Figure 1

Effect of the overexpression of WT and cortactin mutants on pedestal formation and total actin content. (A) Schematic of cortactin domains and mutants under investigation. (B) Cortactin mutants block pedestal formation. Immunofluorescence images of HeLa cells transfected with WT (FL) and cortactin mutants and infected with EPEC for three hours. Mutants used are: Arp2/3 activation mutant (W22A), Erk-phosphorylation-mimicking mutant S405,418D (SD), Erk non-phosphorylatable mutant S405,418A (2A), Src-phosphorylation-mimicking mutant Y421,466,482D (3D), Src non-phosphorylatable mutant Y421,466,482F (3F) and SH3 domain mutant (W525K). GFP staining is shown in green, F-actin is in red, and bacteria and nuclei are in blue. The last column shows merged images of GFP and actin staining. Pictures are at 600× magnification. Scale bar 10 μm. (C) Quantification of pedestal number, cortactin localization and total F-actin content of transfectants. Black bars represent percentages normalized to WT of pedestal formed after 3 hours of infection of HeLa cells expressing GFP-FL and cortactin mutants. White bars represent normalized percentages of localization to pedestals. Grey bars represent the mean fluorescence of total actin content determined by flow cytometry. Experiments were performed at least three times with similar results. (D) Total actin content of HeLa cells transfected with WT and cortactin mutants. Histogram charts of HeLa cells expressing WT and cortactin mutants. Cells were permeabilized and stained with TRITC-phalloidin and total F-actin content was analyzed by flow cytometry. Overlaid histograms show the actin content of cortactin/mutants with respect to GFP transfected control cells (green curve). Pretreatment with cytochalasin D (blue curve) of cells expressing WT cortactin is shown as a control.