PI(3,4)P2/PI(3,4,5)P3 accumulation in sealed phagosomes: role in actin tail formation. (A, B, and D) RAW macrophages stably expressing mCherry-actin were transfected with PH-Akt–GFP and monitored after CR3-mediated uptake of complement-coated RBCs. In B, the complement-coated RBCs were marked with a fluorescent F(ab’)2 (insets) to verify the constancy of the focal plane. (C) Plots of the background-subtracted intensities of PH-Akt–GFP and mCherry-actin as ratios of the F(ab’)2 intensity ± SEM of 29 individual phagosomes. To control for the heterogeneous timing of PI(3,4,5)P3 formation and actin tail formation after internalization, the plots were centered at time 0, which represents the moment of maximal PH-Akt–GFP recruitment to an internalized phagosome. (D) The cells were treated with 100 nM wortmannin immediately after phagocytosis was completed. Numbers indicate time in seconds after formation of phagosomes indicated by arrows. Insets illustrate corresponding differential contrast images. Images in A, B, and D are representative of at least six individual experiments of each kind. Bars, 5 µm. (E) Quantitation of PH-Akt– (left) or actin-positive phagosomes (right) at the indicated times after sealing. The cells were treated with 100 nM wortmannin shortly after phagocytosis was completed. Neither PH-Akt–GFP nor actin were detectable in wortmannin-treated samples after 10 min. Data, expressed as percentages, are means ± SEM of at least five individual experiments of each type; ≥80 phagosomes were counted per experiment.
