WASH is required for exocytosis. (a) Parental AX2 (○) and wshA⁻ (•) cells were incubated with FITC-dextran, and uptake was measured fluorimetrically. The inset shows a longer time course of the same data. (b and c) Parental AX2 (○), wshA⁻ cells (•), and wshA⁻ cells expressing GFP-WASH (△) and GFP-WASHΔVCA (▴) were loaded with FITC-dextran for 2 h and then washed. Expulsion of dextran was measured fluorimetrically. (d) Cells were grown for five generations in medium with 20% dextran. wshA⁻ cells, but not the parental strain, grew large and accumulated multiple dense vesicles. Bars, 5 µm. (e) Cells were grown for five generations in medium with and without 20% dextran. wshA⁻ growth is greatly slowed by the presence of indigestible dextran. (f and g) Transmission electron micrographs of parental AX2 (f) and wshA⁻ (g) cells incubated overnight with BSA–colloidal gold and chased for 2 h. Cells were examined as described in Hagedorn et al. (2009). Arrowheads in g indicate vesicular structures containing gold particles. Bars, 2 µm. (h) C. neoformans–containing vesicles in macrophages are also coated in WASH. Cultured J774 cells (left) were allowed to phagocytose C. neoformans and were then fixed with 4% formaldehyde and stained with anti-WASH (middle) and phalloidin (right). Bar, 10 µm. (i) Impairment of C. neoformans exocytosis caused by WASHΔVCA expression. Cells were transfected with full-length WASH or WASHΔVCA, incubated with C. neoformans for 2 h, and then observed for 24 h. The difference is significant to P = 0.05 (Fisher’s exact test). Error bars in each case show the SD. n = 3 in each case in a–c and e.