title: Signaling in chemotactic amoebae remains spatially confined to stimulated membrane regions.
abstract: Recent work has demonstrated that the receptor-mediated signaling system in chemotactic amoeboid cells shows typical properties of an excitable system. Here, we delivered spatially confined stimuli of the chemoattractant cAMP to the membrane of differentiated Dictyostelium discoideum cells to investigate whether localized receptor stimuli can induce the spreading of excitable waves in the G-protein-dependent signal transduction system. By imaging the spatiotemporal dynamics of fluorescent markers for phosphatidylinositol (3,4,5)-trisphosphate (PIP₃), PTEN and filamentous actin, we observed that the activity of the signaling pathway remained spatially confined to the stimulated membrane region. Neighboring parts of the membrane were not excited and no receptor-initiated spatial spreading of excitation waves was observed. To generate localized cAMP stimuli, either particles that carried covalently bound cAMP molecules on their surface were brought into contact with the cell or a patch of the cell membrane was aspirated into a glass micropipette to shield this patch against freely diffusing cAMP molecules in the surrounding medium. Additionally, the binding site of the cAMP receptor was probed with different surface-immobilized cAMP molecules, confirming results from earlier ligand-binding studies.
authors.affiliation: Institute of Physics and Astronomy, University of Potsdam, Karl-Liebknecht-Strasse 24/25, 14476 Potsdam, Germany.
authors.forename: Matthias
authors.initials: M
authors.lastname: Gerhardt
authors.affiliation: Institute of Physics and Astronomy, University of Potsdam, Karl-Liebknecht-Strasse 24/25, 14476 Potsdam, Germany.
authors.forename: Michael
authors.initials: M
authors.lastname: Walz
authors.affiliation: Institute of Physics and Astronomy, University of Potsdam, Karl-Liebknecht-Strasse 24/25, 14476 Potsdam, Germany beta@uni-potsdam.de.
authors.forename: Carsten
authors.initials: C
authors.lastname: Beta
mesh_terms: D008841:Actin Cytoskeleton / Q000378:metabolism; D002460:Cell Line; D002462:Cell Membrane / Q000187:drug effects* / Q000378:metabolism; D002633:Chemotaxis / Q000187:drug effects*; D000242:Cyclic AMP / Q000494:pharmacology*; D004023:Dictyostelium / Q000187:drug effects* / Q000378:metabolism; D008024:Ligands; D008564:Membrane Potentials; D008856:Microscopy, Fluorescence; D051059:PTEN Phosphohydrolase / Q000378:metabolism; D018408:Patch-Clamp Techniques; D058539:Phosphatidylinositol 3-Kinase / Q000378:metabolism; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism; D011953:Receptors, Cyclic AMP / Q000819:agonists* / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects*; D013997:Time Factors; D014162:Transfection
chemical_list: D008024:Ligands; D018129:Phosphatidylinositol Phosphates; D011953:Receptors, Cyclic AMP; C060974:phosphatidylinositol 3,4,5-triphosphate; D000242:Cyclic AMP; D058539:Phosphatidylinositol 3-Kinase; D051059:PTEN Phosphohydrolase
keywords: Dictyostelium; Excitable dynamics; Micropipette aspiration; PI3K; PIP2; PIP3; PTEN; Patch clamp; Signal transduction; cAMP; cAMP receptor