PtdIns(3,4)P 2 accumulates at the site of particle engagement during phagocytosis, is susceptible to the degradation by INPP4B phosphatases, and its production is downstream of class I PI3Ks and 5′ phosphatases. (A) Confocal sections of RAW 264.7 macrophages expressing GFP-cPHx2 alone (left) or in combination with either BFP-INPP4B-CAAX (gray inset; middle) or BFP-INPP4B(C842A)-CAAX (gray inset; right) during phagocytosis. Asterisks represent sites of phagocytic cup formation. SRBCs are shown in blue. (B) Normalized phagosomal-cPHx2 fluorescence intensity in INPP4B(C842A)-CAAX (n = 101) and INPP4B-CAAX (n = 100). For quantitation, here and elsewhere, phagosomal fluorescence intensity was normalized to plasmalemmal fluorescence intensity. Unpaired t test of individual values across n = 3 independent experiments; data are mean ± SD. (C) Representative confocal micrographs of RAW 264.7 macrophages expressing GFP-cPHx2 and pretreated with either vehicle control (left), 500 nM PI-103 (middle), or 500 nM GDC-0941 (right). Asterisks denote particles. Insets represent the area denoted by the dotted box showing SRBCs. (D) Normalized phagosomal GFP-cPHx2 fluorescence intensity in control (n = 58) and PI-103–treated cells (n = 62). Unpaired t test of individual values across n = 3 independent experiments; data are mean ± SD. (E) Representative confocal micrographs of RAW 264.7 macrophages co-expressing GFP-2xP4M and mCherry-cPHx3 during phagocytosis in either control (top) or GSK-A1–treated (bottom) conditions. Asterisks indicate sites of particle binding. (F) Representative confocal sections of RAW 264.7 macrophages expressing SHIP2 (left), SYNJ2 (middle), and INPP5B (right) phosphatases during phagocytosis. Asterisks indicate sites of particle binding. ****, P < 0.0001.
