DNA breaks do not induce neokinetochore formation. (A–D and F) Time-lapse images of neuroblasts expressing H2A.Z::RFP and Spc105::GFP (A), CenpC::GFP (B), GFP::Nuf2 (C), Mad1::GFP (D), and GFP::Mps1 (F) after I-CreI expression. The white arrowheads point to the I-CreI–induced acentric chromatids. Yellow arrows indicate the localization of the GFP-labeled proteins at the kinetochore. (E) Mad1 is not required for BubR1 localization on broken chromatids. Time-lapse images of a mad1¹ mutant neuroblast expressing I-CreI and labeled with H2A.Z::RFP and GFP::BubR1. The white arrowhead indicates I-CreI–induced acentric chromatids. The yellow arrow and cyan arrowheads indicate the localization of GFP::BubR1 at the kinetochore and tether. (G) Mps1 is not required for BubR1 localization on broken chromatids. Time-lapse images of mps1¹ mutant neuroblast labeled with GFP::BubR1 before and after laser ablation. The yellow circle corresponds to the zone of laser ablation. The cyan arrowheads indicate the appearance of GFP::BubR1 at the site of chromosome damage. Time (given in minutes/seconds) 0:00 corresponds to the first acquisition immediately after laser ablation. (H and I) BubR1 localization on DNA breaks does not require Polo-dependent phosphorylation of the BubR1 KARD motif. Time-lapse images of neuroblasts expressing I-CreI labeled with H2A.Z::RFP. The neuroblasts express either the GFP::BubR1-KARD-D mutant, where the putative Polo-dependent phosphorylation sites in the KARD motif are replaced by aspartate (H), or GFP::BubR1-KARD-A, where the same residues are mutated to alanine (I). The white arrowheads indicate the I-CreI–induced acentric chromatids. Yellow arrows and cyan arrowheads point to the localization of GFP::BubR1-KARD-D and GFP::BubR1-KARD-A on the kinetochore and tether. Cells are delineated with white dotted lines. Bars, 10 µm.