Uniform cAMP stimulation of Dictyostelium cells induces localized patches of signal transduction and pseudopodia.
The chemoattractant cAMP induces the translocation of cytosolic PHCrac-GFP to the plasma membrane. PHCrac-GFP is a green fluorescent protein fused to a PH domain that presumably binds to phosphatydylinositol polyphosphates in the membrane. We determined the relative concentration of PHCrac-GFP in the cytosol and at different places along the cell boundary. In cells stimulated homogeneously with 1microM cAMP we observed two distinct phases of PHCrac-GFP translocation. The first translocation is transient and occurs to nearly the entire boundary of the cell; the response is maximal at 6-8 s after stimulation and disappears after approximately 20 s. A second translocation of PHCrac-GFP starts after approximately 30 s and persists as long as cAMP remains present. Translocation during this second response occurs to small patches with radius of approximately 4-5 microm, each covering approximately 10% of the cell surface. Membrane patches of PHCrac-GFP are both temporally and spatially closely associated with pseudopodia, which are extended at approximately 10 s from the area with a PHCrac-GFP patch. These signaling patches in pseudopodia of homogeneously stimulated cells resemble the single patch of PHCrac-GFP at the leading edge of a cell in a gradient of cAMP, suggesting that PHCrac-GFP is a spatial cue for pseudopod formation also in uniform cAMP.
Department of Biochemistry, University of Groningen, Nijenborgh 4, 9747 AG Groningen, the Netherlands.
Marten
M
Postma
Jeroen
J
Roelofs
Joachim
J
Goedhart
Theodorus W J
TW
Gadella
Antonie J W G
AJ
Visser
Peter J M
PJ
Van Haastert
D000818:Animals; D001692:Biological Transport; D002462:Cell Membrane / Q000378:metabolism* / Q000502:physiology; D002465:Cell Movement / Q000187:drug effects / Q000502:physiology*; D002630:Chemotactic Factors / Q000494:pharmacology; D000242:Cyclic AMP / Q000494:pharmacology; D004023:Dictyostelium / Q000378:metabolism* / Q000502:physiology; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000378:metabolism; D008856:Microscopy, Fluorescence; D011485:Protein Binding; D017434:Protein Structure, Tertiary / Q000502:physiology; D011554:Pseudopodia / Q000378:metabolism* / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000378:metabolism / Q000502:physiology; D015398:Signal Transduction / Q000502:physiology*
D002630:Chemotactic Factors; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D000242:Cyclic AMP