FIGURE 2:

GflB is located at the cell periphery in growing cells and the leading edge of chemotaxing cells. (A) GflB constructs used. (B) Growing WT cells expressing the indicated forms of GflB fused to GFP were observed by fluorescence microscopy. Arrowheads indicate pseudopods. Bar, 10 μm. (C) Full-length GflB and its N-terminal extension (amino acids 361–664) bind to phosphatidylserine in a lipid dot blot assay. Nitrocellulose membranes spotted for the indicated lipids were incubated with cell lysates expressing the indicated FLAG-tagged proteins. Protein–lipid interactions were detected using anti-FLAG primary antibodies and fluorescently labeled secondary antibodies. (D) Differentiated WT cells expressing GFP-GflB and GFP-GflB_1–644 were placed in a cAMP gradient and viewed by fluorescence microscopy. cAMP gradients were formed from the right side of images. (E) Differentiated WT cells expressing the indicated GFP-fusion proteins were uniformly stimulated with cAMP in the presence or absence of the PI3 kinase inhibitor LY294002 and the actin polymerization inhibitor latrunculin A (LatA). (F, G) Quantification of GFP-GflB and GFP-GflB_1–644 localization. GFP intensity at the cell periphery was normalized to GFP intensity in the cytosol; –, basal intensity before addition of cAMP (0 s); +, peak intensity after addition of cAMP (5 s). Values are normalized to the basal intensity and represent the mean ± SEM. At least nine cells were analyzed for each group.