Fig. 1: Lack of Aurora A- and B- mediated phosphorylation of CENP-E promotes premature removal of CENP-E from kinetochores and its accumulation at the spindle poles.
From: CENP-E activation by Aurora A and B controls kinetochore fibrous corona disassembly
a Representative spinning-disk confocal time-series of mitosis in U2OS GFP-CENP-E WT/T422A cells. H2B-RFP and SiR-tubulin are used to visualize DNA and MTs, respectively. Red arrowheads indicate spindle pole accumulation of CENP-E mutant. Time: hour:min. Scale bar: 10 μm. b Immunoblot of inducible expression of GFP-CENP-E along with depletion of endogenous CENP-E. c GFP-CENP-E intensity at KTs over time after NEB in U2OS cells expressing CENP-A-mCherry. Dots and dashed lines represent mean values and SD respectively. The solid thick line represents the fitted curve. N (number of cells, number of independent experiments): GFP-CENP-E WT (10, 4), GFP-CENP-E T422A (10, 4). d Representative time-series of GFP-CENP-E WT/T422A localization in monopolar spindles. Time scale: hour:min. Bottom panel shows GFP-CENP-E WT distribution in Aurora A-inhibited cells. Red arrowhead emphasizes transient spindle pole accumulation of CENP-E WT in the absence of Aurora A activity. Time: hour:min. Scale bars: 10 μm. e Quantification of the percentage of mitotic cells with transient accumulation of GFP-CENP-E WT at the spindle poles. The values are plotted with mean and SEM. N (number of cells, number of independent experiments): CTRL (32, 3) MLN8054 (28, 3). f Maximum intensity projected confocal images of endogenous CENP-E at KTs in U2OS cells undergoing the indicated treatments. Scale bars: 10 μm. g Quantification of endogenous CENP-E intensity normalized to CENP-C intensity at KTs. Violin plots with median (thick dashed lines) and quartiles (light dashed lines) represent the average KT intensities in cells. N (number of KTs, number of cells, number of independent experiments): NOC (1036, 29, 3), NOC + ZM447439 (1010, 29, 3), STLC (1044, 29, 3), STLC + ZM447439 (1063, 29, 3). Statistical significance was determined by the Mann–Whitney U-test (unpaired, two-tailed; no normal distribution). p values are indicated. h Illustrated model of the impact of T422A mutation on CENP-E localization and chromosome congression. Non-phosphorylatable CENP-E (green) is prematurely removed from KTs, and it accumulates at the spindle poles. Cells are arrested in a pseudo-metaphase with uncongressed polar chromosomes.
