Supplemental Materials

This article contains the following supporting material:

• Video 1 - 360° rotation of a 3D-reconstructed HeLa cell (from videomicroscope acquisitions). VacA is associated to cell surface membrane domain above F-actin. HeLa cells were processed as described in Figure 1 A. The top left rotation presents the VacA indirect fluorescence in black and white. The bottom left rotation shows the F-actin fluorescence in black and white. The top right rotation presents the merge between VacA (red fluorescence) and actin (green fluorescence). Each frame represents a clockwise rotation of 5° (72 total frames). The rotation is presented at 10 frames per second.
• Video 2 - 360° rotation of a 3D-reconstructed AGS cell (from videomicroscope acquisitions). VacA is associated to cell surface membrane domains above F-actin. AGS cells were processed as described in Figure 1 B. The top left rotation presents the VacA indirect immunofluorescence in black and white. The bottom left rotation shows F-actin fluorescence in black and white. The top right rotation presents the merge between VacA (red fluorescence) and actin (green fluorescence). Each frame represents a clockwise rotation of 5° (72 total frames). The rotation is presented at 10 frames per second.
• Video 3 - 360° rotation of the 3D-reconstructed part of HeLa cell presented in Figure 2 zoom 3 (from confocal sections). HeLa cell was processed as described in Figure 2. The rotation presents the merge between VacA (red fluorescence) and actin (green fluorescence). Each frame represents a clockwise rotation of 10° (36 total frames). The rotation is presented at 6 frames per second.
• Video 4 - 360° rotation of the 3D-reconstructed part of HeLa cell presented in Figure 2 insert A (from confocal sections). The preparation was processed as described in Figure 2. The rotation presents an enlargement of a typical tubulo-vesicular endocytic structure labeled with VacA (black and white) after 10 min of endocytosis. Each frame represents a clockwise rotation of 10° (36 total frames). The rotation is presented at 6 frames per second.
• Video 5 - 360° rotation of a 3D-reconstructed AGS cell (from videomicroscope acquisitions). VacA is associated to the cell peripheral early intracellular compartment reached by the cytotoxin upon 10 min of transfer of cells to 37°C. VacA is in red and F-actin in green (AGS cells were processed as described in Figure 3). Each frame represents a clockwise rotation of 5° (72 total frames). The rotation is presented at 10 frames per second.
• Video 6 - 360° rotation of a 3D-reconstructed AGS cell (from confocal sections). Upon 10 min of pinocytosis, VacA is associated with the early peripheral intracellular compartment devoid of EEA1 labelling. VacA is in red and EEA1 in blue (AGS cells were processed as described in Figure 5). Each frame represents a clockwise rotation of 10° (36 total frames). The rotation is presented at 6 frames per second.
• Video 7 - 360° rotation of the 3D-reconstructed part of HeLa cell presented in Sup. Figure 1 zoom 1 (from confocal sections). VacA is associated with the early peripheral intracellular compartment devoid of GFP-caveolin 1 labelling. VacA is in red and caveolin 1 in green (HeLa cells were processed as described in Figure Sup. 1). Each frame represents a clockwise rotation of 10° (36 total frames). The rotation is presented at 6 frames per second.
• Video 8 - 360° rotation of the 3D-reconstructed part of AGS cell presented in Sup. Figure 1 zoom 2 (from confocal sections). VacA is associated with the early peripheral intracellular compartment devoid of GFP-caveolin 1 labelling. VacA is in red and caveolin 1 in green (AGS cells were processed as described in Figure Sup. 1). Each frame represents a clockwise rotation of 10° (36 total frames). The rotation is presented at 6 frames per second.
• Supplemental Figure 1 - The VacA rabbit polyclonal antibody IgG 958 does not react by immunofluorescence with cells not treated with VacA, caveolin 1 does not colocalize with cell-bound or endocytosed (30 min) VacA and depletion of membrane cholesterol blocks VacA pinocytosis. HeLa cells (A) or AGS cells (B) were incubated or not with VacA at 4°C for 1 h, washed, fixed, and processed for the detection of VacA or caveolin 1 (shown only in HeLa cells) by confocal microscopy. For the detection of VacA, cells were incubated with the 958 IgG and then with the second fluorescent antibody as described in the Materials section. Caveolin 1 in HeLa cells (intoxicated or not with VacA) is clearly detectable as small dots in the cytoplasm (arrows) and slightly detectable at the level of the plasma membrane (arrowheads). No colocalization is observed between caveolin 1 and VacA in particular along the cell edges. Pictures were taken from full cell reconstructions using confocal sections. (C) HeLa cells were incubated with VacA as in A, but transfered in warm medium (37°C) for 30 min, fixed and processed for the detection of VacA and caveolin 1 (as in A) by confocal microscopy. No colocalization of VacA (arrowheads) and caveolin 1 (arrows) are observed. (D) Hela cells were serum-starved for 20 min, depleted or not of cholesterol by incubation with methyl-Β-cyclodextrin (10 mM for 30 min at 37°C) and depletion was control by monitoring the decrease of membrane fluorescence using the cholesterol-binding molecule filipin (data not shown). Cells were washed and incubated for 1 h with VacA and FITC-TF at 4°C. Cells were subsequently washed and incubated for 10 min at 37°C, fixed and processed for the detection of VacA and FITC-TF by confocal microscopy. For C and D, pictures were taken from single confocal sections. Scale bars: 10 μm.
• Supplemental Figure 2 - VacA is not colocalized with caveolin 1 in cells transfected with GFP-caveolin 1 or with endocytosed cholera toxin B subunit. (A) HeLa cells or AGS were transfected with GFP-caveolin 1. Cells were incubated at 4°C and VacA was added for 1 h at 4°C. Cells were washed, incubated with VacA in warm medium for 10 min and then processed for the detection of VacA (red) and GFP-caveolin 1 (green) by confocal microscopy. Pictures were taken from full cell reconstructions using confocal sections. In zooms, VacA and GFP-caveolin 1 fluorescences are in black and white and the merge pictures in color. (B) Same than in A excepted that HeLa cells were incubated with CTxB (0,2 μg/ml, red) together with VacA (blue) for 1h at 4°C. CTxB and caveolin 1 are colocalized (arrows) without VacA (arrowheads). Pictures were taken from a single confocal section. Scale bars: 10 μm.