Fig. 5. Ezrin recruitment is responsible for actin polymerization. (A) 2 hours prior to infection, HUVECs were microinjected with pCB6-Nter-Ezrin-VSVG, encoding the VSVG-tagged truncated N-terminal domain of ezrin, as a dominant-negative form of ezrin. Cells were infected with the ROU strain of N. meningitidis for 4 hours and then labeled for F-actin, endogenous ezrin and VSVG-tag (Nter-ezrin). In the transfected cells a large amount of VSVG-labeled truncated ezrin was recruited below the bacterial colonies (top right) without actin polymerization (top left). Endogenous ezrin (bottom right) was recruited underneath bacterial colonies in non-transfected cells. Transfection with the dominant-negative form almost completely prevented the recruitment of endogenous ezrin. (B) Bacterial colonies with polymerized actin and/or recruited endogenous ezrin were counted by immunofluorescence analysis in non-transfected cells or cells transfected with a plasmid encoding the VSVG-tagged truncated amino-terminal domain of ezrin (pCB6-ezrin-Nter). These data correspond to the results of at least six independent experiments. Results are expressed in percent of adherent colonies. (C) Bacterial colonies with polymerized actin were counted by immunofluorescence analysis in HUVECs that were transfected with a plasmid encoding full length ezrin (pCB6-ezrin) or the VSVG-tagged truncated amino-terminal domain of ezrin (pCB6-ezrin-Nter). Results are expressed in percent of adherent colonies.