Kinesin-8^Klp67A is required for stable kinetochore–MT attachment: bipolar spindle. (A and B) Time-lapse imaging of spindle MTs (magenta; SiR-tubulin staining), chromosomes (blue; H2B-mCherry), and GFP-Rod (green) after control or kinesin-8^Klp67A RNAi. The behavior of an initially unaligned chromosome (arrows) was dramatically different. (C) Increased frequency (event no. per cell per second) of MT detachment in the absence of kinesin-8^Klp67A (P < 0.001; Welch’s t test). Events were counted when MTs terminated end-on attachment. RNAi and imaging were performed three times, data were quantitatively analyzed twice, and the two datasets were combined. Error bars represent SEM. (D) Sensitivity to MT depolymerization. One or more chromosomes at the metaphase plate became unaligned upon colcemid treatment at much higher frequency in the absence of kinesin-8^Klp67A. Bars, 5 µm.