Figure 2.

Localization of phosphatidylinositol-3,4,5-trisphosphate by using PH_Crac–GFP. (A) Dictyostelium cells were stimulated in a perfusion chamber with a uniform cAMP concentration of 1 μM. In untreated cells, PH_Crac–GFP transiently translocates to the entire membrane in about 8 s, which is followed by persistent translocation of PH_Crac–GFP to small patches. About 10 s after a PH_Crac–GFP patch appears, a pseudopod is formed at the position of a PH_Crac–GFP patch (Postma et al, 2003). In the lower panels, cells were pretreated for 5 min with 1 μM latrunculin A, which induces the depolymerization of actin, the inhibition of pseudopod formation and the rounding up of the cell. A uniform cAMP concentration still induces a localized response with patches of PH_Crac–GFP in these spherical cells. (B) Cells were stimulated locally with cAMP by using a micropipette. The position of the pipette is indicated by an asterisk (data from Parent et al, 1998; Parent & Devreotes, 1999). The multiple patches induced by uniform cAMP have the same size and life-time as the single patch induced by the cAMP gradient, and they closely correlate with pseudopod formation. Crac, cytosolic regulator of adenylyl cyclase; GFP, green fluorescent protein; PH, pleckstrin homology.