WASH and actin polymerization are essential for vesicle neutralization. (a) Vesicle neutralization assay. Cells were loaded with FITC- and TRITC-dextran to distinguish between acidic and neutral vesicles. Neutral postlysosomes (arrows) are absent from WASH-null cells and are rescued by expression of GFP-WASH but not GFP-WASHΔVCA (GFP is not seen under these conditions). (b) Latrunculin A completely but reversibly blocks the neutralization of vesicles. AX2 cells were loaded with a pulse of FITC-dextran, and the appearance of neutral vesicles was monitored in the presence or absence of 10 µg/ml latrunculin A. The figure shows measurements of Videos 1 and 2. The representative curves are from an experiment that was performed at least five times.