Article Figures & Data
Figures
- Figure 1.
A, Color-coded images of the negative (FITC only) and positive (Cy3 anti-FITC) controls. The rainbow scale shows fluorescence lifetime as color; if molecules are closer to each other, donor fluorescence (FITC) lifetime is shorter and the color will be closer to red. The graphs show lifetime distribution collected for every pixel of the images; positive control shows a shift to the left. B, The Cy3 acceptor in one-half of the cell (positive control) was destroyed by photobleaching (outlined area), leading to dequenching of the FITC fluorescence intensity and a shift to a longer lifetime.
- Figure 2.
A, B, Confocal microscope images of the cells that were double immunostained with FITC-labeled PS1 (A), and Cy3-labeled APP (B) antibodies demonstrate predominantly perinuclear localization of the proteins. Scale bar, 10 μm.
- Figure 3.
FLIM analysis of the proximity between APP and PS1 molecules within the cell. A, Intensity image shows standard immunostaining pattern for PS1, similar to that shown in Figure 2 A. B, Color-coded FLIM image shows lifetimes, reflecting proximity between PS1 and APP. The cell regions showing closest proximity between PS1 and APP are in the distal compartments near the cell surface. Colorimetric scale shows fluorescence lifetime in picoseconds. C, D, Enlarged boxed areas from B. The FLIM image is superimposed onto a table with calculated average lifetimes for each pixel of the image. Scale bar, 0.2 μm.
- Figure 4.
Photobleach dequenching FRET between APP and PS1 demonstrates close proximity between C terminus of APP and loop region of PS1 (A). Cy3-labeled APP (emission, 568 nm) and FITC-labeled PS1 (emission, 488 nm) before and after photobleaching the acceptor (Cy3) in a selected area within the cell. DAPT does not prevent close association of APP with PS1 (B). Scale bar, 10 μm.
- Figure 5.
A–D, Confocal microscope images of wtPS1 (A, B) and aspartate mutant (D257A) PS1 (C, D) double stained with biotinylatedγ-secretase inhibitor WPE-III-31C-bi (A, C) and PS1 antibody (B, D). PS1 immunostaining colocalizes with Cy3 streptavidin-labeled WPE-III-31C-bi in the cells expressing wtPS1. WPE-III-31C-bi does not bind to aspartate mutant PS1 holoprotein. Scale bar, 20 μm.
- Figure 6.
Nicastrin is important for the association of APP with PS1–γ-secretase complex. A, Nct RNAi leads to a significant inhibition of nicastrin expression in the cells. B, A decrease in the FITC–PS1 lifetime in APP–PS1 double-immunostained cells is observed in mock-treated cells, indicating a close proximity between the two proteins. This association is eliminated by Nct RNAi treatment because the FITC lifetime becomes the same as in the PS1–FITC alone control.
Tables
Condition FRET donor FITC labeled FRET acceptor Cy3 labeled FITC lifetime (psec) mean ± SD wt or D257A PS1 (control) PS1 loop None 2600 ± 110 (n = 12) wt PS1 PS1 loop APP 770 2290 ± 216** (n = 27) wt PS1 PS1 loop APP 770 + DAPT 2416 ± 118* (n = 9) wt PS1 PS1 loop APP770 + WPE-111-31C 1972 ± 264** (n = 30) D257A PS1 PS1 loop APP770 2255 ± 264** (n = 26) The table shows summary data of the FLIM assay for PS1 loop-APP C-terminus proximity under baseline conditions and in the presence of manipulations to preclude APP γ-secretase cleavage. If there is no interaction, lifetimes on the order of ≈2600 psec are observed. If FRET is detected, a population with a statistically shorter lifetime (approximately < 2400 psec) is observed.
↵* p < 0.05,
↵** p < 0.01, compared with non-FRETing control.
Condition FRET donor FITC labeled FRET acceptor Cy3 labeled FITC lifetime (psec) mean ± SD wt PS1 PS1 NT (X81) None 2630 ± 82 (n = 9) wt PS1 PS1 loop WPE-III-31C-biotin 1815 ± 162* (n = 15) wt PS1 PS1 NT (X81) WPE-III-31C-biotin 2672 ± 44 (n = 14) wt PS1 PS1 CT WPE-III-31C-biotin 2220 ± 110* (n = 12) The degree of the fluorescence lifetime shortening indicates that the γ-secretase transition state analog WPE-III-31C is in the closest proximity to the PS1 loop region and far away from PS1 N terminus (n, indicates number of cells). NT, N terminus; CT, C terminus.
↵* p < 0.001 compared with non-FRETing control.
