Figure 6.
Chromosome oscillation promotes the correction of erroneous kinetochore–microtubule attachments during metaphase through Aurora A activity.(A) Lagging chromosomes in HeLa cells. Cells were treated with either DMSO, BTB-1, or UMK57 for the last 1 h of a 3-h MG132 treatment, then released from the drug treatment for 1.5 h, fixed, and stained with anti-centromere antibodies (ACAs) and DAPI. Anaphase/telophase cells were examined for the presence or absence of lagging chromosomes (inset). Scale bar: 5 µm (white), 500 nm (yellow). (B) Quantification of HeLa cells containing lagging chromosomes in the presence of BTB-1 or UMK57. Cells were treated, fixed, and stained as in A. Frequency of anaphase/telophase cells containing lagging chromosomes was quantified. Error bars represent SD of three independent experiments, in which a minimum of 100 cells were observed for each condition. P values were obtained using the Dunnett’s multiple comparisons test. (C) Quantification of RPE-1 cells containing lagging chromosomes in the presence of AZD1152 or MLN8237 or depleted of Aurora A by the AID system. Cells were treated with either DMSO, AZD1152, or MLN8237 for the last 1 h of a 3-h MG132 treatment, then released from the drug treatment for 1.5 h before fixation. For RPE-1 cells containing Aurora A–mAID-mClover, they were treated with doxycycline and auxinole for 24 h and treated with or without IAA for the last 1 h of a 3-h proTAME and apcin treatment, then released from the drug treatment for 1.5 h before fixation. Cells were stained as in A, and frequency of anaphase/telophase cells containing lagging chromosomes was quantified. Error bars represent SD of three independent experiments, in which a minimum of 100 cells were observed for each condition. P values were obtained using the Dunnett’s multiple comparisons test. (D) Quantification of RPE-1 cells containing lagging chromosomes depleted of endogenous Hec1 expressing Hec1-WT-GFP, Hec1-S55A-GFP, Hec1-S69A-GFP, or Hec1-2A-GFP shown in Fig. 5 E. Cells were stained as in A, and frequency of anaphase/telophase cells containing lagging chromosomes was quantified. Error bars represent SD of three independent experiments, in which a minimum of 100 cells were observed for each condition. P values were obtained using the Tukey–Kramer multiple comparisons test. (E) Model of correction of erroneous kinetochore (KT)–microtubule (MT) attachments by Aurora kinases. Left panels: During prometaphase, Hec1 on kinetochores is phosphorylated by Aurora B (orange), which resides at the inner centromere, as well as by Aurora A (green), which localizes mainly to spindle poles, thus facilitating correction of erroneous kinetochore–microtubule attachments. In metaphase, Hec1 phosphorylation by Aurora B is reduced, while the Aurora A that localizes to the spindle phosphorylates Hec1-S55 when kinetochores approach close to spindle poles during chromosome oscillation, thus contributing to elimination of any remaining erroneous kinetochore–microtubule attachments. Right panels: In nontransformed cell lines, chromosome oscillation facilitates the Hec1-S55 phosphorylation by Aurora A, and this in turn increases the amplitude of chromosome oscillation together with the Hec1-S69 phosphorylation, thereby promoting the correction of erroneous kinetochore–microtubule attachments (red). In cancer cell lines, chromosome oscillation is attenuated, which leads to reduced Hec1-S55 phosphorylation by Aurora A, resulting in inefficient correction of erroneous attachments and increase in chromosome missegregation.

Chromosome oscillation promotes the correction of erroneous kinetochore–microtubule attachments during metaphase through Aurora A activity. (A) Lagging chromosomes in HeLa cells. Cells were treated with either DMSO, BTB-1, or UMK57 for the last 1 h of a 3-h MG132 treatment, then released from the drug treatment for 1.5 h, fixed, and stained with anti-centromere antibodies (ACAs) and DAPI. Anaphase/telophase cells were examined for the presence or absence of lagging chromosomes (inset). Scale bar: 5 µm (white), 500 nm (yellow). (B) Quantification of HeLa cells containing lagging chromosomes in the presence of BTB-1 or UMK57. Cells were treated, fixed, and stained as in A. Frequency of anaphase/telophase cells containing lagging chromosomes was quantified. Error bars represent SD of three independent experiments, in which a minimum of 100 cells were observed for each condition. P values were obtained using the Dunnett’s multiple comparisons test. (C) Quantification of RPE-1 cells containing lagging chromosomes in the presence of AZD1152 or MLN8237 or depleted of Aurora A by the AID system. Cells were treated with either DMSO, AZD1152, or MLN8237 for the last 1 h of a 3-h MG132 treatment, then released from the drug treatment for 1.5 h before fixation. For RPE-1 cells containing Aurora A–mAID-mClover, they were treated with doxycycline and auxinole for 24 h and treated with or without IAA for the last 1 h of a 3-h proTAME and apcin treatment, then released from the drug treatment for 1.5 h before fixation. Cells were stained as in A, and frequency of anaphase/telophase cells containing lagging chromosomes was quantified. Error bars represent SD of three independent experiments, in which a minimum of 100 cells were observed for each condition. P values were obtained using the Dunnett’s multiple comparisons test. (D) Quantification of RPE-1 cells containing lagging chromosomes depleted of endogenous Hec1 expressing Hec1-WT-GFP, Hec1-S55A-GFP, Hec1-S69A-GFP, or Hec1-2A-GFP shown in Fig. 5 E. Cells were stained as in A, and frequency of anaphase/telophase cells containing lagging chromosomes was quantified. Error bars represent SD of three independent experiments, in which a minimum of 100 cells were observed for each condition. P values were obtained using the Tukey–Kramer multiple comparisons test. (E) Model of correction of erroneous kinetochore (KT)–microtubule (MT) attachments by Aurora kinases. Left panels: During prometaphase, Hec1 on kinetochores is phosphorylated by Aurora B (orange), which resides at the inner centromere, as well as by Aurora A (green), which localizes mainly to spindle poles, thus facilitating correction of erroneous kinetochore–microtubule attachments. In metaphase, Hec1 phosphorylation by Aurora B is reduced, while the Aurora A that localizes to the spindle phosphorylates Hec1-S55 when kinetochores approach close to spindle poles during chromosome oscillation, thus contributing to elimination of any remaining erroneous kinetochore–microtubule attachments. Right panels: In nontransformed cell lines, chromosome oscillation facilitates the Hec1-S55 phosphorylation by Aurora A, and this in turn increases the amplitude of chromosome oscillation together with the Hec1-S69 phosphorylation, thereby promoting the correction of erroneous kinetochore–microtubule attachments (red). In cancer cell lines, chromosome oscillation is attenuated, which leads to reduced Hec1-S55 phosphorylation by Aurora A, resulting in inefficient correction of erroneous attachments and increase in chromosome missegregation.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal