期刊
EXPERIMENTAL CELL RESEARCH
卷 313, 期 7, 页码 1496-1507出版社
ELSEVIER INC
DOI: 10.1016/j.yexcr.2007.02.012
关键词
macropinocytosis; ruffling; phosphomositides; phosphoinositide 3-kinase; PH domain; actin cytoskeleton; signal transduction; imaging
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 C delta 1-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. (c) 2007 Elsevier Inc. All rights reserved.
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