Distribution of PI(4,5)P2 and PI4KII during actin tail formation. (A and C) Macrophages expressing the indicated constructs were analyzed by confocal microscopy after uptake of complement- or IgG-coated RBCs. Bars, 5 µm. (A) Cells stably expressing mCherry-actin (right) were transiently transfected with tandem 2PH-PLCδ–GFP (left). The insets show cells stably expressing mCherry-actin (right) transiently transfected with the single PH-PLCδ–GFP (left). (B) Quantitation of 2PH-PLCδ–GFP- and actin-positive phagosomes at the indicated times after sealing for both CR3- and FcγR-mediated phagocytosis. (C) Cells were transiently transfected with GFP-PI4KIIA (left) or GFP-PI4KIIB (right) and monitored after CR3- (top) or FcγR-mediated phagocytosis (bottom). (D) Quantitation of PI4KIIA- and PI4KIIB-positive phagosomes during the first 15 min after uptake of complement- and IgG-coated RBCs. Numbers indicate time in seconds after phagosome formation, and arrows point to the nascent or recently formed phagosome. Data, expressed as percentages, are means ± SEM of at least three individual experiments of each type; ≥50 phagosomes were counted per experiment.
