Comparative analysis of chemotaxis in Dictyostelium using a radial bioassay method: protein tyrosine kinase activity is required for chemotaxis to folate but not to cAMP.
The role of signal transduction during chemotaxis of Dictyostelium discoideum cells to cAMP and folic acid was investigated using a radial bioassay technique. The effects of signalling agonists were assessed by measuring the diameters of visible rings formed by the outward migration of amoebae up radial gradients of chemoattractant. This rapid and simple bioassay method yields chemotactic rates equivalent to more complex assay systems. In support of previous studies, chemotaxis toward both cAMP and folic acid was inhibited in a dose-dependent manner by LaCl3, EDTA, chlorotetracycline and A1F3, supporting the importance of calcium ions and G protein-mediated signalling in both chemotactic events. The work was extended by examining the effects of the protein tyrosine kinase inhibitor genistein. This agent inhibited chemotaxis to folate in a dose-dependent manner but had no observable effect on chemotaxis toward cAMP. The notion that phosphorylation of proteins on tyrosine residues is critical for chemotaxis to folic acid was supported by Western blotting experiments with monoclonal anti-phosphotyrosine antibodies which detected two candidate proteins of M(r) 52,000 and 38,000 in the membranes of folate-responsive amoebae. These two bands disappeared with starvation which leads to the loss of responsiveness of folic acid and the acquisition of responsiveness to cAMP. Time-lapse videomicrography also revealed some unique differences in chemotactic response. Starved cells responded to cAMP as individuals but feeding cells chemoattracted to folic acid on a populational basis. The ability to compare two different types of chemotaxis using a simple, rapid and accurate bioassay system should enhance future studies of chemotaxis in wild-type and mutant strains of D. discoideum.
Browning
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Department of Zoology, Erindale College, University of Toronto, Mississauga, Ontario, Canada.
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D017607:Aluminum Compounds / Q000494:pharmacology; D000818:Animals; D001681:Biological Assay / Q000379:methods; D015153:Blotting, Western; D002614:Chelating Agents / Q000494:pharmacology; D002633:Chemotaxis / Q000187:drug effects / Q000502:physiology*; D002751:Chlortetracycline / Q000494:pharmacology; D000242:Cyclic AMP / Q000494:pharmacology*; D004023:Dictyostelium / Q000187:drug effects / Q000502:physiology*; D004305:Dose-Response Relationship, Drug; D004492:Edetic Acid / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005459:Fluorides / Q000494:pharmacology; D005492:Folic Acid / Q000494:pharmacology*; D019204:GTP-Binding Proteins / Q000378:metabolism; D019833:Genistein; D007529:Isoflavones / Q000494:pharmacology; D007811:Lanthanum / Q000494:pharmacology; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology
D017607:Aluminum Compounds; D002614:Chelating Agents; D004791:Enzyme Inhibitors; D007529:Isoflavones; C028521:lanthanum chloride; D007811:Lanthanum; D005492:Folic Acid; D004492:Edetic Acid; D019833:Genistein; D000242:Cyclic AMP; D011505:Protein-Tyrosine Kinases; D019204:GTP-Binding Proteins; D005459:Fluorides; D002751:Chlortetracycline; C032311:aluminum fluoride