Fig. 5. VirA regulates MT stabilization and membrane ruffling. (A) COS-7 cells were transfected with plasmids encoding GFP–VirA (FL), GFP–VirA-N224 (N224) or vector alone (Vector). Actin (red in MT + Actin) and MT (green in MT + Actin) were visualized. The transfected cells were identified by GFP. COS-7 cells overexpressing VirA showed marked changes in cell shape with membrane ruffles around the periphery and, in addition, the MT networks, with which GFP–VirA was associated, were destabilized. Bar, 10 µm. The percentage of transfected COS-7 cells overexpressing GFP–VirA (FL), GFP–VirA-N315 (N315), GFP–VirA-N224 (N224) or GFP (Vector) showing marked changes in cell shape with membrane ruffles was calculated (graph). ‘n’ is the total number of cells examined in three independent experiments. (B) HeLa cells were transfected with the plasmid encoding GFP–VirA (FL), GFP–VirA-N224 (N224) or the vector alone (Vector). Actin (red in MT + Actin) and MT (green in MT + Actin) were visualized. The transfected cells were identified by GFP. HeLa cells overexpressing VirA showed membrane ruffles (arrowheads). The MT network associated with GFP–VirA beneath the ruffles had undergone alterations (arrow). Bar, 10 µm. The percentage of transfected HeLa cells overexpressing GFP–VirA (FL), GFP–VirA-N224 (N224) or GFP (Vector) showing membrane ruffles was calculated (graph). ‘n’ is the total number of cells examined in three independent experiments. (C) HeLa cells were microinjected (arrow) with VirA and observed by time-lapse video microscopy. Microinjection of VirA into HeLa cells led to membrane ruffling at the point of injection. The time elapsed after microinjection is shown in minutes:seconds. Arrowheads indicate membrane ruffles. Bar, 10 µm.