Restricted accumulation of phosphatidylinositol 3-kinase products in a plasmalemmal subdomain during Fc gamma receptor-mediated phagocytosis

Phagocytosis is a highly localized and rapid event, requiring the generatio n of spatially and temporally restricted signals. Because phosphatidylinosi tol 3-kinase (PI3K) plays an important role in the innate immune response, we studied the generation and distribution of 3' phosphoinositides (3'PIs) in macrophages during the course of phagocytosis, The presence of 3'PI was monitored noninvasively in cells transfected with chimeras of green fluores cent protein and the pleckstrin homology domain of either Akt, Btk, or Gab1 , Although virtually undetectable in unstimulated cells, 3'PI rapidly accum ulated at sites: of phagocytosis. This accumulation was sharply restricted to the phagosomal cup, with little 3'PI detectable in the immediately adjac ent areas of the plasmalemma. Measurements of fluorescence recovery after p hotobleaching were made to estimate the mobility of lipids in the cytosolic monolayer of the phagosomal membrane. Stimulation of phagocytic receptors induced a marked reduction of lipid mobility that likely contributes to the restricted distribution of 3'PI at the cup. 3'PI accumulation during phago cytosis was transient, terminating shortly after sealing of the phagosomal vacuole. Two factors contribute to the rapid disappearance of 3'PI: the dis sociation of the type I PI3K from the phagosomal membrane and the persisten t accumulation of phosphoinositide phosphatases.