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Fig. 5. AICD derived from full-length APP translocates to the nucleus and generates AFT-complexes. (A) Cells expressing APP fused to a C-terminal HA tag (red) followed in tandem by citrine (yellow). As expected, full-length APP was localized in the ER/Golgi and in vesicles in the processes and somata, but not in nuclei. Staining of APP with antibodies directed against the C-terminal HA tag colocalized with citrine fluorescence in extranuclear localizations. Confocal xz-scans through nuclei confirmed the extranuclear localization of full-length APP. (B) Cells co-expressing full-length APP together with Fe65 and Tip60 formed nuclear AFT-complexes with spherical spot morphology. (C) In contrast to the extranuclear colocalization of HA antibody staining with citrine fluorescence, HA antibodies failed to detect the nuclear AFT-complexes that emitted strong citrine fluorescence signals. (D) FRET measurements showed close molecular association of APP-derived AICD with Tip60 in nuclear AFT-complexes. Antibodies against the C-terminal HA tag of APP, stained with Cy5 (blue), consistently failed to detect AICD in AFT-complexes. Bar, 10 µm in A to C, 20 µm in D upper row and 5 µm in D lower row.
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