Figure 9.

The binding of VacA to the cell surface is regulated by Rac1 but not by Cdc42. (A) Association of VacA with Cdc42 T17N transfected cells. HeLa cells were incubated with VacA at 4°C for 1 h, washed, and incubated for 0 or 10 min at 37°C. Cells were fixed, permeabilized, processed for the detection of VacA and Cdc42 T17N immunofluorescences, and observed by confocal microscopy. At 0 min, VacA is associated with membrane extensions (arrowhead) in both control and transfected cells (stars). After 10 min at 37°C, VacA remained mostly associated with the membrane extensions (arrowhead) of Cdc42 T17N-transfected cells (stars) but was clearly internalized in the GEECs in nontransfected cells (arrow). The large pictures show a negative black and white total cell reconstruction. The small pictures show one medial confocal section of the corresponding fields of the large pictures for Cdc42 T17N and VacA fluorescences. (B) HeLa cells were transfected with either the dominant-negative form of Rac1 (Rac T17N), Cdc42 (Cdc42 T17N), or by the dominant-positive form of Rac1 (Rac Q61L). Cells were incubated with VacA for 1 h at 4°C. Cells were washed, fixed, and processed for immunofluorescence of actin, VacA, Rac1, or Cdc42. The pictures were taken from full cell reconstructions using confocal sections. The histogram represents the quantification of VacA fluorescence associated with the whole cell surface. For this purpose cells were reconstructed totally using confocal sections. Each cell area was determined using the fluorescence pattern of F-actin. This area was used to delimit the VacA fluorescence at the surface of each cell. The total fluorescence level of VacA was quantified. The results are expressed as a difference between VacA fluorescence level on transfected cells and that of surrounded untransfected cells (results are express in %, untransfected cells being taken as the 100% for each transfected construction). Quantifications were performed using the Metamorph software. Results represent the account average of three independent experiences. Scale bars, 10 μm.