Figure 5. CENP-Ei promotes rotational... | Rockefeller University Press

Figure 5.

$CENP-Ei promotes rotational movements in 3D. (a) Experimental regime. HeLa cells were exposed to MG132 (10 µM) 1 hour before imaging and SiR-Tubulin (100 nM) before imaging. Either DMSO (control) or 30 nM CENP-E inhibitor (CENP-Ei) as indicated were added during imaging. (b) Representative maximum projection live-cell images of a HeLa cell expressing H2B-GFP (green) and stained with SiR-Tubulin dye (magenta) for 60 min prior to imaging. Cells were imaged for over 1 h with images taken every 3 min. Yellow circles indicate uncongressed chromosomes. (c) Cartoon shows a 3D spindle (gray) with the corresponding rotation angles α (spindle tumbling), β (spindle rolling), and γ (spindle rotation) along its principal spindle axes x,y,z, respectively. (d) Violin plots show 3D angle distribution for α spindle tumbling, β rolling, and γ rotation. Corresponding colored dots represent measurements from all time points of the same cell. The white marker within the box refers to the median, the shaded area refers to the estimated kernel probability density, and the box indicates the interquartile range of the data, respectively. (e) Violin plots show fractions of longitudinal, equatorial, and axial spindle displacement. Corresponding colored dots represent measurements from all time points of the same cell. The white marker within the box refers to the median, the shaded area refers to the estimated kernel probability density, and the box indicates the interquartile range of the data, respectively. Statistical significance was determined by Generalized Linear Model (GLM) and Mann–Whitney U test (in d and e) after a pre-analysis of the underlying distribution. N = 38 control and N = 43 CENP-Ei cells from three experiments. Scale bars: 5 µm.$

CENP-Ei promotes rotational movements in 3D. (a) Experimental regime. HeLa cells were exposed to MG132 (10 µM) 1 hour before imaging and SiR-Tubulin (100 nM) before imaging. Either DMSO (control) or 30 nM CENP-E inhibitor (CENP-Ei) as indicated were added during imaging. (b) Representative maximum projection live-cell images of a HeLa cell expressing H2B-GFP (green) and stained with SiR-Tubulin dye (magenta) for 60 min prior to imaging. Cells were imaged for over 1 h with images taken every 3 min. Yellow circles indicate uncongressed chromosomes. (c) Cartoon shows a 3D spindle (gray) with the corresponding rotation angles α (spindle tumbling), β (spindle rolling), and γ (spindle rotation) along its principal spindle axes x,y,z, respectively. (d) Violin plots show 3D angle distribution for α spindle tumbling, β rolling, and γ rotation. Corresponding colored dots represent measurements from all time points of the same cell. The white marker within the box refers to the median, the shaded area refers to the estimated kernel probability density, and the box indicates the interquartile range of the data, respectively. (e) Violin plots show fractions of longitudinal, equatorial, and axial spindle displacement. Corresponding colored dots represent measurements from all time points of the same cell. The white marker within the box refers to the median, the shaded area refers to the estimated kernel probability density, and the box indicates the interquartile range of the data, respectively. Statistical significance was determined by Generalized Linear Model (GLM) and Mann–Whitney U test (in d and e) after a pre-analysis of the underlying distribution. N = 38 control and N = 43 CENP-Ei cells from three experiments. Scale bars: 5 µm.