Behavior of kinetochores with severed K-fibers in flattened RPE cells. (A) High temporal-resolution (10 frames/s, 108-nm pixels) kymograms of kinetochore movements for a control chromosome (i), a chromosome with one intact (K2) and one severed (K1) K-fiber (ii), and a chromosome with a single kinetochore (K1). The sister kinetochore (K2) is ablated by laser (iii). Yellow crosses mark time and position of the laser beam. Notice that trajectories of kinetochores with intact K-fibers are smooth, whereas the kinetochore with severed K-fiber exhibits frequent poleward jumps. (B) Tukey plot characterization of the displacement from the original position for the classes of kinetochores presented in A. Control chromosomes (i) and chromosomes with severed K-fibers (ii) remain bioriented, whereas ablation of a single kinetochore results in a gradual displacement of the chromosome poleward (iii). (C) Dynamics of centromere stretching for the chromosomes shown in A (i and ii). Notice the difference between the relatively slow and smooth changes in the interkinetochore distance of the control chromosome (i) versus rapid transitions between stretching and relaxation of the centromere with one intact and one severed K-fiber. (D) Decrease in the interkinetochore distance induced by severing a K-fiber fits an exponential function, whereas restoration of centromere stretching is driven by rapid linear movements of the kinetochore attached to the severed K-fiber. Each panel presents a detailed view of short periods in C (ii). Time 0 marks laser irradiation. (E) Tukey plot characterization of the time from the K-fiber severing and initiation of the first poleward movement and the duration of the first poleward movement. (F) Distribution of kinetochore velocities during brief poleward movements of kinetochores with severed K-fibers. Error bars are 95% confidence intervals for binomial distribution.