Expression of pleckstrin-2 in Jurkat cells localizes to the cellular membrane upon stimulation of cell-surface receptors and enhances lamellipodia formation. (A) Jurkat cells expressing GFP alone or GFP-labeled pleckstrin-2 variants were plated upon OKT3 or fibronectin. This figure shows that WT pleckstrin-2 associates with the cell membrane when cells are plated upon OKT3 or fibronectin. Images were captured at ×40 magnification using IP Lab imaging software. (B) Jurkat cells expressing GFP fused to the amino-terminus of wild-type pleckstrin-2 were plated on fibronectin. Real-time confocal imaging of live cells was performed. Shown is a cell expressing pleckstrin-2 before and after addition of 10% FBS. This figure shows that wild-type pleckstrin-2 associates with the membrane and membrane projections rapidly (about 30 seconds) with the addition of serum. Images were captured using an UtraVIEW-LCI confocal scanner at ×100 magnification. (C) Jurkat cells expressing GFP alone or GFP-labeled pleckstrin-2 variants were plated on OKT3. This figure demonstrates that membrane localization is dependent on the pleckstrin-2 PH domains. Similar to WT pleckstrin-2, the DEP domain mutant associates with the cell membrane upon stimulation by the T-cell receptor–activating antibody but does not form membrane ruffles. Images were captured using an UtraVIEW-LCI confocal scanner at × 100 magnification.