FIG. 5.

Expression of RhoAV14 but not of Rac1V12 prevents ExoT-mediated actin cytoskeleton disassembly and cell rounding. HeLa cells plated on coverslips were transfected with 1 μg of either pMyc-RhoAV14 (A and B) or pMyc-Rac1V12 (C and D) for 14 h prior to fixation; 1 μg of pHA-T was cotransfected in panels B and D. All samples were stained with anti-Myc MAb 9E10 (green) and anti-HA MAb 3F10 (red) to identify cells expressing RhoAV14 or Rac1V12 and ExoT, respectively. The actin cytoskeleton was visualized with Bodipy 650/655-conjugated phalloidin (blue). Cells were imaged with a Bio-Rad 1024 confocal microscope equipped with KrAg and HeNe lasers and fluorescein isothiocyanate, Texas red, and Cy5 filter sets using a 60× objective. Z sections were acquired sequentially and processed using NIH Image 1.62b software and were then merged and pseudocolored in Adobe Photoshop 5.0. Cells doubly transfected with ExoT and RhoAV14 (B), which appear yellow, maintained a spread-out shape and showed some stress fibers; cotransfection with Rac1V12, however, did not prevent cell rounding and complete stress fiber loss (yellow-stained cells, panel D).