Figure 6.

Dynamics of endosomal clustering and vacuole formation. (A-C) HeLa cells transiently transfected with pEGFP-Rab7 were incubated with VacA for 20 h, resulting in perinuclear aggregation of Rab7-positive vesicles. The medium overlying VacA-treated cells then was supplemented with 5 mM ammonium chloride. The formation of EGFPRab7-labeled vacuoles is shown at 15 min (A), 30 min (B), and 1 h (C) after addition of ammonium chloride. (D-F) Cells transfected with pEGFP-Rab7 were treated with 5 μg/ml acid-activated VacA in the absence of supplemental ammonium chloride. Intracellular localization of EGFP-Rab7 was analyzed before VacA treatment (D), at 3 h (E), and 8 h (F) after addition of VacA. (G-I) EGFP-Rab7-expressing cells were incubated with VacA for 3 h, resulting in formation of cytoplasmic clusters of Rab7-positive vesicles similar to E. The medium then was supplemented with 5 mM ammonium chloride. The formation of EGFP-Rab7-labeled, progressively enlarging vacuoles is shown at 15 min (G), 30 min (H), and 1 h (I) after addition of ammonium chloride. J, three-dimensional reconstructed image of H. Insets are magnified to show the development of vacuoles from the vesicle clusters, and the attachment of small vesicles to the nascent enlarging vacuoles within the clusters. Bar, 10 μm.