Figure 5.
Aurora A–dependent Hec1 phosphorylation promotes chromosome oscillation.(A) Trajectories of kinetochores in RPE-1 cells arrested in metaphase in the presence or absence of Aurora inhibitors. Cells were treated with AZD1152 or MLN8237 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 AZD1152 (upper) or MLN8237 (lower), plotted as the distance from the spindle equator. See also Video 5. (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 values were obtained using the Dunnett’s multiple comparisons test. (C) Trajectories of kinetochores in RPE-1 cells arrested in metaphase depleted of Aurora A by the AID system. RPE-1 cells containing Aurora A–mAID-mClover were treated as in Fig. S3 A with or without IAA and observed by live-cell imaging. The blue and red trajectories show the movements of a pair of sister kinetochores in nontreated cells (upper) or IAA-treated cells (lower), plotted as the distance from the spindle equator. See also Video 5. (D) DAP measurements of kinetochore position in RPE-1 cells treated as in C. Error bars represent SD of 10 kinetochore pairs from three cells. P value was obtained using the Student’s t test. (E) Trajectories of kinetochores in RPE-1 cells arrested in metaphase depleted of endogenous Hec1 and expressing EGFP-tagged WT Hec1 or Hec1 mutants, in which phosphorylation sites are mutated to alanine. Cells expressing Hec1-WT, Hec1-S55A, or S69A, a single phosphorylation site mutant; Hec1-2A, in which both S55 and S69 are mutated to alanine; or Hec1 mutants in which all phosphorylation sites are mutated to alanine except for S55 (8A55WT), S69 (8A69WT), or both (7A2WT) were arrested in metaphase 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 expressing each Hec1 construct, plotted as the distance from the spindle equator. See also Video 6. (F) DAP measurements of kinetochore position in RPE-1 cells treated as in E. Error bars represent SD of 10 kinetochore pairs from three cells. P values were obtained using the Tukey–Kramer multiple comparison test.

Aurora A–dependent Hec1 phosphorylation promotes chromosome oscillation. (A) Trajectories of kinetochores in RPE-1 cells arrested in metaphase in the presence or absence of Aurora inhibitors. Cells were treated with AZD1152 or MLN8237 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 AZD1152 (upper) or MLN8237 (lower), plotted as the distance from the spindle equator. See also Video 5. (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 values were obtained using the Dunnett’s multiple comparisons test. (C) Trajectories of kinetochores in RPE-1 cells arrested in metaphase depleted of Aurora A by the AID system. RPE-1 cells containing Aurora A–mAID-mClover were treated as in Fig. S3 A with or without IAA and observed by live-cell imaging. The blue and red trajectories show the movements of a pair of sister kinetochores in nontreated cells (upper) or IAA-treated cells (lower), plotted as the distance from the spindle equator. See also Video 5. (D) DAP measurements of kinetochore position in RPE-1 cells treated as in C. Error bars represent SD of 10 kinetochore pairs from three cells. P value was obtained using the Student’s t test. (E) Trajectories of kinetochores in RPE-1 cells arrested in metaphase depleted of endogenous Hec1 and expressing EGFP-tagged WT Hec1 or Hec1 mutants, in which phosphorylation sites are mutated to alanine. Cells expressing Hec1-WT, Hec1-S55A, or S69A, a single phosphorylation site mutant; Hec1-2A, in which both S55 and S69 are mutated to alanine; or Hec1 mutants in which all phosphorylation sites are mutated to alanine except for S55 (8A55WT), S69 (8A69WT), or both (7A2WT) were arrested in metaphase 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 expressing each Hec1 construct, plotted as the distance from the spindle equator. See also Video 6. (F) DAP measurements of kinetochore position in RPE-1 cells treated as in E. Error bars represent SD of 10 kinetochore pairs from three cells. P values were obtained using the Tukey–Kramer multiple comparison test.

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