cGMP potentiates receptor-stimulated Ca2+ influx in Dictyostelium discoideum

AbstractBinding of extracellular cAMP to surface receptors induces at least two responses in Dictyostelium discoideum, the G-protein-dependent activation of guanylyl cyclase, and the opening of a plasma membrane Ca2+ channel. Some experiments suggest that intracellular cGMP opens the Ca2+ channel, while others demonstrate that the channel can open in the absence of functional G-proteins (and thus in the absence of cGMP formation). We have analysed 45Ca2+ uptake in three mutants with altered cGMP formation. Mutant stmF shows a prolonged cGMP response due to deletion of an intracellular phosphodiesterase. Uptake of receptor-stimulated 45Ca2+ is enhanced about two-fold in this mutant if compared to wild-type cells, suggesting that cGMP regulates the opening of the channel. Mutant KI-7 has very low levels of surface cAMP receptors, but nevertheless an enhanced receptor-stimulated cGMP response due to a defect in the turn-off of guanylyl cyclase. This mutant shows poor receptor-stimulated 45Ca2+ uptake, suggesting that cGMP alone is not sufficient to open the Ca2+ channel. Finally, mutant KI-8 has no cGMP due to the absence of nearly all guanylyl cyclase activity. The mutant shows significant but reduced 45Ca2+ uptake (19% of wild-type; 60% if corrected for the reduced level of surface cAMP receptors), suggesting that the channel can open in the absence of cGMP. Taken together, the results demonstrate that receptor-stimulated Ca2+ influx is not directly induced by cGMP formation; it can occur in the absence of cGMP, but is potentiated two- to four-fold by cGMP

微生物に由来する新規ガン抑制因子の作用機構の解析

科学研究費助成事業 研究成果報告書：挑戦的萌芽研究2016-2017課題番号 : 16K1513

Chemoattractant receptors activate, recruit and capture G proteins for wide range chemotaxis

The wide range sensing of extracellular signals is a common feature of various sensory cells. Eukaryotic chemotactic cells driven by GPCRs and their cognate G proteins are one example. This system endows the cells directional motility towards their destination over long distances. There are several mechanisms to achieve the long dynamic range, including negative regulation of the receptors upon ligand interaction and spatial regulation of G proteins, as we found recently. However, these mechanisms are insufficient to explain the 105-fold range of chemotaxis seen in Dictyostelium. Here, we reveal that the receptor-mediated activation, recruitment, and capturing of G proteins mediate chemotactic signaling at the lower, middle and higher concentration ranges, respectively. These multiple mechanisms of G protein dynamics can successfully cover distinct ranges of ligand concentrations, resulting in seamless and broad chemotaxis. Furthermore, single-molecule imaging analysis showed that the activated Gα subunit forms an unconventional complex with the agonist-bound receptor. This complex formation of GPCR-Gα increased the membrane-binding time of individual Gα molecules and therefore resulted in the local accumulation of Gα. Our findings provide an additional chemotactic dynamic range mechanism in which multiple G protein dynamics positively contribute to the production of gradient information

Evidence that differentiation-inducing factor-1 controls chemotaxis and cell differentiation, at least in part, via mitochondria in D.discoideum

Differentiation-inducing factor-1 [1-(3,5-dichloro-2,6-dihydroxy-4-methoxyphenyl)hexan-1-one (DIF-1)] is an important regulator of cell differentiation and chemotaxis in the development of the cellular slime mold Dictyostelium discoideum. However, the entire signaling pathways downstream of DIF-1 remain to be elucidated. To characterize DIF-1 and its potential receptor(s), we synthesized two fluorescent derivatives of DIF-1, boron-dipyrromethene (BODIPY)-conjugated DIF-1 (DIF-1-BODIPY) and nitrobenzoxadiazole (NBD)-conjugated DIF-1 (DIF-1-NBD), and investigated their biological activities and cellular localization. DIF-1-BODIPY (5 µM) and DIF-1 (2 nM) induced stalk cell differentiation in the DIF-deficient strain HM44 in the presence of cyclic adenosine monosphosphate (cAMP), whereas DIF-1-NBD (5 µM) hardly induced stalk cell differentiation under the same conditions. Microscopic analyses revealed that the biologically active derivative, DIF-1-BODIPY, was incorporated by stalk cells at late stages of differentiation and was localized to mitochondria. The mitochondrial uncouplers carbonyl cyanide m-chlorophenylhydrazone (CCCP), at 25–50 nM, and dinitrophenol (DNP), at 2.5–5 µM, induced partial stalk cell differentiation in HM44 in the presence of cAMP. DIF-1-BODIPY (1–2 µM) and DIF-1 (10 nM), as well as CCCP and DNP, suppressed chemotaxis in the wild-type strain Ax2 in shallow cAMP gradients. These results suggest that DIF-1-BODIPY and DIF-1 induce stalk cell differentiation and modulate chemotaxis, at least in part, by disturbing mitochondrial activity

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

BackgroundIn 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 FindingsTo 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/SignificanceOur 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

Comparative genome and transcriptome analyses of the social amoeba Acytostelium subglobosum that accomplishes multicellular development without germ-soma differentiation

Background Social amoebae are lower eukaryotes that inhabit the soil. They are characterized by the construction of a starvation-induced multicellular fruiting body with a spore ball and supportive stalk. In most species, the stalk is filled with motile stalk cells, as represented by the model organism Dictyostelium discoideum, whose developmental mechanisms have been well characterized. However, in the genus Acytostelium, the stalk is acellular and all aggregated cells become spores. Phylogenetic analyses have shown that it is not an ancestral genus but has lost the ability to undergo cell differentiation. Results We performed genome and transcriptome analyses of Acytostelium subglobosum and compared our findings to other available dictyostelid genome data. Although A. subglobosum adopts a qualitatively different developmental program from other dictyostelids, its gene repertoire was largely conserved. Yet, families of polyketide synthase and extracellular matrix proteins have not expanded and a serine protease and ABC transporter B family gene, tagA, and a few other developmental genes are missing in the A. subglobosum lineage. Temporal gene expression patterns are astonishingly dissimilar from those of D. discoideum, and only a limited fraction of the ortholog pairs shared the same expression patterns, so that some signaling cascades for development seem to be disabled in A. subglobosum. Conclusions The absence of the ability to undergo cell differentiation in Acytostelium is accompanied by a small change in coding potential and extensive alterations in gene expression patterns

Derivatives of Differentiation-Inducing Factor 1 Differentially Control Chemotaxis and Stalk Cell Differentiation in <i>Dictyostelium discoideum</i>

Differentiation-inducing factors 1 and 2 (DIF-1 and DIF-2) are small lipophilic signal molecules that induce stalk cell differentiation but differentially modulate chemotaxis toward cAMP in the cellular slime mold Dictyostelium discoideum; DIF-1 suppresses chemotactic cell movement in shallow cAMP gradients, whereas DIF-2 promotes it. The receptor(s) for DIF-1 and DIF-2 have not yet been identified. We examined the effects of nine derivatives of DIF-1 on chemotactic cell movement toward cAMP and compared their chemotaxis-modulating activity and stalk cell differentiation–inducing activity in wild-type and mutant strains. The DIF derivatives differentially affected chemotaxis and stalk cell differentiation; for example, TM-DIF-1 suppressed chemotaxis and showed poor stalk-inducing activity, DIF-1(3M) suppressed chemotaxis and showed strong stalk-inducing activity, and TH-DIF-1 promoted chemotaxis. These results suggest that DIF-1 and DIF-2 have at least three receptors: one for stalk cell induction and two for chemotaxis modulation. In addition, our results show that the DIF derivatives can be used to analyze the DIF-signaling pathways in D. discoideum