FIGURE 6:

Recruitment of the p85 domain of PtdIns3K to the plasma membrane increases macropinocytic activity of GM/IFN-γ/LPS–cultured macrophages. (A) Schematic showing the recruitment of p85-CFP to the plasma membrane via the rapamycin-inducible heterodimerization system. (B, C) GM/IFN-γ/LPS– and M/IL4-cultured macrophages were cotransfected with FKBP-p85-CFP, Lyn₁₁-FRB, and PH(Akt)-RFP. Transfected cells were imaged (B) 24 h posttransfection either before (left) and after (right) addition of rapamycin (1 µM, 15 min; the same cell is shown on the left and right), and the activation PtdIns3K was quantified as redistribution of PH(Akt)-RFP, a biosensor of PtdIns(3,4,5)P₃, to the plasma membrane (C), quantified as described for PH(Akt)-GFP in Figure 5B. Typical images (B) and quantifications (C; means ± SEM) are representative of 20–30 cells from three to four independent experiments. (D, E) GM/IFN-γ/LPS– and M/IL4-cultured macrophages were cotransfected with FKBP-p85-CFP and Lyn₁₁-FRB. After a 24-h transfection, the cells were incubated with fluorescently labeled 70 kDa dextran (TMR-dextran, 125 µg/ml) for 15 min at 37°C, with or without concurrent treatment with rapamycin (1 µM). Cells were then washed, fixed, and imaged immediately (D). Cells positive for CFP were selected for measurements of macropinocytosis, which was quantified as described for Figure 2C (E). Representative images (D) and quantification (means ± SEM; E) of 30–50 cells from three to five independent experiments. Scale bars, 15 µm.