Figure 4.

H₂O₂ gradients in cell polarization. NIH-3T3 cells transiently expressing HyPer7-LifeAct were imaged during spontaneous migration. (A) Images of a migrating cell at different time points. Upper and middle rows: fluorescent images excited by 405 nm and 488 nm. Lower row: ratiometric images. Addition of PEG-catalase (PEG-Cat) destroys the gradient and leads to retraction of the protrusions. Lines 1 and 2 were used to build kymographs. Scale bar shows 10 μm. (B) Kymographs of the HyPer7-LifeAct ratio along lines 1 and 2 representing the leading edge and the side of the cell from images shown in panel A, respectively. Notice that a more extensive gradient makes the protrusion more stable. (C, D) Correlation plots between the protrusion lifetime and either HyPer-LifeAct ratio gradient (C) or the mean HyPer-LifeAct ratio in the cell demonstrates positive correlation between protrusion stability and the intensity of the H₂O₂ gradient from the protrusion to the cell body (correlation coefficient 0.37), but not the mean H₂O₂ levels (correlation coefficient −0.06).