FIG 6.

Map targeting of mitochondria is essential for ADAM10-EGFR-MAPK activation. (A) Schematic presentation of Map_wt and the Map_ΔMTS-EspH_1–25 and Map_Δ101–152 mutants used in this study. (B) Map translocation. HeLa cells were infected with the indicated EPEC strains and the translocation assay was applied to evaluate Map translocation into the host cells, as described in Materials and Methods. (C) Map localization to mitochondria. HeLa cells were infected with the indicated EPEC strains, and cells were immunostained with anti-HA (Map) and anti-Hsp60 (mitochondria) antibodies. Cells were also stained with DAPI for visualizing the DNA of the host nucleus and bacteria, respectively. Cells were then analyzed by confocal microscopy, and representative images are shown. Arrows indicate infecting EPEC microcolonies. Bar = 5 μm. The degree of Map-mitochondrion colocalization was determined as described in Materials and Methods and the caption of Fig. S1I. (D and E) Effects of translocated Map on pERK and pEGFR levels. HeLa cells were infected with the indicated EPEC strains and the levels of pERK and pEGFR were determined, as before. Results are means and SE from 3 independent experiments. (F) Effects of translocated Map on ADAM10 sheddase activity. HeLa cells transfected with the BTC-AP-encoding plasmid were infected with the indicated EPEC strains and the level of ADAM10 sheddase activity was determined, as before. Results are means and SE from 3 independent experiments. ***, P < 0.0005; **, P < 0.005; *, P < 0.05; ns, nonsignificant (P > 0.05).