Localized increases in PI3K activity are sufficient to reorient neutrophil polarity. (A) Schematic illustrating the experiments shown in B as well as in Fig. S1 (B and C). A square box (∼225 µm²) of red light was positioned over approximately the rear third of an actively migrating dHL-60 cell to locally activate PIP₃ production while inhibiting it elsewhere with IR light (left). If localized PIP₃ production is sufficient to drive neutrophil polarization, the cell should reorient its polarity axis and begin migrating into the box (center). Alternatively, if localized PIP₃ production is insufficient, the cell should continue its trajectory away from the box (right). (B) Migrating dHL-60 cells in squeeze chambers were initially exposed to a pattern of red/IR light as depicted in A (top, representative cell). After 5 min, this pattern was “inverted” to suppress opto-PI3K activity in the 225 µm² box but stimulate it everywhere else (middle). The mean cell area lying within the 225-µm² box was plotted over time (bottom). Red/gray shading behind the plot indicates portions of the experiment where opto-PI3K activity was either restricted to or excluded from the 225-µm² box, respectively. Each point represents a mean of 19 different cells measured on three different days. Bar, 20 µm. Error bars indicate SEM.