![Figure 5. Rapamycin-induced recruitment of PIP5KI during FcγR-mediated phagocytosis. (A) Experimental strategy: RAW macrophages were transfected with two complementary rapamycin-binding domains (LDR, which includes the membrane-targeting N-terminal sequence of Lyn and either PIP5KIγ-FKBP–YFP [YF-PIP5K] or FKBP-YFP [YF]). Addition of rapamycin (black dot) after FcγR-mediated phagocytosis of IgG-coated RBCs induces translocation of the FKBP domain-containing constructs to the phagosome. The star indicates PIP5K activity on the phagosomal membrane. (B) PH-PLCδ–RFP, PIP5KIγ-FKBP–YFP, and LDR were coexpressed transiently, and phagocytosis of IgG-coated RBCs was initiated. Images acquired immediately before (top) and 315 s after addition of 1 µM rapamycin are shown. Arrows point to the nascent or recently formed phagosomes. (C) PH-Akt–RFP, PIP5KIγ-FKBP–YFP, and LDR were coexpressed transiently, and phagocytosis was initiated as in B. Images were acquired 240 s after addition of rapamycin. (D) PIP5KIγ-FKBP–YFP and LDR were coexpressed transiently in RAW cells stably expressing mCherry-actin, and phagocytosis of IgG-coated RBCs was initiated. Images of mCherry-actin acquired immediately before (left) and 204 s after addition of 1 µM rapamycin are shown in the main panels. (E) PIP5KIγ-FKBP–YFP and LDR were coexpressed transiently in RAW cells stably expressing mCherry-actin, and phagocytosis of IgG-coated RBCs was initiated. Shortly after ingestion, the cells were treated with 100 µM LY294002 and then with 1 µM rapamycin. Images acquired 208 s after addition of rapamycin are shown. (F) PH-Akt–RFP, PIP5KIγ-FKBP–YFP, and LDR were coexpressed transiently. Shortly after ingestion, the cells were treated with 100 µM LY294002 and then with rapamycin. Images of mCherry-actin acquired 240 s after addition of rapamycin are shown in the main panels. In C–F, the insets show the distribution of PIP5KIγ-FKBP–YFP before (0 s) and at the indicated times after addition of rapamycin. Images in B–F are representative of five separate experiments, each analyzing ≥40 phagosomes. Bars, 3 µm. (G) Quantification of multiple experiments like those in C–F. Data are means ± SEM of five separate experiments.](https://cdn.rupress.org/rup/content_public/journal/jcb/191/5/10.1083_jcb.201004005/5/jcb_201004005_gs_fig5.jpeg?Expires=1773568866&Signature=H7oJ1fkm5Cechm6DieElQrXlYuFR7z6z9XANXWDn1pUBhA8u57136l1wM3o~BicDGyqW5vaaHRkJCdQm5VK9xEuqlE6-xxL2GZ~vdqD-IBTSrCKbsXxajZOstP9Ji7Q333QDrNYsAuRwLlDygbMYpokxnIjfo4P6Evi4cQwOJVUqOP1w3ZbgVsEwKEbDNEjsHv1XYCf0JtmzoOxcegeYnFL0zdf0E69ic3mB5dARbXLc0O2M3a4owSAYy1fcIUByC87bWhOuVNZcwQ5U6o3CIS6uO8hEAq4qShoxZloQ6yI8fnsxAO6TFi5oJEk1BAKZaE06pqWYGPaTu36t-jkRlQ__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Rapamycin-induced recruitment of PIP5KI during FcγR-mediated phagocytosis. (A) Experimental strategy: RAW macrophages were transfected with two complementary rapamycin-binding domains (LDR, which includes the membrane-targeting N-terminal sequence of Lyn and either PIP5KIγ-FKBP–YFP [YF-PIP5K] or FKBP-YFP [YF]). Addition of rapamycin (black dot) after FcγR-mediated phagocytosis of IgG-coated RBCs induces translocation of the FKBP domain-containing constructs to the phagosome. The star indicates PIP5K activity on the phagosomal membrane. (B) PH-PLCδ–RFP, PIP5KIγ-FKBP–YFP, and LDR were coexpressed transiently, and phagocytosis of IgG-coated RBCs was initiated. Images acquired immediately before (top) and 315 s after addition of 1 µM rapamycin are shown. Arrows point to the nascent or recently formed phagosomes. (C) PH-Akt–RFP, PIP5KIγ-FKBP–YFP, and LDR were coexpressed transiently, and phagocytosis was initiated as in B. Images were acquired 240 s after addition of rapamycin. (D) PIP5KIγ-FKBP–YFP and LDR were coexpressed transiently in RAW cells stably expressing mCherry-actin, and phagocytosis of IgG-coated RBCs was initiated. Images of mCherry-actin acquired immediately before (left) and 204 s after addition of 1 µM rapamycin are shown in the main panels. (E) PIP5KIγ-FKBP–YFP and LDR were coexpressed transiently in RAW cells stably expressing mCherry-actin, and phagocytosis of IgG-coated RBCs was initiated. Shortly after ingestion, the cells were treated with 100 µM LY294002 and then with 1 µM rapamycin. Images acquired 208 s after addition of rapamycin are shown. (F) PH-Akt–RFP, PIP5KIγ-FKBP–YFP, and LDR were coexpressed transiently. Shortly after ingestion, the cells were treated with 100 µM LY294002 and then with rapamycin. Images of mCherry-actin acquired 240 s after addition of rapamycin are shown in the main panels. In C–F, the insets show the distribution of PIP5KIγ-FKBP–YFP before (0 s) and at the indicated times after addition of rapamycin. Images in B–F are representative of five separate experiments, each analyzing ≥40 phagosomes. Bars, 3 µm. (G) Quantification of multiple experiments like those in C–F. Data are means ± SEM of five separate experiments.
