Fig. 2. Activated PAK4 induces cell rounding. (i) MDCK cells were microinjected with a plasmid encoding PAK4GBD and incubated for 3 hours in 0.2% FCS prior to a further incubation in 0.2% FCS for 20 hours (A) or stimulation with HGF for 20 hours (B). Cells were then fixed and stained for HA-tagged PAK4 (green) and F-actin (red). Bar, 10 µm. Apical sections (at the level of F-actin-containing microvilli) are shown of PAK4GBD expression (B) in order to visualise clearly any rounded-up PAK4-expressing cells (arrows). In this plane non-expressing rounded cells are primarily mitotic cells. (ii) MDCK cells were microinjected with a plasmid encoding the kinase domain of PAK4, PAK4 (A,C), or a kinase-inactive PAK4 kinase domain, PAK4M350 (B), and incubated for 3 hours in 0.2% FCS prior to stimulation for 20 hours with HGF (A,B) or further incubation in the absence of HGF (C). Cells were fixed and stained for HA-tagged PAK4 (A-C) and F-actin (D-F). Bar, 10 µm. (iii) Selected frames (time=0 or 4 hours) are shown from a time-lapse video recording (Movie 1) of MDCK cells expressing activated PAK4 (PAKGBD) (arrows) and stimulated at t=0 with HGF. Bar, 20 µm.