Fig. 1: CENP-E activity is required for the rapid initiation of congression but not for the subsequent movement of polar chromosomes.
From: CENP-E initiates chromosome congression by opposing Aurora kinases to promote end-on attachments
a Lattice light sheet microscopy (LLSM) image of RPE-1 cells expressing centrin1-GFP and CENP-A-GFP (color coded for depth), treated with 80 nM CENP-E inhibitor (GSK-923295) for 3 h. Boxed areas highlight mitotic cells. b Schematic of the experimental approach to acutely modulate CENP-E activity. c Representative LLSM time-lapse images showing: RPE-1 cell expressing centrin1-GFP and CENP-A-GFP (color coded by depth) after CENP-E depletion (left); U2OS cell with CENP-A-GFP (color coded by depth) and polar chromosomes (arrowheads) during early anaphase (top right); and U2OS cell expressing GFP-CENP-E-T422 (red) after CENP-E depletion and SPY650-DNA staining (yellow) (bottom right). All images are maximum projections. Time 0 is mitosis onset. d Number of polar chromosomes in RPE-1 (left) and U2OS (right) cells (top), distance to the nearest spindle pole of polar kinetochore pairs in RPE-1 cells (middle) over time from NEBD or inhibitor reactivation and their distance from the equator until time of successful alignment (bottom). Thick lines; means; shaded areas, standard deviations. e Chromosome congression velocity in the final 6 min (RPE-1) or 4 min (U2OS) before alignment for indicated treatments. Colored points represent individual cells; black lines show the mean, with light and dark gray areas marking 95% confidence intervals for the mean and standard deviation, respectively. f Percentage of polar kinetochore pairs that aligned (white) or remained polar (gray) within 30 min post-spindle elongation in RPE-1 under indicated conditions. g Schematic illustrating two hypotheses: CENP-E is required for congression initiation (1) or for continued movement (2). The supported hypothesis is indicated with a tick. Numbers: (d and e) control (15 cells, 32 kinetochores), reactivated (18, 86), depleted (18, 69), inhibited (12, 65) (RPE-1), (d) T422 (35 cells), WT (35), depleted (21) (U2OS), (e): control (30 cells, 64 kinetochore pairs), reactivated (10, 38), depleted (10, 52) (U2OS), (f) reactivated (23 cells), depleted (38), inhibited (14) (RPE-1), all from ≥3 independent biological replicates. Statistics: two-tailed ANOVA with post hoc Tukey’s HSD test (e), pairwise two-proportion z-test, two-tailed (f). Symbols: n.s., P > 0.05; inh., inhibited; depl., depleted; chrom., chromosomes; siRNA, small interfering RNA; NEBD, nuclear envelope breakdown. Source data are provided as a Source Data file.
