Figure 1. Mad1 loss at individual... | Rockefeller University Press

Figure 1.

$Figure 1. Mad1 loss at individual mammalian kinetochores is a switch-like process with robust stereotypical single exponential kinetics. (A) Time-lapse imaging (maximum-intensity projections) of representative SAC satisfaction kinetics (EYFP-Mad1; green) at individual kinetochores (CenpC-mCherry; magenta) during spindle assembly in a PtK2 cell. Full circles identify the first (K1; blue) and second (K2; orange) sisters in a pair to lose Mad1, and white dashed circles identify spindle poles. t = 0 indicates the start of Mad1 loss on K1. See also Video 1. (B) Mad1 (solid green) and CenpC (dashed magenta) intensities for K1 in panel A around SAC satisfaction. Time lapse (bottom) of K1 at 13-s intervals. Bars: (A, main images) 3 µm; (A, insets, and B) 1 µm. (C) Individual traces, means, and SEM of the Mad1/CenpC intensity ratio (I) over time (t) around SAC satisfaction with traces synchronized at t = 0 (n = 46 kinetochores). (D) Distribution of the fractional position along the pole-to-pole axis ((k-polenear)/(pole–pole)) where kinetochores start to lose Mad1. Dashed lines indicate the mean position for each sister. The first sister (K1; n = 17) loses Mad1 close to its pole, and the second sister (K2; n = 29) near the metaphase plate. (E) Mean and SEM (top) of the Mad1/CenpC intensity ratio with t = 0 Mad1 loss start, and distribution (bottom) of times to reach a threshold intensity ratio, in bipolar cells (n = 46 kinetochores), different kinetochores (K1 and K2; n = 17 and 29) in these cells, in monopolar spindles (STLC-treated; Video 2; n = 20 kinetochores) that have lower tension, and in Hec1-9A–expressing cells, which have higher tension and attachment levels (n = 20 kinetochores). Vertical lines on box plots indicate the minimum value (except outliers), 25th percentile, median value, 75th percentile, and maximum value (except outliers). Red crosses indicate outlier values >1.5× the interquartile range. In all cases, Mad1 loss kinetics are indistinguishable from controls (NS; P > 0.05; two-sided Mann-Whitney U test).$

Mad1 loss at individual mammalian kinetochores is a switch-like process with robust stereotypical single exponential kinetics. (A) Time-lapse imaging (maximum-intensity projections) of representative SAC satisfaction kinetics (EYFP-Mad1; green) at individual kinetochores (CenpC-mCherry; magenta) during spindle assembly in a PtK2 cell. Full circles identify the first (K1; blue) and second (K2; orange) sisters in a pair to lose Mad1, and white dashed circles identify spindle poles. t = 0 indicates the start of Mad1 loss on K1. See also Video 1. (B) Mad1 (solid green) and CenpC (dashed magenta) intensities for K1 in panel A around SAC satisfaction. Time lapse (bottom) of K1 at 13-s intervals. Bars: (A, main images) 3 µm; (A, insets, and B) 1 µm. (C) Individual traces, means, and SEM of the Mad1/CenpC intensity ratio (I) over time (t) around SAC satisfaction with traces synchronized at t = 0 (n = 46 kinetochores). (D) Distribution of the fractional position along the pole-to-pole axis ((k-pole_near)/(pole–pole)) where kinetochores start to lose Mad1. Dashed lines indicate the mean position for each sister. The first sister (K1; n = 17) loses Mad1 close to its pole, and the second sister (K2; n = 29) near the metaphase plate. (E) Mean and SEM (top) of the Mad1/CenpC intensity ratio with t = 0 Mad1 loss start, and distribution (bottom) of times to reach a threshold intensity ratio, in bipolar cells (n = 46 kinetochores), different kinetochores (K1 and K2; n = 17 and 29) in these cells, in monopolar spindles (STLC-treated; Video 2; n = 20 kinetochores) that have lower tension, and in Hec1-9A–expressing cells, which have higher tension and attachment levels (n = 20 kinetochores). Vertical lines on box plots indicate the minimum value (except outliers), 25th percentile, median value, 75th percentile, and maximum value (except outliers). Red crosses indicate outlier values >1.5× the interquartile range. In all cases, Mad1 loss kinetics are indistinguishable from controls (NS; P > 0.05; two-sided Mann-Whitney U test).