Uniform cAMP Stimulation of Dictyostelium Cells Induces Localized Patches of Signal Transduction and Pseudopodia

The chemoattractant cAMP induces the translocation of cytosolic PH _crac-GFP to the plasma membrane. PH_crac 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 PH_crac-GFP in the cytosol and at different places along the cell boundary. In cells stimulated homogeneously with 1 μM cAMP we observed two distinct phases of PH_crac-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 ∼20 s. A second translocation of PH_crac-GFP starts after ∼30 s and persists as long as cAMP remains present. Translocation during this second response occurs to small patches with radius of ∼4-5 μm, each covering ∼10% of the cell surface. Membrane patches of PH _crac-GFP are both temporally and spatially closely associated with pseudopodia, which are extended at ∼10 s from the area with a PH _crac-GFP patch. These signaling patches in pseudopodia of homogeneously stimulated cells resemble the single patch of PH _crac-GFP at the leading edge of a cell in a gradient of cAMP, suggesting that PH_crac-GFP is a spatial cue for pseudopod formation also in uniform cAMP.