Phosphoinositide metabolism during membrane ruffling and macropinosome formation in EGF-stimulated A431 cells

Inhibitors of phosphoinositide 3-kinase (PI3K) were found to perturb macropinosome formation without affecting the membrane ruffling and actin polymerization in epidermal growth factor-stimulated A431 cells. Live-cell imaging and quantitative image analysis of the fluorescence intensity ratio of the YFP-tagged phospholipase Cδ1-pleckstrin homology domain (YFP-PLC-PH) relative to membrane-targeted CFP (CFP-Mem) demonstrated that the concentration of PI(4,5)P2 in the membrane ruffles forming macropinocytic cups increased to more than double that in planar plasma membranes. The PI(4,5)P2 level in the membrane reached its maximum just before macropinosome closure and rapidly fell as the macropinocytic cups closed. In contrast, the PI(3,4,5)P3 concentrations visualized based on the YFP-Akt-PH or YFP-Bruton's tyrosine kinase (Btk)-PH/CFP-Mem ratio increased locally at the site of macropinosome formation and peaked at the time of macropinosome closure. The kinetics of PI(4,5)P2 and PI(3,4,5)P3 appeared to be mechanistically linked to actin remodeling during macropinocytosis. From the pharmacological data using inhibitors and synthetic phosphoinositides and other data, it could be concluded that both PI(4,5)P2 elimination and PI(3,4,5)P3 production by PI3K might be crucial for macropinosome formation from membrane ruffles. This study emphasizes that locally controlled levels of phosphoinositides are important for regulating the function of actin-binding proteins which effect changes in the membrane architecture.