Figure 5. cAMP is excluded from extending pseudopods in randomly migrating and chemotaxing neutrophils.

A. Basal FRET efficiency images represented in pseudo-color for differentiated NS shRNA and AC9 shRNA. Also see Fig. S5A. At low cAMP levels observed in AC9 KD cells, the probe remains in a close state and the FRET response is maximal. In the presence of cAMP, a conformational change occurs and the FRET response is lost.

B. Average basal FRET efficiency in differentiated NS shRNA and AC9 shRNA cells, n=8. * indicates p<0.01 compared to NS shRNA cells.

C. Time course of FRET efficiency following a uniform stimulation with 1 μM fMLP in NS shRNA and AC9 shRNA cells expressing the WT FRET sensor and in NS shRNA cells expressing a mutated FRET sensor (R96E). The left panel shows the area where the FRET efficiency was measured. The right panel depicts FRET efficiency images at different time points for NS shRNA and AC9 shRNA cells expressing the WT FRET sensor. Also see Fig. S5B & Movie S7.

D. Differentiated NS shRNA cells were exposed to a micropipette containing 1 μM fMLP. The position of the micropipette is indicated by the star. The pseudocolor image was taken from a representative experiment and represents FRET efficiency. Also see Movie S7.

E. Graph depicting FRET intensities as a function of the position along the long axis of the cell for three consecutive time points acquired at 15 secs interval (depicted in red, blue and black lines). Each point is an average over a smoothing region 5.6 μm across the cell width and 2.1 μm along the long axis of the cell (dashed square indicates the size of the sliding smoothing region). The top arrow indicates the direction of cell migration. The green line depicts the intensity of CFP fluorescence across the cell.