sequence similarity within the catalytic domains. DdPDE3 is the first cGMP-selective PDE identified in lower eukaryotes.

MFE1, a Member of the Peroxisomal Hydroxyacyl Coenzyme A Dehydrogenase Family, Affects Fatty Acid Metabolism Necessary for Morphogenesis in Dictyostelium spp.

ABSTRACT β-Oxidation of long-chain fatty acids and branched-chain fatty acids is carried out in mammalian peroxisomes by a multifunctional enzyme (MFE) or d-bifunctional protein, with separate domains for hydroxyacyl coenzyme A (CoA) dehydrogenase, enoyl-CoA hydratase, and steroid carrier protein SCP2. We have found that Dictyostelium has a gene, mfeA, encoding MFE1 with homology to the hydroxyacyl-CoA dehydrogenase and SCP2 domains. A separate gene, mfeB, encodes MFE2 with homology to the enoyl-CoA hydratase domain. When grown on a diet of bacteria, Dictyostelium cells in which mfeA is disrupted accumulate excess cyclopropane fatty acids and are unable to develop beyond early aggregation. Axenically grown mutant cells, however, developed into normal fruiting bodies composed of spores and stalk cells. Comparative analysis of whole-cell lipid compositions revealed that bacterially grown mutant cells accumulated cyclopropane fatty acids that remained throughout the developmental stages. Such a persistent accumulation was not detected in wild-type cells or axenically grown mutant cells. Bacterial phosphatidylethanolamine that contains abundant cyclopropane fatty acids inhibited the development of even axenically grown mutant cells, while dipalmitoyl phosphatidylethanolamine did not. These results suggest that MFE1 protects the cells from the increase of the harmful xenobiotic fatty acids incorporated from their diets and optimizes cellular lipid composition for proper development. Hence, we propose that this enzyme plays an irreplaceable role in the survival strategy of Dictyostelium cells to form spores for their efficient dispersal in nature.

Chemotactic and osmotic signals share a cGMP transduction pathway in Dictyostelium discoideum

In the ameboid eukaryote Dictyostelium discoideum, chemotactic stimulation by cAMP induces an increase of intracellular cGMP and subsequently the phosphorylation of myosin heavy chain II. Resistance to high osmotic stress also requires transient increases of intracellular cGMP and phosphorylation of myosin heavy chain II, although the kinetics is much slower than for chemotaxis. To examine if chemotaxis and osmotic stress share common signaling components we systematically analyzed the osmotic cGMP response and survival in chemotactic mutants with altered cGMP signaling. Null mutants with deletions of cell surface cAMP receptors or the associated GTP‐binding proteins Gα2 and Gβ show no cAMP‐induced cGMP response and chemotaxis; in contrast, osmotic stress induces the normal cGMP accumulation and survival. The same result was obtained with the non‐chemotactic mutant KI‐10, which lacks the activation of guanylyl cyclase by cAMP. This indicates that these components are required for chemotaxis but not osmotic cGMP signaling and survival. The potential guanylyl cyclase null mutant KI‐8 shows no chemotaxis, no osmotic cGMP increase and reduced survival in high osmolarity. Two types of cGMP‐binding protein mutants, KI‐4 and KI‐7, also show reduced tolerance during high osmotic stress. Taken together, these observations clarify that chemotactic and osmotic signals are detected by different mechanisms, but share a cGMP signaling pathway.

Differentiation-Inducing Factor-1 and -2 Function also as Modulators for Dictyostelium Chemotaxis

Background In the early stages of development of the cellular slime mold Dictyostelium discoideum, chemotaxis toward cAMP plays a pivotal role in organizing discrete cells into a multicellular structure. In this process, a series of signaling molecules, such as G-protein-coupled cell surface receptors for cAMP, phosphatidylinositol metabolites, and cyclic nucleotides, function as the signal transducers for controlling dynamics of cytoskeleton. Differentiation-inducing factor-1 and -2 (DIF-1 and DIF-2) were originally identified as the factors (chlorinated alkylphenones) that induce Dictyostelium stalk cell differentiation, but it remained unknown whether the DIFs had any other physiologic functions. Methodology/Principal Findings To further elucidate the functions of DIFs, in the present study we investigated their effects on chemotaxis under various conditions. Quite interestingly, in shallow cAMP gradients, DIF-1 suppressed chemotaxis whereas DIF-2 promoted it greatly. Analyses with various mutants revealed that DIF-1 may inhibit chemotaxis, at least in part, via GbpB (a phosphodiesterase) and a decrease in the intracellular cGMP concentration ([cGMP]i). DIF-2, by contrast, may enhance chemotaxis, at least in part, via RegA (another phosphodiesterase) and an increase in [cGMP]i. Using null mutants for DimA and DimB, the transcription factors that are required for DIF-dependent prestalk differentiation, we also showed that the mechanisms for the modulation of chemotaxis by DIFs differ from those for the induction of cell differentiation by DIFs, at least in part. Conclusions/Significance Our findings indicate that DIF-1 and DIF-2 function as negative and positive modulators for Dictyostelium chemotaxis, respectively. To our knowledge, this is the first report in any organism of physiologic modulators (small molecules) for chemotaxis having differentiation-inducing activity.