The APC/C synthetic substrate GFP::CycBNt::Bub3 is maintained on the tether during early anaphase in a BubR1 KEN box–dependent manner. (A) Scheme of the APC/C synthetic substrate GFP::CycBNt::Bub3. The N terminus (including the amino acids 1–246) of Cyclin B (CycB_1–246) was fused on its N terminus to GFP and on its C terminus to full-length Bub3. The CycBNt sequence was flanked with a 4× glycine–alanine linker (L). (B) Time-lapse images of WT and bubR1-KEN mutant cells after I-CreI induction labeled with H2A.Z::RFP and GFP::CycBNt::Bub3. The kinetochore and tether localization of GFP::CycNt::Bub3 are indicated with yellow arrows and cyan arrowheads, respectively. Bar, 10 µm. (C) Quantitative analysis of the disappearance of the GFP::CycBNt::Bub3 signal on kinetochores and tethers. The graph shows the fluorescence intensity of GFP signal on kinetochores and tethers over time (see Fig. S5 for raw data and Materials and methods section Image analysis for details on the quantification). The fluorescence intensities were normalized to the fluorescence intensity measured at the time point −1 min. The 0 of the x axis corresponds to anaphase onset as defined by the onset of sister chromatid separation. The signal was measured every 20 s. (D) Scatter dot plot showing the time of complete disappearance of the signal of GFP::CycBNt::Bub3 in WT and bubR1-KEN mutant cells expressing I-CreI. The time starts at anaphase onset. The black lines correspond to mean ± 95% confidence interval. A Mann-Whitney nonparametric test was used to calculate p-values (***, P < 0.001). A.U., arbitrary units.