KT relaxed length depends on the stretched length in PtK1 cells, but not in S. pombe. (A–D) Comparison of the relaxed KT length after laser ablation, NOC treatment, and control unstretched KTs. As control KTs, we measured PtK1 anaphase KTs that were attached to a K-fiber emanating from only one spindle pole and S. pombe KTs that were not lagging during anaphase. Length of control KTs was determined by using automated software to detect the KT and to count pixels with KT-specific signal. These measurements are influenced by the imaging conditions and they may not represent the absolute length of KTs, but they can be used to compare merotelic and control KTs. Relaxed length after severing one K-fiber for the outer KT, Hec1/Ndc80, is shown in green and for inner KT, CenpA/Cnp1, in magenta. The relaxed length of the outer KT after severing both K-fibers is shown in blue. The relaxed length of KTs in PtK1 cells after 9-µM NOC treatment is shown in white. The lengths are calculated as mean of the values measured in the last 30 s and 15 s of the experiment for PtK1 cells and fission yeast, respectively. The tops and bottoms of each box are the 25% and 75% of the sample, respectively. The line in the middle of each box is the sample median. Hec1-single ablation, n = 25; Hec1-double ablation, n = 9; Hec1-NOC treatment, n = 9; Hec1-control KTs, n = 43; CenpA-ablation, n = 15; CenpA-control KTs, n = 67; Ndc80-single ablation, n = 18; Ndc80-double ablation, n = 10; Ndc80-control KTs, n = 11; Cnp1-ablation, n = 24; and Cnp1-control KTs, n = 11. There was a statistically significant difference between the relaxed and control KT length in all cases in A–C (**, P < 0.001; ***, P < 0.0001), but no significant difference (NS, P > 0.05) in D. NOC was added at 9-µM concentration for 15–25 min. (E and F) Correlation between initial stretched KT length and relaxed length. The initial stretched length was calculated as a mean of the values measured before ablation. Solid colored lines represent linear fit for each specific condition.