Neutrophils lacking phosphoinositide 3-kinase γ show loss of directionality during N-formyl-Met-Leu-Phe-induced chemotaxis

Confocal imaging and time-lapsed videomicroscopy were used to study the directionality, motility, rate of cell movement, and morphologies of phosphoinositide 3-kinase γ (PI3K)γ(−/−) neutrophils undergoing chemotaxis in Zigmond chambers containing N-formyl-Met-Leu-Phe gradients. Most of the PI3Kγ(−/−) neutrophils failed to translocate up the cheomotactic gradient. A partial reduction in cell motility and abnormal morphologies were also observed. In the wild-type neutrophils, the pleckstrin homology domain-containing protein kinase B (AKT) and F-actin colocalize to the leading edge of polarized neutrophils oriented toward the gradient, which was not observed in PI3Kγ(−/−) neutrophils. In PI3Kγ(−/−) neutrophils, AKT staining consistently failed to perfectly overlap with the F-actin. This failure was observed as an F-actin-filled region of 2.3 ± 0.5 μm between AKT and the cell membrane. These data suggest that PI3Kγ regulates neutrophil chemotaxis primarily by controlling the direction of cell migration and the intracellular colocalization of AKT and F-actin to the leading edge.