APLP1 expression prevents localization of AICD to AFT complexes.

<p>(A) Confocal fluorescence images of HEK cells transfected with HA-Fe65, CFP-Tip60 and Cit-AL1ICD (top row) or Cit-AL2ICD (bottom row). Note the nuclear localization of Al1ICD to nuclear complexes. (B) Confocal fluorescence images of HEK cells transfected with HA-Fe65, Myc-Tip60 and cotransfected with APP-Cit (top row) APLP1-Cer (middle row) or both (bottom row). Note that AFT complex formation (arrowhead) was ablated in cells expressing APP as well as APLP1 (arrows). Scale bars represent 13 µm.</p

N-terminal residues of APP family ICDs are crucial for nuclear signaling capability.

<p>(A) Confocal fluorescence images of HEK cells transfected with HA-Fe65, CFP-Tip60 and cotransfected with APP-Cit or the indicated APP-Cit mutation constructs. Scale bar represents 13 µm. (B) Co-immunoprecipitation of SBP-tagged APP or APP(VML646LLR) together with HA-tagged Fe65 using Dynabeads. (C) Western blot analysis of HEK cells transfected with APP-Cit, APLP1-Cit or APP(VML646LLR)-Cit constructs after 24 hour treatment with the γ-secretase inhibitor DAPT. AICD-Cit transfected cell lysate was loaded to identify ICD bands and the membrane was probed with anti-GFP antibody.</p

APP and APLP2 have a higher protein turnover than APLP1.

<p>(A) Western blot analysis of HEK cells transfected with C-terminally HA-tagged APP/APLPs after indicated times of protein synthesis inhibition with cycloheximide (CHX). Western blots were probed with anti-HA antibody. Note the strong accumulation of APLP1 as compared to APP and APLP2. (B) APP/APLP full-length levels from A were normalized to α-tubulin. Mean ± SEM of n = 3 are shown for each time point. Data was fitted to exponential functions by the least square approach. R²_(APP) = 0.99; R²_(APLP1) = 0.82; R²_(APLP2) = 0.98.</p

ICDs derived from APP and APLP2, but not APLP1, form nuclear AFT complexes.

<p>(A) Confocal fluorescence images of HEK cells transfected with HA-Fe65, CFP-Tip60, and APP-Cit (top row), HA-Fe65, CFP-Tip60, and APLP1-Cit (middle row), HA-Fe65, CFP-Tip60, and APLP2-Cit (bottom row). (B) Confocal fluorescence images of N2a cells transfected with HA-Fe65, CFP-Tip60, and APP-Cit (top row), HA-Fe65, CFP-Tip60, and APLP1-Cit (middle row), HA-Fe65, CTP-Tip60, and APLP2-Cit (bottom row). Note the formation of spherical AFT complexes in the nucleus of cells transfected with APP or APLP2. In contrast expression of APLP1 resulted in accumulation of Fe65 and APLP1 in extranuclear compartments and at the plasma membrane, whereas Tip60 localized to nuclear speckles. Scale bars represent 13 µm.</p

Nuclear signaling capability of APP family members is mediated by the intracellular domain.

<p>(A) Schematic representation of wildtype and chimeric APP/APLP1 constructs. (B) Confocal fluorescence pictures of HEK cells cotransfected with HA-Fe65, CFP-Tip60 and the chimeric constructs APP-AL1ICD-Cit (top row) or APLP1-AICD-Cit (bottom row). Note that AFT complexes are formed in cells expressing APLP1-AICD but not APP-AL1ICD. Scale bar represents 13 µm. (C) Western blot analysis of HEK cells transfected with wildtype or chimeric APP/APLP1 constructs after 24-hour treatment with the γ-secretase inhibitor DAPT. Western blots were probed with anti-HA antibody and accumulation of CTFs was observed with all constructs. (D) Western blot analysis of HEK cells transfected with C-terminally HA-tagged APP/APLP after indicated times of protein synthesis inhibition with cycloheximide (CHX).</p

Nuclear localization of APP family ICDs is regulated by different proteasomal degradation rates.

<p>(A) Western blot analysis of HEK cells transfected with APP-Cit followed by 6 h treatment with indicated concentration of MG-132 or epoxomicin. APP-Cit transfected HEK cells treated with DMSO or DAPT and AICD-Cit transfected cells were loaded to identify CTFs and AICD bands. Membranes were probed with anti-GFP antibody. Note that MG-132 inhibits the proteasome and at higher concentrations also γ-secretase, whereas epoxomicin is a specific proteasome inhibitor. (B) Western blot analysis of HEK cells transfected with APP-Cit, APLP1-Cit or APP(VML646LLR)-Cit constructs followed by 6 hours of proteasome inhibition with epoxomicin or DMSO control treatment. The membrane was probed with anti-GFP antibody and GAPDH was used as a loading control. In the absence of proteasome inhibition ICDs generated from APP are clearly visible. (C) Quantification of ICD levels from B. Mean ± SEM of n = 3 are shown (p<0.05, t-test). (D) Confocal fluorescence images of HEK cells transfected with HA-Fe65, CFP-Tip60 and cotransfected with APP(VML646LLR)-Cit (upper rows) or chimeric APP-AL1ICD-Cit (bottom rows) mutation constructs with 6 hours epoxomicin or DMSO control treatment. Note that AFT complexes are formed after epoxomicin treatment. Scale bar represents 13 µm.</p

APP and APLP2 differ in their subcellular localization from APLP1.

<p>(A) Schematic presentation of APP/APLP-expression constructs with N-terminal 3 myc and C-terminal 3HA tags. The 3-myc tag is preceded by the APP signal peptide (SP) to ensure membrane insertion. (B) Confocal fluorescence images of HEK cells expressing APP (top row), APLP1 (middle row), or APLP2 (bottom row) and stained with anti-myc and anti-HA antibodies. APP and APLP2 showed a more prominent intracellular localization to vesicular structures, whereas APLP1 mostly localized to the cell membrane. (C) Confocal fluorescence images of HEK cells expressing APP (top row), APLP1 (middle row) or APLP2 (bottom row) after live antibody incubation. Incubation of cells with anti-myc antibody at 4°C for 10 minutes results in surface labeling, especially of APLP1. After 30 minutes of anti-myc antibody incubation, cell surface signals of APP and APLP2 reached a similar strength as APLP1. Cells were counter-stained with anti HA antibody after fixation. Scale bars represent 13 µm.</p

APP family members show high sequence homology.

<p>Schematic presentation of APP family ICD sequences and APP/APLP1 mutations. c.s.: cleavage site; b.s.: binding site.</p