Figure 1
β-Cleaved carboxy-terminal fragment of APP (βCTF) activates rab5 on endosomes and increases endosome size. (a) Rab5 activation, reflected by the rate of fluorescence recovery after photobleaching (FRAP), is measured as the rate at which photobleached GTP-bound rab5 (activated rab5) on individual endosomes is replaced by fluorescent cytosolic GDP-rab5 (inactive rab5). The rate of FRAP for endosomal GFP-rab5 is significantly decreased when βCTF levels are increased by overexpressing wild-type APP (APPwt) in N2a cells (N2a APPwt) or blocking βCTF cleavage with a 10 μm γ-secretase inhibitor L685 458 (N2a+L685), as compared with control cells expressing βCTF at endogenous levels (N2a) or transfected with an APP mutant construct unable to be cleaved to βCTF (N2a APPmv). The FRAP rate after transfecting a dominant-active mutant GFP-rab5 Q79L25 as a positive control is extremely reduced, indicating persistent rab5 activation. The summary graph reflects by the percent of average fluorescence recovery at 270 s after photobleaching in each condition (n=20 endosomes, one endosome per cell, total 20 cells, mean±s.e.m., one-way analysis of variance (ANOVA), Tukey’s test, ***P<0.001). (b) βCTF activates rab5 in human embryonic kidney 293 (HEK293) cells. APPwt overexpression increases the levels of active rab5 (GTP-bound rab5) detected with a GTP-rab5-specific antibody (first lane), whereas APPmv mutant has no effect (second lane), as compared with untransfected control (ctrl) cells (third lane). A bar graph presents mean immunoreactive GTP-rab5 signal±s.e.m. for three separate immunoblot experiments (representative blot shown) (**P<0.01, one-way ANOVA, Tukey’s test). Ten percent of cell lysates is used as total rab5. (c) βCTF induces endosomal enlargement in N2a cells. Cross-sectional area of rab5-positive endosomes is increased by APPwt overexpression or 10μM L685 458 (lanes 2 and 3), which raise αCTF and βCTF levels compared with those in ctrl cells (lane 1). APPmv mutant expression elevates αCTF levels but not βCTF levels (lane 4) and does not enlarge endosomes. Each graph bar indicates the number of measured endosomes from 20 cells (*P<0.05 and **P<0.01, respectively, mean±s.e.m., one-way ANOVA, Tukey’s test).
