Inter-KT hyperstretching caused by reduced kMT detachment rates can be rescued by simultaneous reduction of MT poleward flux. (A–D) Model simulation results of the distribution of inter-KT distances under different conditions (n = 4 KT pairs in all cases). (E–H) Experimentally determined distribution of the inter-KT distances under different conditions. Reduction of MT poleward flux was achieved by microinjection of anti-Kif2a antibodies (F, n = 12 KT pairs); reduction of kMT detachment rate was achieved by microinjection of anti-Hec1 antibodies (G, n = 7 KT pairs); simultaneous reduction of MT poleward flux and kMT detachment rate was achieved by coinjection of the two antibodies (H, n = 12 KT pairs); HEK buffer injection (E, n = 8 KT pairs) was used as a control. (I–L) Time series of 19 frames (acquired at 20-s intervals) displaying individual KT pairs from videos of HEC1-GFP PtK1 cells microinjected as indicated in the figure labels. The last frame in each series represents anaphase onset. (M–P) Box plots of inter-KT distances in cells microinjected as indicated in the figure labels. Each dot in the graphs represents the median value of inter-KT distances obtained for different KT pairs (n = 8, 12, 7, and 12 KT pairs for M, N, O, and P, respectively) from 4–7 cells at the same time point; the boxes represent the 25th-75th percentile range; the whiskers extend to the most extreme data points not considered outliers. All the graphs display data for a 10-min interval preceding anaphase (up to 1 min before anaphase onset). Examples of microinjected cells can be viewed in Video 2.