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

Darren D Browning, Tony The, Danton H. O'Day

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21 Citations (Scopus)

Abstract

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 AIF3, 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 Mr 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 to 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.

Original languageEnglish (US)
Pages (from-to)481-489
Number of pages9
JournalCellular Signalling
Volume7
Issue number5
DOIs
StatePublished - Jan 1 1995

Fingerprint

Dictyostelium
Chemotaxis
Folic Acid
Biological Assay
Protein-Tyrosine Kinases
Amoeba
Chlortetracycline
Video Microscopy
Phosphotyrosine
Aptitude
Genistein
Chemotactic Factors
Protein Kinase Inhibitors
Starvation
GTP-Binding Proteins
Edetic Acid
Tyrosine
Anti-Idiotypic Antibodies
Signal Transduction
Proteins

Keywords

  • Chemotaxis
  • G proteins
  • cAMP
  • calcium
  • folate
  • phosphorylation
  • protein tyrosine kinase
  • signal transduction

ASJC Scopus subject areas

  • Cell Biology

Cite this

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title = "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",
abstract = "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 AIF3, 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 Mr 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 to 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.",
keywords = "Chemotaxis, G proteins, cAMP, calcium, folate, phosphorylation, protein tyrosine kinase, signal transduction",
author = "Browning, {Darren D} and Tony The and O'Day, {Danton H.}",
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AU - O'Day, Danton H.

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N2 - 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 AIF3, 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 Mr 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 to 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.

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