Fig. 5. Clusters in endosomes cause a cholesterol storage defect. (A) Cells were transfected with myc-p25SS, incubated with anti-myc antibodies, as in Fig. 2B, and processed for electron microscopy to reveal the distribution of antibody-tagged p25SS clusters (arrows). The membrane of typical late endosomes is outlined by arrowheads. (B) HeLa cells were transfected with myc-p25SS and then treated with 0.5 µg/ml ASSP mutant of aerolysin for 16 hours at 37°C. The distribution of antibody-tagged p25SS was then compared to that of Lamp1, as in Fig. 4. The toxin mutant causes late endosome vacuolation. (C) Cells were transfected with myc-p25SS or myc-p23-TMCD4 and processed for indirect immunofluorescence using anti-myc antibodies, and filipin to reveal cholesterol distribution. The arrow points to cholesterol accumulation in a p25SS-transfected cell. Scale bars: 10 µm.