Bub3 and BubR1 localize on I-CreI– and laser-induced DNA breaks. (A) Bub3 localizes on the I-CreI–induced tether (also see Video 3). Time-lapse images of WT neuroblasts expressing H2A.Z::GFP and RFP::Bub3 after I-CreI induction. The yellow arrow and cyan arrowheads indicate the localization of RFP::Bub3 on kinetochores and tethers. (B) Bub3 does not localize to the kinetochore or the tether in bubR1¹ mutants. Time-lapse images of bubR1¹ mutant neuroblasts expressing H2A.Z::GFP and RFP::Bub3 after I-CreI induction. (C) BubR1 does not localize to the kinetochore or the tether in 56% and 78% of bub3¹ mutant cells (n = 9). Time-lapse images of bub3¹ mutant neuroblasts expressing H2A.Z::RFP and GFP::BubR1 after I-CreI induction. The white arrowheads point to the I-CreI–induced acentric chromatids. (D and E) Bub3 (D) and BubR1 (E) localize on laser-induced DNA damage during mitosis. Time-lapse images of neuroblasts expressing H2A.Z::RFP and GFP::Bub3, or GFP::BubR1, before and after laser ablation (also see Video 4). The yellow circles correspond to the zones of laser ablation. The cyan arrows point to the accumulation of Bub3 or BubR1 at the site of the chromosome damage. The yellow arrowheads indicate the laser-induced damage. Time (given in minutes/seconds) 0:00 corresponds to the first acquisition immediately after laser ablation. The cells are delineated with dotted lines. Bars, 10 µm.