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. 2008 Aug 27;3(8):e3068. doi: 10.1371/journal.pone.0003068

Figure 2. Synthetic activation of endogenous PI3K, but not of endogenous Rac, induces neutrophil polarization and migration.

Figure 2

Dual color fluorescent images of differentiated HL-60 cells are shown every 3min before and after iRap addition. (A) Neutrophils polarize and begin to migrate in response to synthetic activation of PI3K. (B) Neutrophils induce non-polar pseudopods in response to synthetic activation of endogenous Rac and do not migrate. Synthetic activation of Rac was based on the forced translocation of catalytic Rac GEF activity (from the exchange factor TIAM). FKBP-YFP-TIAM construct (YF-Tiam1) was expressed instead of the YF-iSH construct. In both image series, the top rows shows the YFP-tagged activation peptides (YF-iSH for PI3K or YF-Tiam1 for Rac), and the bottom images show DyeCycle-labeled nuclei. Arrowheads in images of the nuclei point to the transfected cells, whereas asterisks mark untransfected cells. (C) Migration paths of 10 representative cells for each condition. The initial location was normalized to the origin. Cells after synthetic activation PI3K (left), untransfected control cells in the same field of view (middle), and cells after synthetic activation of Rac (right). (D) Average velocity of cells before and after PI3K activation or Rac activation. Graph shows the velocity at 1.5 min before and 6 min after iRap addition. Error bars, S.E.M. (n = 20 from four independent experiments). Scale bars, 10 µm.