Figure 5.

NPC1-FP organelles participate in late events in the endocytic pathway. Pulse-chase experiments at 37°C with DiI LDL revealed the kinetics of entry into the NPC1-containing compartment. CT60 cells transiently transfected with NPC1-GFP or VAMP7-GFP were labeled with DiI-LDL (30 μM) for 3 min and then fixed following a 3-, 10-, 30-, and 120-min chase period. The fraction of all DiI-labeled structures that colocalized with NPC1-GFP, VAMP7-GFP, or only the enlarged VAMP7-GFP organelles was quantified for 10 cells at each time point (A). DiI shows significant colocalization with NPC1-GFP or VAMP7-GFP at 10 min and later time points, indicative of NPC1-FP being present in late endosomes and the terminal endocytic compartment. The enlarged VAMP-GFP organelles colocalize with DiI beginning at 30 min, but the bulk of the DiI has not reached these organelles until 2 h (C). In living cells, the fraction of all DiI-labeled structures engaged in vectorial movement (displacement >1.5 μm) was quantified at 10, 30, and 120 min (B). Similar amounts of movement were seen early in the endocytic pathway (10 and 30 min) regardless of the presence of NPC1. Following these normal early movements, loss of NPC1 prevents further DiI movement (2 h) and results in the sequestration of DiI in the cholesterol-laden organelles. Bar, 5 μm.