FcγR-stimulated accumulation of PtdIns(3,4,5)P3 at forming phagosomes is regulated by 5-phosphatase activity. RAW264.7 macrophages cotransfected with GFP-ARNO/PH and (A) vector, (B) HA-72-5ptase, or (C) HA-72-D480N5ptase were stimulated with eIgG to initiate phagocytosis, and GFP fluorescence was monitored using confocal microscopy. (A-C) GFP-ARNO/PH expression is shown. Images represent a time course of phagocytosis of eIgG. Insets show enlargements of boxed areas. The time of closure was arbitrarily designated as time 0. Arrowheads indicate sites of GFP-ARNO/PH accumulation. Scale bar represents 20 μm. The complete sequences from which these images were taken are shown in Videos S2, S3, and S4. (D) Results shown represent the fluorescence intensity of GFP-ARNO/PH at phagosomes normalized to the cytosolic fluorescence. To allow for comparison between experiments, each RFU was normalized to the maximum RFU obtained for each experiment. ---●--- represents vector; ▴, HA-72-5ptase; and ▒, HA-72-D480N5ptase. Data represents mean (± SEM) of 3 to 5 independent determinations. *P < .05 relative to vector-expressing cells. (E) siCON or si72-5ptase-transfected RAW264.7 macrophages were further transfected with GFP-ARNO/PH 48 hours after transfection and stimulated with eIgG or SOZ, and phagocytosis was monitored using confocal microscopy. Results shown represent the fluorescence intensity of GFP-ARNO/PH at phagosomes normalized to the cytosolic fluorescence. To allow for comparison between experiments each RFU was normalized to the maximum RFU obtained for each experiment. • represent siCON, and ⧫ represent si72-5ptase. Data represents mean (± SEM) from 3 to 7 independent determinations. *P < .05 relative to siCON-expressing cells.
