Structure of BubR1-KEN and BubR1-KD mutations and their effects on spindle function. (A) Alignment of the N-terminal KEN box (left) and the kinase domain (right), indicating in red the residues altered in bubR1-KEN and the conserved lysine altered in bubR1-KD. (B–E) Thin K-fibers and unstable spindle length in bubR1-KD cells. (B) WT neuroblast in mitosis. Within a few minutes of NEB, it has established a stable spindle, developed robust K-fibers, and aligned chromosomes on the metaphase plate. Anaphase (ana) occurs at ∼7 min after NEB. See Video 1. (C–E) Three different bubR1-KD neuroblasts displaying prolonged prometaphase. Chromosomes have difficulty congressing and remaining at the metaphase plate. The spindles appear diffuse, the K-fibers are poorly defined, and spindle length varies (compare the second and third frames of each series). Despite this aberrant behavior, each cell eventually enters anaphase, and the chromosomes segregate normally. See Fig. S1 and Videos 2 and 3. Videos in B–E are wide-field microscopy images of GFP-tubulin and mRFP1-Rod. (F) A bubR1-KEN mutant neuroblast with normal spindle morphology and dynamics. See Video 5. Spinning disk confocal microscope images of GFP-tubulin and mRFP1–BubR1-KEN marking kinetochores (which become difficult to detect as anaphase approaches) are shown. Bar, 5 µm.