PI(3)P is required for actin tail formation. Macrophages expressing the indicated constructs were analyzed by confocal microscopy after CR3-mediated uptake of complement-coated RBCs. Insets show corresponding differential interference contrast images. Numbers indicate time after phagosome formation. Bars, 5 µm. (A) Cells stably expressing mCherry-actin (bottom) were transiently transfected with 2FYVE-GFP (top). (B) Quantitation of 2FYVE-positive phagosomes at the indicated times after sealing. The cells were treated with 100 nM wortmannin shortly after ingestion of the RBCs. Data, expressed as percentages, are means ± SEM of at least six individual experiments of each type; ≥70 phagosomes were counted per experiment. (C) Macrophages stably expressing mCherry-actin (right) were transiently transfected with a construct consisting of five tandem FYVE domains coupled to GFP (left). The dotted lines delineate the cell outlines on the left. (D) Macrophages stably expressing GFP-actin (right) were loaded with anti-Vps34 antibody in the presence of rhodamine-conjugated dextran (left), which was used to identify the cells that were transiently permeabilized by the bead-loading procedure. The dotted lines delineate the outlines of unloaded cells displaying actin-labeled phagosomes. Arrows point to the nascent or recently formed phagosomes. Images in A, C, and D are representative of at least four individual experiments. (E) Quantification of actin- or PH-Akt–RFP-positive phagosomes in cells transiently transfected with 5FYVE-GFP. (F) Quantification of actin-positive phagosomes in cells loaded with an anti-Vps34 antibody (+) or subjected to the permeabilization protocol and either unloaded or loaded with an irrelevant antibody. Data in E and F are means ± SEM of at least four individual experiments; ≥25 phagosomes were counted per experiment.