Figure 1.
AFT complex formation and the BiFC principle.
(A) Spherical nuclear AFT complexes in HEK293 cells cotransfected with APP-Citrine, CFP-Tip60 and HA-Fe65 (arrow). Cells that lack CFP-Tip60 accumulate neither AICD-Citrine nor HA-Fe65 in the nucleus (arrowhead). Nuclei were counterstained with DAPI. Length of bar: 20 µm. (B) Schematic depiction of the BiFC-based AFT complex detection system, where APP and Tip60 are fused to YFP halves. Since Fe65 serves as an adaptor between APP and Tip60, fluorescence complementation only occurs in the presence of all three proteins. (C) Fluorescence maturation in AFT-BiFC. Fluorescence maturation at 30°C was allowed for increasing time periods before FC quantification (n=3 per timepoint, error bars represent SEM). If samples were maintained at 37°C, no maturation occurred (samples labeled 37°C).
Figure 2.
Subcellular localization of BiFC signals.
HEK293 cells were imaged by confocal microscopy. (A) Cotransfection of bFos-YC and bJun-YN generates a BiFC signal distributed throughout the cell nucleus. (B) Cotransfection of APP-YC, HA-Fe65 and Tip 60-YN results in multiple spherical nuclear BiFC signals from AFT complexes. (C) In some cells, cotransfection also results in formation of extranuclear BiFC signals (arrow), which is consistent with an interaction of Tip60 and AICD/Fe65 also outside the nucleus as previously reported by us [8]. (D) Cotransfection with APP-YC and APP-YN results in perinuclear fluorescence (E+F). Colocalization of the APP/APP-BiFC signal with calnexin and TGN46 is consistent with the localization of APP/APP-dimers in the ER/Golgi. Nuclei were counterstained with DAPI. Length of bar: 13 µm (A, C, E, F) or 10 µm (B, D).
Figure 3.
AFT-BiFC requires the presence of Fe65.
(A) After cotransfection of APP-YC, Tip 60-YN and HA-Fe65 the BiFC signal can be detected in a subset of cells in the confocal microscope. In contrast, cotransfection of APP-YC and Tip 60-YN alone does not lead to fluorescence complementation, which indicates absence of direct interaction between AICD and Tip60. Transfection of APP-YC and Tip 60-YN together with the AICD-binding protein MINT1/X11 that traps AICD in the cytosol also does not generate a BiFC signal. Lower panels show BiFC overlay with DAPI nuclear staining. Length of bar: 60 µm. (B) Representative BiFC-FC scatter plots of individual samples. Percentages refer to gated cells. Fluorescence intensity and forward scatter are depicted in arbitrary units. (C) BiFC-FC quantification of HEK293 cells cotransfected with APP-YC and Tip 60-YN together with or without HA-Fe65 or with MINT1/X11 (n=6 vs. 5 vs. 6, error bars represent SEM, *** p<0.005, U-test). The approximately 1% BiFC-positive cells in the APP-Tip60 and APP-X11-Tip60 conditions are most likely due to background autofluorescence of HEK cells as well as fluorescence complementation mediated by endogenous Fe65.
Figure 4.
Quantitative AFT-BiFC applications.
BiFC-FC quantification of HEK293 cells cotransfected with HA-Fe65, Tip 60-YN and different APP-YC constructs. (A) Swedish APP-YC increases nuclear AFT complex formation compared to wildtype APP (n=23 vs. 21 from two independent experiments, FC counts corrected for APP/βActin). (B) Mutation of tyrosine 682 to phenylalanine results in more AICD nuclear signaling (n=23 vs. 22 from two independent experiments, FC counts corrected for APP/βActin). (C) Oxidative challenge with 50 µM H2O2 does not affect nuclear AFT complex formation. Data were analyzed with or without correction for APP/GAPDH expression levels to correct for putative confounding toxic effects of H2O2 (n=22 vs. n=22 from two independent experiments). (C’) Representative Western blot of BiFC samples showing similar APP and GAPDH levels in both conditions indicating no toxic effect of H2O2 at this concentration. (D) Oxidative challenge with 200 µM H2O2 decreases nuclear AFT complex formation with or without correction for APP/GAPDH levels (n=20 vs. 20 from two independent experiments). (D’) Representative Western blot of BiFC samples showing decreased APP and GAPDH levels in the 200 µM H2O2 condition indicative of oxidative stress-induced cell death. For (A–D) Data were pooled from two independent experiments by setting the mean of wildtype APP-YC to 100%. Lipofectamine treatment alone (0.5% fluorescent cells) was used for gating. Error bars represent SEM, * p<0.05, *** p<0.001 (A, D) or p<0.005 (B), n.s. non-significant, t-test.