Fig. 3: The initiation of chromosome congression near centrosomes after CENP-E reactivation coincides with chromosome biorientation.
From: CENP-E initiates chromosome congression by opposing Aurora kinases to promote end-on attachments
a Representative STED images of spindles in cells expressing CENP-A-GFP and centrin1-GFP (color coded for depth, color bar in Fig. 1c), either after CENP-E inhibition or 15 min post-reactivation, immunostained for α-tubulin (gray). Maximum projections (left) and insets showing kinetochore pairs with microtubules in different attachment categories, with corresponding diagrams (right). Percentages reflect distribution of each attachment type in CENP-E-inhibited cells. Percentage of kinetochores in the indicated attachment categories for polar (b) and aligned (c) kinetochores across treatment groups (legend in e). d Interkinetochore distance versus distance from the nearest pole by attachment category (legend in e) following CENP-E reactivation. e Interkinetochore distances per attachment category in CENP-E-inhibited spindles. 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 Representative images of CENP-A-GFP and centrin1-GFP (red) cells immunostained for CENP-E (gray) and Astrin (cyan) after indicated treatments, with merged projections (left) and enlarged kinetochore views (right). Astrin (g) and CENP-E (h) levels on kinetochores, normalized to CENP-A and the aligned group mean within each cell, plotted versus pole distance normalized to aligned group mean. Gray area indicates aligned region; large dots show treatment means ± SEM for each group. i Astrin and CENP-E levels, normalized to CENP-A, plotted against each other across the indicated treatments. j Schematic summarizing that lateral-to-end-on attachment transition precedes major part of congression and occurs near spindle poles following CENP-E reactivation or in its absence. All images are maximum projections. Numbers: (b–d) 69 cells, 380 kinetochore pairs; (e) 20 cells, 185 kinetochore pairs; (g–i) 80 cells, 505 kinetochores, all from ≥3 independent biological replicates. Statistics: two-tailed ANOVA with post hoc Tukey’s HSD test. Symbols: n.s., P > 0.05; ****, P ≤ 0.0001; arb,. arbitrary; inh., inhibited; react., reactivated. Source data are provided as a Source Data file.
