Figure 4.

VacA endocytosis is an actin-dependent but clathrin- and dynamin 2-independent mechanism. (A) HeLa or AGS cells were treated with cytochalasin D for 60 min and then rinsed. VacA was added for 1 h at 4°C with FITC-TF. Cells were rinsed and incubated at 37°C for 30 min in the presence of cytochalasin D and then washed, fixed, permeabilized, processed for the detection of VacA, transferrin and actin (TRITC-phalloidin) by immunofluorescence, and observed by confocal microscopy. Small arrows in the zoom show that VacA (blue fluorescence) remained at the surface of cytochalasin D-depolymerized F-actin cells (red, short arrows) at difference with transferrin (green, long arrows). (B and C) HeLa and AGS cells were transfected with the dominant-negative form of Eps15 (ED95/295) or with the dominant-negative form of dynamin 2 (Dyn 2 K44A). VacA was added for 1 h at 4°C together with FITC-transferrin. Cells were rinsed and incubated at 37°C for 30 min, washed, fixed, permeabilized, processed for the detection of VacA and transferrin by immunofluorescence, and then observed by confocal microscopy. In the zooms, stars: transfected cells. All pictures were taken from two confocal sections (HeLa) or a total cell reconstruction from confocal sections (AGS). Scale bars, 10 μm.