Fig. 2

PHAKT-EGFP Translocates to the Leading Edge When Neutrophils Come To and Leave Laser-Induced Wounds

(A) Time-lapse ratiometric imaging (PHAKT-EGFP/mCherry) reveals PI(3,4,5)P₃-PI(3,4)P₂ localization at the leading edge during attraction to a laser wound in the tail fin (>Movie S2A). The white dots and arrows indicate the position of the wound and direction of migration respectively. Scale bar = 10 μm.

(B) Reversal of PI(3,4,5)P₃-PI(3,4)P₂ (ratiometric imaging of PHAKT-EGFP/mCherry) when a neutrophil leaves the laser wound (Movie S2B). Note loss of PI(3,4,5)P₃-PI(3,4)P₂ polarity at the wound, followed by reversal of polarity to the opposite pole away from the wound when the neutrophil leaves the wound (green line, tracking of a neutrophil; yellow thunder, position of the laser wound; white arrows, direction of migration; illustration with white line, morphology of a neutrophil). Data are representative of more than five time-lapse movies from a minimum of three separate experiments. The numerical values of ratiometric analysis are shown in the scales. Scale bar = 10 μm.