Development of opto-PI3K for probing neutrophil signaling. (A, top) Domain layout of PhyB fusion proteins used in this study. “PhyB” is a truncation containing the first 917 residues of PhyB, and “short-PhyB” contains residues 1–621 of PhyB lacking the PAS₂ domain. Both PhyB truncations are fused to mCherry-CAAX, as shown in Fig. 1 B. (Bottom) PhyB and short-PhyB expression were determined by measuring mCherry fluorescence via flow cytometry. Each bar represents the mean ratio of PhyB expression in differentiated versus undifferentiated neutrophil-like HL-60 cells. Short-PhyB was much better expressed than PhyB in dHL-60 cells. Data were obtained from at least three independent experiments. Error bars indicate SEM. ***, P < 0.001 by unpaired t test. (B) Schematic of experiments shown in C. Whole-field illumination of cells using IR light maintains PhyB in the “OFF” state, causing opto-PI3K (shown in blue) to localize to the cytosol. In contrast, whole-field illumination using red light photoconverts PhyB to the “ON” state, and opto-PI3K translocates to the plasma membrane. This process is reversible (gray arrows). (C) dHL-60s expressing optogenetic components were plated in squeeze chambers. Cells were exposed to alternating rounds of red or IR light as indicated. Images depicting opto-PI3K localization are single focal planes obtained by spinning-disk confocal microscopy. Translocation of opto-PI3K from the cytosol to the plasma membrane was completely reversible, occurred within seconds of exposure to red/IR light, and could be repeated multiple times within the same cell. Cyan arrowheads indicate protrusions formed upon recruitment of opto-PI3K to the plasma membrane. (D) Quantification of percentages of dHL-60s with pseudopods when illuminated with either red or IR light. Cells were prepared as in C. Each point represents the percentage of cells with pseudopods at the indicated time pooled from four independent experiments. Error bars indicate SEM. **, P < 0.01 by paired t test. (E) dHL-60s expressing opto-PI3K components were either unstimulated (left), stimulated with 10 nM fMLP for 4 min (center), or treated with red light for 6 min (right) and then fixed and stained for F-actin with phalloidin. Images are single focal planes obtained via spinning-disk confocal microscopy. Bars, 20 µm. (F) Quantification of F-actin polarization phenotypes in dHL-60 cells prepared as described in E. Cells stimulated with opto-PI3K yield a similar degree of polarity as cells stimulated with chemoattractant. Error bars indicate SD. *, P < 0.05 by unpaired t test.