Effects of CenpE inhibition on kinetochore movements during early prometaphase. (A) Kymograms generated from aligned 3D volumes of RPE1 cells with GFP-labeled kinetochores (CenpA) and centrioles (Centrin1). In this view, the center of spindle remains stationary and the centrosomes move symmetrically along the horizontal axis so that the pattern of movement with respect to the spindle poles is not obscured by rocking of the spindle within the cell. Arrowheads mark extended rapid movements toward the centrosomes. Arrows point at the extended linear movements that lead to monoorientation of some chromosomes in cells treated with GSK923295 or depleted of CenpE via siRNA (see Videos 6, 7, and 8 for recordings used to generate the kymograms). (B) Representative 30 s of kinetochore movements (selected from 0–2 and 2–4 min after NEB). Crosses mark positions of centrosomes. Arrowheads mark rapid movements toward centrosomes sustained for 30 s. (C) Histograms of SD values calculated for frame-to-frame (5-s) displacements of individual kinetochores within 30-s rolling windows spanning 0–2 min (top) and 2–4 min (bottom) after NEB. Low SD values (<10 × 10−2 μm) correspond with stationary kinetochores, whereas higher SDs (>20 × 10−2 μm) indicate jerky movements.
