Figure 3.

IFN-γ induced apical vacuoles are derived from the apical plasma membrane. Epithelial monolayers were incubated with 100 U/ml IFN-γ for 48 h. followed by fixation and double labeling for F-actin and either villin or syntaxin 3 and syntaxin 4. (A) In IFN-γ-treated T84 cells, reconstructed images in the x-z plane reveal colocalization of villin with apical F-actin-coated vacuoles (arrows) and accumulation of syntaxin 3 but not syntaxin 4 inside the vacuoles (arrows). (B) T84 cells were preincubated for 36 h with IFN-γ followed by selective biotinylation of either apical or basolateral plasma membranes. Cells were subsequent incubated in IFN-γ-containing medium for an additional 4 h. Note the presence of biotin label in the VACs (arrows) only if biotin was applied apically. (C) T84 cells were preincubated for 36 h with IFN-γ followed by either apical or basolateral application of 0.1% (wt/vol) tannic acid for additional 4 h. Note that selective inhibition of endocytosis from the apical plasma membrane prevents formation of F-actin-coated vacuoles (arrows), whereas inhibition of basolateral endocytosis is ineffective. Scale bar, 10 μm.