The Ska complex is required for the maintenance of biorientation during congression. (a) Example image sequence of a sister kinetochore pair labeled with eGFP-CENP-A congressing to the metaphase plate in a cell treated with control siRNA (left) or initiating congression toward the metaphase plate in a Ska1-depleted cell, but then rotating through 90° relative to the spindle axis (flipping; right). Red and yellow arrows, P and AP kinetochores; red and yellow stars, position of the kinetochore pair at t = 0 (control) and preflip (Ska1 siRNA); pink stars, postflip position of the Ska1-depleted kinetochore pair; dotted blue line, metaphase plate periphery. Bar, 2 µm. (b) Quantification of unaligned bioriented kinetochore-pair behavior in eGFP-CENP-A–expressing cells treated with control or Ska1 siRNA. Error bars ± SD; n (control) = 157 KT from 90 cells; n (Ska1) = 114 KT from 67 cells. (c, left) Quantification of postflip kinetochore behavior in cells treated with Ska1 siRNA. Error bars ± SD, n = 114 KT from 67 cells. (right) Quantification of postflip kinetochore P-movement velocity in cells treated with Ska1 siRNA. (d, left) Proportion of unaligned sister-pairs oriented along their local microtubule path that are bioriented in cells treated with control or Ska1 siRNA. Error bars ± SD; n (control) = 93 KT from 59 cells; n (Ska1) = 138 KT from 82 cells. (right) Images of a prometaphase HeLa cell expressing eGFP-CENP-A depleted of Ska1 and stained with DAPI and an antibody against α-tubulin. Zoom boxes depict an unaligned kinetochore pair that is bioriented. Bars: (left) 5 µm; (right) 1 µm.