Phosphatidylinositol 3-phosphate is generated in phagosomal membranes

Abstract

Phagocytic cells such as neutrophils and macrophages engulf and destroy invading microorganisms. After internalization, material captured within the phagosomal membrane is destroyed by a complex process of coordinated delivery of digestive enzymes and reactive oxygen species. Several endosomal, lysosomal, and oxidase components expected to participate in these events have recently been shown to bind PtdIns3P, suggesting that this lipid may play a role in this process. We used live, digital fluorescence imaging of RAW 264.7 cells stably expressing either a PtdIns3P binding GFP-PX domain or a GFP-FYVE domain to visualize changes in the levels and subcellular localization of PtdIns3P during phagocytic uptake of IgG-opsonized zymosan particles. Very similar results were obtained using both PtdIns3P probes. The basal distribution of each PtdIns3P probe was partially cytosolic and partially localized to EEA-1-positive endosomal structures. Within about 2-3 min of zymosan attachment and concomitant with the closure of the phagosomal membrane, GFP-positive vesicles moved toward and attached to a localized area of the phagosome. A dramatic, transient accumulation of GFP probe around the entire phagosome rapidly ensued, accompanied by a transient drop in cytosolic GFP fluorescence. The magnitude and timing of this rise in PtdIns3P clearly suggest that it is an ideal candidate for controlling the early stages of phagosomal maturation.

A Regulated Adaptor Function of p40phox: Distinct p67phoxMembrane Targeting by p40phoxand by p47phox

“…This phenomenon is not observed constantly (Յ10%), but it occurred only occasionally when followed by time-lapsed photography ( Figure 2A and Supplemental Video 1). We then used GFP-p40 phox (PX) for further studies, because it was reported to bind specifically to PI(3)P. Consistent with a previous report (Ellson et al, 2001a), GFP-p40 phox (PX) is localized faintly in the cytoplasm and predominantly on vesicular structures ( Figure 2B), which could be colabeled with antibodies against early endosomal antigen (EEA)-1 ( Figure 2C), a marker of the early endosome that is enriched in PI(3)P (Gillooly et al, 2000) ( Figure 2B). The vesicular structures and free-form of GFP-p40 phox (PX) seems to fuse readily with phagosomes in later stages of Fc␥R-mediated phagocytosis, after the phagosome is sealed ( Figure 2B and Supplemental Video 2).…”

“…However, Nox2 activity can be reconstituted in vitro in the absence of p47 phox , when p67 phox and Rac1 are provided in excess (Freeman and Lambeth, 1996;Koshkin et al, 1996) or when p67 phox is adapted with the membrane-binding sequences from Rac1, although GTP-bound Rac is still required for oxidase activation (Gorzalczany et al, 2000;Alloul et al, 2001), indicating p67 phox and Rac1 are minimum essential cytoplasmic components in the Nox2 system. p40 phox also has a PX domain that specifically binds to phosphatidylinositol 3-phosphate [PI(3)P] (Bravo et al, 2001;Kanai et al, 2001), a phospholipid enriched in the early endosome (Gillooly et al, 2000) and produced in the phagosomal membrane during phagocytosis (Ellson et al, 2001a;Gillooly et al, 2001). Thus, p40 phox is also thought to serve as an adaptor component that recruits p67 phox to phagosomal membranes (Kuribayashi et al, 2002).…”

“…Finally, we show that AA has dramatic affects in unmasking the PX domain, thereby enabling p40 phox to bind these vesicles along with p67 phox . We suggest two mechanisms for the targeting p40 phox to phagosomes during Fc␥R-mediated phagocytosis: 1) p40 phox can bind initially to PI(3)P-enriched early endosomes, followed by their fusion to phagosomes, and/or 2) p40 phox can target directly to phagosomes, where PI(3)P is transiently produced during phagosytosis (Ellson et al, 2001a;Gillooly et al, 2001). Because the cytosolic phox proteins exist as a ternary complex (Kuribayashi et al, 2002;Lapouge et al, 2002), we propose a model for Nox2 complex assembly involving these spatiotemporal factors during the Fc␥R-mediated respiratory burst ( Figure 11): 1) in early stages, before phagosome sealing, p47 phox functions as the predominant carrier, mediating translocation of the ternary complex to newly forming phagosomes, 2) in later stages Model of activation and assembly of Nox2 system during phagocytosis.…”

Phosphoinositides in Constitutive Membrane Traffic

“…Clathrin binding enhanced the affinity of AP2 for internalization signals to a similar extent, but the effect of clathrin and PtdIns(3)P were not additive (286). However, when fluorescent proteins that bind PtdIns(3)P are expressed in cells, they label endosomes and not the plasma membrane (32,92,318), suggesting that there is little free PtdIns(3)P at the cell surface. There is a recent report that PtdIns3KIIC2␣ localizes to the nucleus rather than the plasma membrane (77).…”

“…At higher expression levels, they will interfere with the normal regulation of PIP production. With these limitations in mind, PtdIns(3)P has been detected primarily on endocytic membranes (32,92,318), PtdIns(4,5)P 2 at the plasma membrane (148,149,219,234,365) and on internal membranes enriched in lipid rafts (27,297), and PtdIns(4)P on the Golgi (13,201,372). PtdIns4K activity has been detected in cell fractions enriched in lysosomes (7,54,56).…”