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First published online 26 June 2003
doi: 10.1242/jcs.00643

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Demonstration by FRET of BACE interaction with the amyloid precursor protein at the cell surface and in early endosomes

Alzheimer Disease Research Laboratory, Massachusetts General Hospital, Harvard Medical School, Charlestown, MA 02129, USA

View larger version (23K): [in a new window]   Fig. 1. Reagents used in this study. This scheme shows the constructs and antibodies used in this study. Membrane-spanning regions are shown in black. All APP constructs are tagged at the C-terminus with myc, V5 or EGFP; wild-type APP770, APP with Swedish mutant, APP delta49 which lacks the entire cytoplasmic domain. BACE constructs are either N-terminally tagged (with myc) or C-terminally tagged with V5. For labeling the ectodomain regions of APP and BACE, 8E5 and anti-myc antibody are used, respectively. For amyloid-ß ELISA, biotinylated 6E10 and BA27 are used to capture amyloid-ß.

View larger version (131K): [in a new window]   Fig. 2. Localization of APP and BACE on the cell surface and in the endocytic pathway. APP and BACE-co-transfected H4 cells were labeled by antibodies against extracellular regions (8E5 for APP, anti-myc for BACE) as described in the Materials and Methods section, and allowed to undergo endocytosis. They were fixed and permeabilized after 0, 15, 30 or 60 minutes, and the secondary antibodies were applied to detect the localization of the primary antibodies. At time 0 minutes, only the cell surface was labeled. Localization of APP and BACE are found in nearly identical clusters on the cell surface as shown in the bottom-right panel (APP in red, BACE in green). At 15-30 minutes, most signal was found in the endosomes, showing complete co-localization of APP and BACE signals. However, at 60 minutes, APP tended to localize in lysosomes, whereas BACE did not. Bar, 20 µm.

View larger version (130K): [in a new window]   Fig. 3. Triple immunostaining of APP, BACE and endosomal (EEA1) (A)/lysosomal (Lamp-1) (B) markers. (A) Co-localization of APP and BACE in endosomes at 15 minutes. At 15 minutes, most signal was found in the endosomes (confirmed by EEA1 staining), showing complete co-localization of APP and BACE. (B) Distinct localization of APP and BACE at 60 minutes. APP signal was found mostly in lysosomes (confirmed by Lamp1), however BACE signal was not. Bar, 20 µm.

View larger version (58K): [in a new window]   Fig. 4. Example of FRET reflecting the interaction between APP and BACE in the endosomes. (A-D) Example of FRET between APP-BACE in the endosomes. H4 cells were co-transfected with BACE-V5 (A,B) and APP-myc (C,D), immunostained with anti-APP antibody (labeled by Cy3) and anti-BACE antibody (labeled by FITC), both of which are directed against ectodomains. After labeling on ice, cells were incubated at 37°C for 15 minutes. Most of the APP signal was found in endosomes (confirmed by anti-EEA antibody), and co-localized with the BACE signal. Photobleaching of the Cy3 label of APP in the endosomes (D, boxed area) led to a marked increase in the BACE fluorescent signal within the photobleached area, demonstrating FRET (B, boxed area). Bar, 10 µm. (E) FRET ratio increases of FlD2/FlD1 after photobleaching between APP and BACE in the secretory pathway, cell surface and endocytic pathway are shown. FRET measurements are shown for ectodomain interactions at 0 minutes after labeling (cell surface), 15-30 minutes after labeling (endosomes) and 60 minutes after labeling (lysosomes). FRET increases on the cell surface and in the endosomes were significantly above 1.0 (P<0.0001). FRET was not detected in the lysosomes.

View larger version (16K): [in a new window]   Fig. 5. Detection of amyloid-ß peptide during endocytosis by ELISA. H4 cells transiently transfected with various APP constructs were pulse-labeled with biotinylated-6E10 and chased for the indicated time points in the absence or presence of DAPT for 4 hours. The conditioned media was collected and the biotinylated-6E10-labeled amyloid-ß peptide was measured by ELISA using BA27 as the capture antibody and streptavidin-HRP for detecting labeled 6E10. (A) The time-course of amyloid-ß detected from the conditioned media of wild-type APP770 and APP770sw-transfected H4 cells is illustrated. Data shown are typical of 3 separate experiments. (B) Comparison of the amyloid-ß levels detected from various conditions (wt, Swedish, delta49, DAPT) for the first 30 minutes. A significant difference (P<0.001) in the amount of amyloid-ß levels compared to that of the wild-type is shown by asterisk.