Distinct roles of class I and class III phosphatidylinositol 3-kinases in phagosome formation and maturation

Abstract

Phagosomes acquire their microbicidal properties by fusion with lysosomes. Products of phosphatidylinositol 3-kinase (PI 3-kinase) are required for phagosome formation, but their role in maturation is unknown. Using chimeric fluorescent proteins encoding tandem FYVE domains, we found that phosphatidylinositol 3-phosphate (PI[3]P) accumulates greatly but transiently on the phagosomal membrane. Unlike the 3'-phosphoinositides generated by class I PI 3-kinases which are evident in the nascent phagosomal cup, PI(3)P is only detectable after the phagosome has sealed. The class III PI 3-kinase VPS34 was found to be responsible for PI(3)P synthesis and essential for phagolysosome formation. In contrast, selective ablation of class I PI 3-kinase revealed that optimal phagocytosis, but not maturation, requires this type of enzyme. These results highlight the differential functional role of the two families of kinases, and raise the possibility that PI(3)P production by VPS34 may be targeted during the maturation arrest induced by some intracellular parasites.

Figure 1.

Distribution and quantification of PI(3)P during phagocytosis. (A–G) Phagocytosis of IgG-opsonized RBC by RAW macrophages expressing 2FYVE–GFP. The time elapsed after addition of the RBCs is indicated (in min). Arrows point to phagosomes formed in the 4–5-min interval. DIC image (A). Bar, 10 μm. (H) HPLC quantification of PI(3)P in RAW cells before (control) and 12 min after phagocytosis of IgG-opsonized 3-μm latex beads. The PI(3)P data were normalized to the counts of the phosphatidylinositol peak and are expressed as percent of the untreated control, to facilitate comparison between experiments. The data are means ± SE of three determinations.

Figure 2.

Effects of wortmannin on phagosomal acquisition of PI(3)P, EEA1, and LAMP-1. RAW cells were either left untreated (A, C, and open symbols in E) or pretreated with 100 nM wortmannin for 30 min (B, D, and solid symbols in E), before exposure to IgG-opsonized latex beads (3 μm diam). In A and B, the cells were transfected with 2FYVE–GFP and allowed to internalize beads for 7 min before fixation. In C and D, phagocytosis was allowed to occur for 7 min, followed by an additional 30 min after washing unbound beads. Subsequently, cells were immunostained for LAMP-1. Confocal fluorescence microscopy is shown in the main panels and DIC in the insets. Bars, 10 μm. (E) Quantification of the effect of wortmannin on the fraction of phagosomes containing EEA1 (triangles) or LAMP-1 (squares). Cells were pretreated with or without wortmannin and allowed to internalize beads for 7 min, as above. After washing unbound beads, incubation at 37°C proceeded for the indicated times, followed by immunostaining with antibodies to EEA1 or LAMP-1. Data are means ± SE of five experiments, each with at least 100 phagosomes counted.

Figure 3.

Phagosomal maturation in wild-type and type I PI 3-kinase–deficient fibroblasts. Fibroblasts were obtained from wild-type (A, C, E, and solid bars in G) or from double knockout mice lacking the α and β isoforms of the p85 subunit of type I PI 3-Kinase (B, D, F, and open bars in G). These cells were transfected with epitope-tagged FcγRIIA to confer phagocytic capacity, and then exposed to IgG-opsonized RBC (A, B, and second set of columns in G) or latex beads (C–F and leftmost bars in G). In A and B the cells were cotransfected with Akt-PH-GFP to monitor 3′-polyphosphoinositides and were allowed to internalize RBC for 6 min. Arrows indicate sites of attachment of RBCs. (C and D) Cells were cotransfected with 2FYVE–GFP to monitor PI(3)P distribution and were allowed to internalize beads for 25 min. (E and F) Cells were allowed to internalize beads for 20 min followed by a 60-min incubation after removing unbound beads. Cells were then fixed and immunostained for LAMP-1. (C–F) Arrows point to phagosomes. Bars, 10 μm. (G) Quantification of the phagocytic index using beads (PI, Beads) or RBC (PI, RBCs) (left axis, in particles/100 cells) and of the fraction of latex bead phagosomes containing EEA1 or LAMP-1 (right axis, as percent of total phagosomes). EEA1 and LAMP-1 were quantified 25 min and 80 min after phagocytosis, respectively, as described. Data are means ± SE of five experiments, each with at least 100 phagosomes counted.

Figure 4.

Recruitment and role of VPS34 in phago–lysosome fusion. (A and B) Distribution of 2FYVE–GFP and VPS34 during phagocytosis. COS-7 cells expressing FcγRIIA were cotransfected with 2FYVE–GFP and VPS34. After exposure to opsonized particles for 15 min, the cells were fixed and 2FYVE–GFP (A) and VPS34 (B) were visualized directly or by immunostaining, respectively. (C and D) Effect of anti-VPS34 antibodies on phagosomal distribution of 2FYVE–GFP. CHO cells transfected with 2FYVE–GFP were injected with nonimmune rabbit IgG (C) or with anti-hVPS34 antibody (D). After a 2-h period, the cells were allowed to internalize beads for 10 min. Arrows point to phagosomes. The insets identify the microinjected cells, stained with Cy3-labeled anti–rabbit IgG antibodies. (E–I) CHO cells expressing FcγRIIA were injected with either nonimmune rabbit IgG (E and F) or with anti-hVPS34 antibody (G and H). Phagocytosis of opsonized 3-μm latex beads was allowed to proceed for 20 min, followed by 50 min of maturation after removal of unbound beads, and ultimately stained for LAMP-1. (E and G) Identification of microinjected cells by staining with labeled anti-rabbit IgG. F and H: LAMP-1 staining of the cells shown in E and G, respectively. Full white arrows and open arrowheads point to injected and uninjected cells, respectively. Bars, 10 mm. (I) Quantification of phagosome acquisition of LAMP-1 in cells injected with nonimmune rabbit IgG (control) or with anti-hVPS34 antibody. Data are means ± SE of four separate experiments, each with at least 100 injected cells.