Phosphatidylinositol 3-phosphate is generated in phagosomal membranes

Phagocytic cells such as neutrophils and macrophages engulf and destroy inv ading microorganisms. After internalization, material captured within the p hagosomal membrane is destroyed by a complex process of coordinated deliver y of digestive enzymes and reactive oxygen species. Several endosomal, lyso somal, 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 [1-5]. We used live, digital fluorescence imaging of RAW 264.7 cells stably expressing either a PtdIns3P binding GFP-PX domain o r a GFP-FYVE domain to visualize changes in the levels and subcellular loca lization of PtdIns3P during phagocytic uptake of IgG-opsonized zymosan part icles. Very similar results were obtained using both PtdIns3P probes, The b asal distribution of each PtdIns3P probe was partially cytosolic and partia lly localized to EEA-1-positive endosomal structures. Within about 2-3 min of zymosan attachment and concomitant with the closure of the phagosomal me mbrane, GFP-positive vesicles moved toward and attached to a localized area of the phagosome. A dramatic, transient accumulation of GFP probe around t he entire phagosome rapidly ensued, accompanied by a transient drop in cyto solic GFP fluorescence. The magnitude and timing of this rise in PtdIns3P c learly suggest that it is an ideal candidate for controlling the early stag es of phagosomal maturation.