Kinetochores are under metaphase-level tension before Mad1 loss, but tension across the kinetochore is insufficient to initiate Mad1 loss. (A) Time-lapse imaging (maximum-intensity projection) of SAC satisfaction kinetics (EYFP-Mad1) concurrently with K–K distance (CenpC-mCherry) showing representative changes in centromere tension (ruler) relative to Mad1 loss (start at t = 0, followed by arrows) on K1 (blue) and K2 (orange) from the PtK2 cell in Fig. 1 A. See also Video 3. Bars, 1 µm. (B) K–K distance over time for the pair in A with smoothing over a three-time-point window and t = 0 (dashed lines) indicating the start of Mad1 loss for K1 (blue) and K2 (orange). (C) Means and SEM of K–K distance over time plotted relative to Mad1 loss start (t = 0) for each of K1 (left; n = 17 pairs) and K2 (right; n = 29 pairs). (D) K–K distance at all time points before t = −2 min for K1 (n = 10 pairs) and K2 (n = 19 pairs) at t = 0 for K1 (n = 10 pairs) and K2 (n = 19 pairs) and at reference points in separate experiments (metaphase kinetochores [n = 40 pairs] and 10 µM nocodazole [n = 24 pairs]). Measurements for K2’s t = −2 min do not include any data before K1’s t = 0. Error bars indicate SEM (*, P < 0.05; two-sided Mann-Whitney U test). (E) Fraction of pairs (n = 17) with a K–K distance crossing >1.5 µm as time evolves relative to Mad1 loss start at K1 (t = 0).