Fig. 6. Cholesterol accumulation and motility inhibition. (A) Cells were transfected with mycp25SS, incubated with anti-myc antibodies, as in Fig. 2B, and then processed for triple-channel immunofluorescence. The distribution of antibody-tagged p25SS (red) was compared to that of Lamp1 (green) and cholesterol (blue, directly revealed with filipin) in a merged image. A high magnification view of the outlined area is shown in color for each compound, emphasizing the differences in the distribution of p25SS, Lamp1 and cholesterol. (B) Cells were transfected with CD63-GFP alone or together with myc-p25SS. Transfected cells were identified with anti-myc antibodies added to the living cells (not shown). The motility of late endosomes containing CD63-GFP was analyzed (Lebrand et al., 2002) by collecting images (200 mseconds exposure time) every second over a 25-second period. Then, all images were stacked. When represented in this manner, a moving object shifts position, thus creating a series of overlapping or closely associated spots that reveals its track (left panels), as in the control cells. Initial and final positions were color-coded after electronic conversion of the first and last pictures in the sequence to red and green, respectively, so that a moving object appears both red and green, and an immobile object yellow (middle panels). Examples of the traces of individual elements are shown, and highlighted by boxes in the right panels. (C) In cells co-transfected with CD63GFP and p25SS, as in B, the direct distances between initial and final positions of CD63GFP-labeled vesicles were quantified after 25 seconds (and not the actual trajectory followed by vesicles), as indicated (700 vesicles were analyzed and standard deviations are shown). Scale bars: (A,B) 10 µm.