Protein and phospholipid methylation during chemotaxis in Dictyostelium discoideum and its relationship to calcium movements (original) (raw)
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Proceedings of the National Academy of Sciences, 1980
In Dictyostelium discoideum, the chemoattractant cyclic AMP activates the enzyme guanylate cyclase, giving a brief up to 10-fold increase in the intracellular cyclic GMP content. The addition of physiological cyclic GMP concentrations to a homogenate of D. discoideum cells markedly increased the incorporation of the 3H-labeled methyl group from S-adenosyl-L[me hyl-3HImethionine into monoand dimethylated phosphatidylethanolamine and phosphatidylcholine.
Cyclic AMP-induced pH changes in Dictyostelium discoideum and their control by calcium
Biochimica et Biophysica Acta (BBA) - General Subjects, 1978
During differentiation of Dictyostelium discoideum the extracellular pH was measured in unbuffered cell suspensions. 1. Stimulation of the ceils by cyclic AMP pulses in the nanomolar range resulted in two successive increases of the extracellular proton concentration. 2. Cyclic AMP analogs were effective in the order of their chemotactic activity, indicating that the pH changes were mediated by cyclic AMP receptors present at the cell surface. 3. Cyclic AMP binding to the receptors was, however, not sufficient to elicit the pH changes; for glutaraldehyde-treated cells which exhibit normal binding did not respond. Moreover, the proton : cyclic AMP ratio was in the order of 3 • 10 ~'. 4. The second proton peak was not due to cyclic AMP secretion or subsequent hydrolysis and persisted in the presence of dithiothreitol, an inhibitor of cyclic AMP phosphodiesterases. 5. Removal of extracellular calcium increased the proton peaks, particularly the first one, up to 10-fold. A half maximal response was obtained with 3-10-1° M cyclic AMP. 6. Only at cyclic AMP doses higher than 0.1 laM did hydrolysis of the added cyclic AMP contribute significantly to the pH changes.
FEBS Letters, 1982
In Dictyostelium discoideum, extracellular CAMP induces chemotaxis and cell aggregation. Suspensions of CAMP-sensitive cells are shown to respond to a 10e6M CAMP-pulse with increased methylation of 4 proteins with app. Mr 110000, 46000, 28000 and 16000. The kf, 110000 and 28000 proteins show a triphasic response with maxima 15, 60 and 150-180s after stimulation. The responses of the Mr46000 and 16000 proteins are monophasic, maxima being reached 3 and 15 s after stimulation, respectively. Optimal responses of methylation are observed over 10-7-10-6M CAMP. The methylation reaction may be involved in the processing of the chemotactic signal. Protein carboxymethylation Chemotaxis Signal transduction 2.1. Materials L-[methyl-3H]Methionine (15 Ci/mmol) was obtained from Amersham International (Bucks). 2.2. Organism Dictyostelium discoideum NC-4 (H) was used for all experiments. Cells were grown on a solid 266
Ca2+ chemotaxis in Dictyostelium discoideum
Journal of Cell Science, 2010
Using a newly developed microfluidic chamber, we have demonstrated in vitro that Ca 2+ functions as a chemoattractant of aggregationcompetent Dictyostelium discoideum amoebae, that parallel spatial gradients of cAMP and Ca 2+ are more effective than either alone, and that cAMP functions as a stronger chemoattractant than Ca 2+ . Effective Ca 2+ gradients are extremely steep compared with effective cAMP gradients. This presents a paradox because there is no indication to date that steep Ca 2+ gradients are generated in aggregation territories. However, given that Ca 2+ chemotaxis is co-acquired with cAMP chemotaxis during development, we speculate on the role that Ca 2+ chemotaxis might have and the possibility that steep, transient Ca 2+ gradients are generated during natural aggregation in the interstitial regions between cells. (2006). Stem cell engraftment at the endosteal niche is specified by the calcium-sensing receptor. Nature 439, 599-603. Aguirre, A., Gonzalez, A., Planell, J. and Engel, E. (2010). Extracellular calcium modulates in vitro bone marrow-derived Flk-1+ CD34+ progenitor cell chemotaxis and differentiation through a calcium-sensing receptor. Biochem. Biophys. Res. Commun. 393, 156-161. Alcantara, F. and Monk, M. (1974). Signal propagation during aggregation in the slime mould Dictyostelium discoideum. J. Gen. Microbiol. 85, 321-334. Andrew, N. and Insall, R. (2007). Chemotaxis in shallow gradients is mediated independently of PtdIns 3-kinase by biased choices between random protrusions. Nat. Cell Biol. 9, 193-200. Bandyopadhyay, S., Jeong, K. H., Hansen, J. T., Vassilev, P. M., Brown, E. M. and Chattopadhyay, N. (2007). Calcium-sensing receptor stimulates secretion of an interferon-gamma-induced monokine (CXCL10) and monocyte chemoattractant protein-3 in immortalized GnRH neurons. J. Neurosci. Res. 85, 882-895. Bangerth, F. (1979). Calcium-related physiological disorders of plants. Annu. Rev. Phytopathol. 17, 97-122. Bohme, R., Bumann, J., Aeckerle, S. and Malchow, D. (1987). A high-affinity plasma membrane Ca 2+ -ATPase in Dictyostelium discoideum: its relation to cAMP-induced Ca 2+ fluxes. Biochim. Biophys. Acta 904, 125-130. Bonner, J. T. (1949). The demonstration of acrasin in the later stages of the development of the slime mold Dictyostelium discoideum. J. Exp. Zool. 110, 259-271. Boyden, S. (1962). The chemotactic effect of mixtures of antibody and antigen on polymorphonuclear leucocytes.
Cell Motility and the Cytoskeleton, 2009
Extracellular Ca ++ , a ubiquitous cation in the soluble environment of cells both free living and within the human body, regulates most aspects of amoeboid cell motility, including shape, uropod formation, pseudopod formation, velocity and turning in Dictyostelium discoideum. Hence it affects the efficiency of both basic motile behavior and chemotaxis. Extracellular Ca ++ is optimal at 10 mM. A gradient of the chemoattractant cAMP generated in the absence of added Ca ++ only affects turning, but in combination with extracellular Ca ++ , enhances the effects of extracellular Ca ++ . Potassium, at 40 mM, can substitute for Ca ++ . Mg ++ , Mn ++ , Zn ++ and Na + cannot. Extracellular Ca ++ , or K + , also induce the cortical localization of myosin II in a polar fashion. The effects of Ca ++ , K + or a cAMP gradient do not appear to be similarly mediated by an increase in the general pool of free cytosolic Ca ++ . These results suggest a model, in which each agent functioning through different signaling systems, converge to affect the cortical localization of myosin II, which in turn effects the behavioral changes leading to efficient cell motility and chemotaxis.
Nonpolar lipid and phospholipid methylation during development of Dictyostelium discoideum
Biochimica et Biophysica Acta (BBA) - Lipids and Lipid Metabolism, 1985
The life-cycle of the cellular slime mold Dictyostelium discoideum is characterized by development from a vegetative stage of solitary amoebae via cell aggregation and a 'slug' stage to a fruiting body. It has been suggested that phospholipid methylation is involved in CAMP-induced CAMP-synthesis, which is the mechanism underlying cell aggregation. Therefore, we have examined changes in lipid methylation during development. Individual amoebae incorporated the methyl group of [ methyl-3H]methionine both in phospholipid and certain nonpolar lipid components. Nonpolar methyl acceptors, characterized by thin-layer chromatography, were probably A**-stigmasten-g-01, A**-stigmasten-w-01 fatty acid ester and ubiquinone. The activities of nonpolar and phospholipid methyltransferases, as assayed in a homogenate with S-adenosyl-[methyL3H]methionine as a methyl donor, showed a strong decline from the vegetative stage to the slug stage, which was not followed by a subsequent rise during the final stage. When the activities of both enzymes were measured in intact cells during development, the decline of the amount of enzyme was masked by a 60-fold increase of the specific activity of intracellular S-adenosyl(methyL3H]methionine during cell differentiation. We conclude that the activities of phospholipid iV-methyltransferase and adenylate cyclase (or the number of cyclic AMP receptors on the cell surface) are not regulated in parallel during development.
Acidocalcisomes Are Functionally Linked to the Contractile Vacuole of Dictyostelium discoideum
Journal of Biological Chemistry, 2002
The mass-dense granules of Dictyostelium discoideum were shown to contain large amounts of phosphorus, magnesium, and calcium, as determined by x-ray microanalysis, either in situ or when purified using iodixanol gradient centrifugation. The high phosphorus content was due to the presence of pyrophosphate and polyphosphate, which were also present in the contractile vacuoles. Both organelles also possessed a vacuolar H ؉ -ATPase, an H ؉ -pyrophosphatase, and a Ca 2؉ -ATPase, as determined by biochemical methods or by immunofluorescence microscopy. The H ؉ -pyrophosphatase activity of isolated mass-dense granules was stimulated by potassium ions and inhibited by the pyrophosphate analogs aminomethylenediphosphonate and imidodiphosphate and by KF and N-ethylmaleimide in a dosedependent manner. The mass-dense granules and the contractile vacuole appeared to contact each other when the cells were submitted to hyposmotic stress. Acetazolamide inhibited the carbonic anhydrase activity of the contractile vacuoles and prolonged their contraction cycle in a dose-dependent manner. Similar effects were observed with the anion exchanger inhibitor 4,4-diisothiocyanatodihydrostilbene-2, 2-disulfonic acid and the vacuolar H ؉ -ATPase inhibitor bafilomycin A 1 . Together, these results suggest that the mass-dense granules of D. discoideum are homologous to the acidocalcisomes described in protozoan parasites and are linked to the function of the contractile vacuole.
Involvement of intracellular calcium in protein secretion in Dictyostelium discoideum
Journal of Cell Science, 1992
We reported previously that Ca2+ depletion of Dictyostelium discoideum cells severely inhibits extracellular cyclic nucleotide phosphodiesterase (PD) synthesis at a post-transcriptional step. In this study, further experiments were performed to learn more about the nature of this phenomenon. Examination of the polysomal distribution of PD transcripts in control cells and in cells depleted of Ca2+ by incubation with EGTA and A23187 (EA) suggested that inhibition of PD production does not involve translational control. Kinetic analysis of this inhibitory process revealed that soluble, intracellular PD activity, synthesized from either the 2.4 or 1.9 kb PD mRNA, decreased very rapidly upon addition of EA. Furthermore, this decrease in activity was accompanied by the preferential loss of PD-related polypeptides, indicating a proteolytic event. EA-induced PD degradation required cellular energy and concomitant protein synthesis but was unaffected by most of the lysosomotropic agents tested. Therefore, PD proteolysis might not occur in the lysosome. In cell fractionation experiments, the EA-sensitive, intracellular PD activity comigrated with a rough ER marker in Percoll/KCl gradients. In addition to its effect on the PD, EA were also observed to inhibit production and rapidly lower the intracellular levels of another secreted glycoprotein, the PD inhibitor. Together, these results suggest that depletion of some intracellular Ca2+ store(s) in Dictyostelium, possibly the ER, disrupts the normal function of the secretory pathway, resulting in selective degradation of certain proteins.
Journal of cell science, 1991
Dictyostelium cells use extracellular cyclic AMP both as a chemoattractant and as a morphogen inducing cell-type-specific gene expression. Cyclic AMP binds to surface receptors, activates one or more G-proteins, and stimulates adenylate cyclase, guanylate cyclase and phosphoinositidase C. Mutant fgdC showed aberrant chemotaxis, and was devoid of cyclic AMP-induced gene expression and differentiation. Both the receptor- and G-protein-mediated stimulation of adenylate cyclase and guanylate cyclase were unaltered in mutant fgdC as compared to wild-type cells. In wild-type cells phosphoinositidase C was activated about twofold by the cyclic AMP receptor. In mutant fgdC cells, however, the enzyme was inhibited by about 60%. These results suggest that phosphoinositidase C is regulated by a receptor-operated activation/inhibition switch that is defective in mutant fgdC. We conclude that activation of phosphoinositidase C is essential for Dictyostelium development.