Fig. 4. HDLp colocalizes with LDL in early endocytic vesicles. CHO(iLR) cells were allowed to simultaneously internalize OG-HDLp (A) and DiI-LDL (B) in incubation medium for 30 minutes at 18°C. Fixed cells were analyzed using confocal laser microscopy to visualize the colocalization of the ligands in endosomes by overlaying the two images (C). Overlapping fluorescently labeled endosomes stain yellow after merging the layers. The HDLp-positive juxtanuclear compartment is depleted of LDL. CHO(iLR) cells were simultaneously preincubated with OG-HDLp and DiI-LDL. After the preincubation, the cells were transferred to an aluminium chamber and incubated in chase medium at 37°C. At 10 minutes, large amounts of HDLp concentrated in the juxtanuclear region (D), whereas LDL remained spatially distributed throughout the entire cell interior (F). Within a defined area (squares in D and F), the relative fluorescent intensity of the juxtanuclear-positioned structure was plotted on a relative scale (from 0 to 255, indicated by the vertical bar) for OG-HDLp (E) and DiI-LDL (G). Internalized HDLp accumulates in a non-lysosomal juxtanuclear compartment. CHO cells stably expressing iLR were preincubated with OG-HDLp, rinsed in HEPES buffer and mounted in an aluminium chamber. The cells were subsequently incubated at 37°C in chase medium that was supplemented with LT. Images were generated with multicolour imaging, using confocal laser microscopy to spatially visualize internalized HDLp and LT, simultaneously, in living cells. After a chase of 15 minutes, OG-HDLp-positive endocytic vesicles were highly concentrated in the juxtanuclear region (H), which was depleted of LT (I). Partial colocalization with LT was visualized by merging the two images (J). To enhance the visibility of the spatial distribution of HDLp and LT, a bright-field image of the observed cells was overlayed with fluorescent images. Additionally, detailed images of a single juxtanuclear structure were taken to visualize the minimal colocalization (K,L,M). Intracellular transport of ligands by iLR is microtubule-dependent. CHO(iLR) cells were preincubated with fluorescently-labeled ligand in the presence of 5 µM nocodazole. The cells were subsequently incubated for an additional 30 minutes at 37°C in chase medium supplemented with 5 µM nocodazole. Fixed cells were observed with confocal laser microscopy and showed a peripheral localization of vesicles that contained LDL (N), HDLp (O) or RAP (P). Bars, 20 µm.