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

• Published in: Experimental cell research 2007-04, Vol.313 (7), p.1496-1507
• Main Authors: Araki, Nobukazu, Egami, Youhei, Watanabe, Yasuo, Hatae, Tanenori
• Format: Article
• Language: English
• Subjects: Actin cytoskeleton; Animals; Cell Line, Tumor; Cell Membrane - drug effects; Cell Membrane - metabolism; Cell Membrane - physiology; Cellular biology; Epidermal Growth Factor - pharmacology; Imaging; Macropinocytosis; Metabolism; PH domain; Phosphatidylinositol 3-Kinases - metabolism; Phosphatidylinositol 4,5-Diphosphate - metabolism; Phosphatidylinositol Phosphates - metabolism; Phosphatidylinositols - metabolism; Phosphoinositide 3-kinase; Phosphoinositides; Pinocytosis; Proteins; Ruffling; Signal transduction; Transfection
• ISSN: 0014-4827; 1090-2422
• DOI: 10.1016/j.yexcr.2007.02.012

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)P 2 in the membrane ruffles forming macropinocytic cups increased to more than double that in planar plasma membranes. The PI(4,5)P 2 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)P 3 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)P 2 and PI(3,4,5)P 3 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)P 2 elimination and PI(3,4,5)P 3 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.