Figure 3

LRP10 colocalizes with APP and modulates its intracellular distribution. HeLa cells were transfected with GFP-APP and control vector pcDNA3 (A, D), wild-type LRP10-HA, (LRP10^wt-HA; B, E), or HA-tagged-LRP10 in which two DXXLL motifs (that bind the clathrin adaptors GGAs and AP1/2) in the cytoplasmic tail were mutated (LRP10^2DXXAA-HA, C, F). Cells were fixed, permeabilized, and immunostained with anti-GFP (green), anti-HA (red), and anti-TGN46 or EEA1 (blue) antibodies. The labeled cells were examined by confocal fluorescence microscopy. (A, D) In control pcDNA3 cells, APP-GFP was detected mainly in the juxtanuclear region and surrounding vesicles where it partially overlapped with TGN46 (A, inset, arrowheads) and EEA1 (D, inset, arrowheads). (B, E) LRP10^wt-HA was detected in the juxtanuclear region and surrounding vesicles and partially overlapped with TGN46 (B) and EEA1 (E). In these cells, GFP-tagged APP was detected mainly in the Golgi region, where it partially overlapped with LRP10^wt-HA (B). The merged images show a partial overlap between LRP10^wt-HA and GFP-APP in the Golgi cisternae labeled by TGN46 (B, inset, arrowheads) and surrounding endosomes labeled by EEA1 (E, inset, arrowheads). (C, F) HA-tagged LRP10^2DXXAA was redistributed to the plasma membrane (arrows) and peripheral early endosomes (F, inset). In these cells, GFP-APP was also detected on the plasma membrane (F, inset, arrow), Golgi (C, inset) and in peripheral endosomes labeled by EEA1 (F, inset), where it colocalized with LRP10^2DXXAA-HA (F, arrowheads, inset). Scale bar, 10 μm.