Figure 4.
Chromosome oscillation facilitates Aurora A–dependent phosphorylation of Hec1-S55 during metaphase. (A) Trajectories of kinetochores in RPE-1 cells arrested in metaphase in the presence or absence of taxol. Cells were treated with or without 10 nM taxol for the last 1 h of a 2-h MG132 treatment and observed by live-cell imaging. The blue and red trajectories show the movements of a pair of sister kinetochores in cells treated with taxol, plotted as the distance from the spindle equator. See also Video 4. (B) DAP measurements of kinetochore position in RPE-1 cells treated as in A. Error bars represent SD of 10 kinetochore pairs from three cells. P value was obtained using the Welch’s t test. (C) Hec1-S55 phosphorylation in RPE-1 cells arrested in metaphase in the presence of taxol. Cells were treated with 10 nM taxol with either DMSO or AZD1152 and/or MLN8237 for the last 1 h of a 3-h MG132 treatment and then fixed and stained with anti-Hec1 (green) and anti–phospho-Hec1-S55 (red) antibodies. DNA was stained with DAPI (blue). Boxed regions in the panels are shown as magnified images in insets. Scale bar: 5 µm (white), 500 nm (yellow). (D) Quantification of the Hec1-S55 phosphorylation signal in RPE-1 cells treated as in C. Relative intensity of the phospho-Hec1-S55 signal was calculated and displayed as in Fig. 2 B. The median of DMSO-treated cells was set as 1. The data represent a minimum of 242 kinetochores from five cells for each condition. P values were obtained using the Steel multiple comparisons test. (E) Trajectories of kinetochores in HeLa cells arrested in metaphase with or without Kif18A depletion. Cells were treated with or without a Kif18A siRNA for 36 h, followed by 2-h MG132 treatment, and observed by live-cell imaging. The blue and red trajectories show the movements of a pair of sister kinetochores in cells depleted of Kif18A, plotted as the distance from the spindle equator. See also Video 4. (F) DAP measurements of kinetochore position in HeLa cells treated as in E. Error bars represent SD of 10 kinetochore pairs from three cells. P value was obtained using the Student’s t test. (G) Hec1-S55 phosphorylation in HeLa cells arrested in metaphase depleted of Kif18A. Cells were treated with a Kif18A siRNA for 36 h, followed by treatment with either DMSO or AZD1152 and/or MLN8237 for the last 1 h during 3-h MG132 treatment, and then fixed and stained with anti-Hec1 (green) and anti–phospho-Hec1-S55 (red) antibodies. DNA was stained with DAPI (blue). Boxed regions in the panels are shown as magnified images in insets. Scale bar: 5 µm (white), 500 nm (yellow). (H) Quantification of the Hec1-S55 phosphorylation signal in HeLa cells treated as in G. Relative intensity of phospho-Hec1-S55 signal was calculated and displayed as in Fig. 2 B. The median of cells treated with the Kif18A siRNA and DMSO was set as 1. The data represent a minimum of 206 kinetochores from five cells for each condition. P values were obtained using the Steel multiple comparisons test. (I) Trajectories of kinetochores in HeLa cells arrested in metaphase in the presence or absence of UMK57, an MCAK potentiator. Cells were treated with or without UMK57 for the last 1 h of a 2-h MG132 treatment and observed by live-cell imaging. The blue and red trajectories show the movements of a pair of sister kinetochores in cells treated with UMK57, plotted as the distance from the spindle equator. See also Video 4. (J) DAP measurements of kinetochore position in HeLa cells treated as in I. Error bars represent SD of 10 kinetochore pairs from three cells. P value was obtained using the Student’s t test. (K) Hec1-S55 phosphorylation in HeLa cells during metaphase in the presence of UMK57. Cells were treated with UMK57 and either DMSO or AZD1152 and/or MLN8237 for the last 1 h of a 3-h MG132 treatment, then fixed and stained with anti-Hec1 (green) and anti–phospho-Hec1-S55 (red) antibodies. DNA was stained with DAPI (blue). Cells arrested in metaphase are shown. Boxed regions in the panels are shown as magnified images in insets. Scale bar: 5 µm (white), 500 nm (yellow). (L) Quantification of the Hec1-S55 phosphorylation signal in HeLa cells treated as in K. Relative intensity of phospho-Hec1-S55 signal was calculated and displayed as box and dot plots as in Fig. 2 B. The median of UMK57-treated cells without Aurora inhibitors was set as 1. The data represent a minimum of 213 kinetochores from five cells for each condition. P values were obtained using the Steel multiple comparisons test. Kif18Asi, treatment with an siRNA for Kif18A.

Chromosome oscillation facilitates Aurora A–dependent phosphorylation of Hec1-S55 during metaphase. (A) Trajectories of kinetochores in RPE-1 cells arrested in metaphase in the presence or absence of taxol. Cells were treated with or without 10 nM taxol for the last 1 h of a 2-h MG132 treatment and observed by live-cell imaging. The blue and red trajectories show the movements of a pair of sister kinetochores in cells treated with taxol, plotted as the distance from the spindle equator. See also Video 4. (B) DAP measurements of kinetochore position in RPE-1 cells treated as in A. Error bars represent SD of 10 kinetochore pairs from three cells. P value was obtained using the Welch’s t test. (C) Hec1-S55 phosphorylation in RPE-1 cells arrested in metaphase in the presence of taxol. Cells were treated with 10 nM taxol with either DMSO or AZD1152 and/or MLN8237 for the last 1 h of a 3-h MG132 treatment and then fixed and stained with anti-Hec1 (green) and anti–phospho-Hec1-S55 (red) antibodies. DNA was stained with DAPI (blue). Boxed regions in the panels are shown as magnified images in insets. Scale bar: 5 µm (white), 500 nm (yellow). (D) Quantification of the Hec1-S55 phosphorylation signal in RPE-1 cells treated as in C. Relative intensity of the phospho-Hec1-S55 signal was calculated and displayed as in Fig. 2 B. The median of DMSO-treated cells was set as 1. The data represent a minimum of 242 kinetochores from five cells for each condition. P values were obtained using the Steel multiple comparisons test. (E) Trajectories of kinetochores in HeLa cells arrested in metaphase with or without Kif18A depletion. Cells were treated with or without a Kif18A siRNA for 36 h, followed by 2-h MG132 treatment, and observed by live-cell imaging. The blue and red trajectories show the movements of a pair of sister kinetochores in cells depleted of Kif18A, plotted as the distance from the spindle equator. See also Video 4. (F) DAP measurements of kinetochore position in HeLa cells treated as in E. Error bars represent SD of 10 kinetochore pairs from three cells. P value was obtained using the Student’s t test. (G) Hec1-S55 phosphorylation in HeLa cells arrested in metaphase depleted of Kif18A. Cells were treated with a Kif18A siRNA for 36 h, followed by treatment with either DMSO or AZD1152 and/or MLN8237 for the last 1 h during 3-h MG132 treatment, and then fixed and stained with anti-Hec1 (green) and anti–phospho-Hec1-S55 (red) antibodies. DNA was stained with DAPI (blue). Boxed regions in the panels are shown as magnified images in insets. Scale bar: 5 µm (white), 500 nm (yellow). (H) Quantification of the Hec1-S55 phosphorylation signal in HeLa cells treated as in G. Relative intensity of phospho-Hec1-S55 signal was calculated and displayed as in Fig. 2 B. The median of cells treated with the Kif18A siRNA and DMSO was set as 1. The data represent a minimum of 206 kinetochores from five cells for each condition. P values were obtained using the Steel multiple comparisons test. (I) Trajectories of kinetochores in HeLa cells arrested in metaphase in the presence or absence of UMK57, an MCAK potentiator. Cells were treated with or without UMK57 for the last 1 h of a 2-h MG132 treatment and observed by live-cell imaging. The blue and red trajectories show the movements of a pair of sister kinetochores in cells treated with UMK57, plotted as the distance from the spindle equator. See also Video 4. (J) DAP measurements of kinetochore position in HeLa cells treated as in I. Error bars represent SD of 10 kinetochore pairs from three cells. P value was obtained using the Student’s t test. (K) Hec1-S55 phosphorylation in HeLa cells during metaphase in the presence of UMK57. Cells were treated with UMK57 and either DMSO or AZD1152 and/or MLN8237 for the last 1 h of a 3-h MG132 treatment, then fixed and stained with anti-Hec1 (green) and anti–phospho-Hec1-S55 (red) antibodies. DNA was stained with DAPI (blue). Cells arrested in metaphase are shown. Boxed regions in the panels are shown as magnified images in insets. Scale bar: 5 µm (white), 500 nm (yellow). (L) Quantification of the Hec1-S55 phosphorylation signal in HeLa cells treated as in K. Relative intensity of phospho-Hec1-S55 signal was calculated and displayed as box and dot plots as in Fig. 2 B. The median of UMK57-treated cells without Aurora inhibitors was set as 1. The data represent a minimum of 213 kinetochores from five cells for each condition. P values were obtained using the Steel multiple comparisons test. Kif18Asi, treatment with an siRNA for Kif18A.

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