Figure 3.
Hec1-S55 phosphorylation during metaphase is mainly dependent on Aurora A localizing to the spindle.(A) Schematic diagram of tagging endogenous Aurora A with mAID-mClover tag by the CRISPR/Cas–based method. (B) Depletion of Aurora A–mAID-mClover by adding IAA in RPE-1 cells arrested in metaphase. Cells were treated as in Fig. S3 A with or without IAA, then fixed and stained with an anti–Aurora A (red) antibody. DNA was stained with DAPI (blue). The mClover signal (green) was acquired by fluorescence microscopy. Cells arrested in metaphase are shown. Scale bar: 5 µm. (C) Hec1-S55 phosphorylation in RPE-1 cells arrested in metaphase depleted of Aurora A using the AID system. Cells treated as in Fig. S3 A with or without AZD1152 and/or MLN8237 were fixed and stained with anti-Hec1 (blue, shown as green) and anti–phospho-Hec1-S55 (red) antibodies. The mClover signal (green, shown as monochrome) was acquired by fluorescence microscopy. 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). (D) Quantification of the Hec1-S55 phosphorylation signal in RPE-1 cells arrested in metaphase depleted of Aurora A using the AID system. Relative intensity of the phospho-Hec1-S55 signal in cells treated as in C 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 205 kinetochores from five cells for each condition. P values were obtained using the Steel–Dwass multiple comparisons test. (E) Quantification of distance between spindle pole and Hec1 signal on kinetochores depending on the Hec1-S55 phosphorylation. A schematic of measurement is shown at the top, and the result from 227 kinetochores from five cells is shown as box and dot plots as in Fig. 2 B. P value was obtained using the Mann–Whitney U test. (F and G) Phosphorylation of Hec1-S55 (F) or S69 (G) along the metaphase plate. Upper panels: Metaphase RPE-1 cells expressing Aurora A–mAID-mClover treated as in Fig. S3 A in the absence of IAA were fixed and stained with anti-GFP (green), anti–phospho-Hec1-S55 (F), or S69 (G; blue) and anti-centromere (ACA; red) antibodies. Scale bars: 5 µm. Lower graphs: Intensities of Aurora A–mAID-mClover (green), phospho-Hec1-S55 (F), or S69 (G; blue) and ACA (red) signals in the cells shown in the upper panels were plotted against relative distance from the spindle equator, where the distance from the spindle equator to a spindle pole was set as 5. (H) Replacement of endogenous TPX2 with WT TPX2 (WT), TPX2-F307E F334E F335E (3E), or the N-terminal fragment (aa 1–43) of TPX2 (N). RPE-1 cells containing mAID-mClover-TPX2 and expressing mCherry-TPX2 (WT, 3E, or N) were treated as in Fig. S3 A, then fixed and stained with anti-mCherry (red) or anti–Aurora A (blue). The mClover signal (green) was acquired by fluorescence microscopy. Cells arrested in metaphase are shown. Scale bar: 5 µm. (I) Hec1-S55 phosphorylation in RPE-1 cells arrested in metaphase expressing TPX2-WT, 3E, or N. Cells explained in H were treated as in Fig. S3 A, 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). (J) Quantification of the Hec1-S55 phosphorylation in RPE-1 cells arrested in metaphase expressing TPX2-WT, 3E, or N. Relative intensity of the phospho-Hec1-S55 signal in cells treated as in I 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 188 kinetochores from five cells for each condition. P values were obtained using the Steel–Dwass multiple comparisons test. AURKA, Aurora kinase A; Neo, neomycin resistance gene; Dox, doxycycline.

Hec1-S55 phosphorylat ion during metaphase is mainly dependent on Aurora A localizing to the spindle. (A) Schematic diagram of tagging endogenous Aurora A with mAID-mClover tag by the CRISPR/Cas–based method. (B) Depletion of Aurora A–mAID-mClover by adding IAA in RPE-1 cells arrested in metaphase. Cells were treated as in Fig. S3 A with or without IAA, then fixed and stained with an anti–Aurora A (red) antibody. DNA was stained with DAPI (blue). The mClover signal (green) was acquired by fluorescence microscopy. Cells arrested in metaphase are shown. Scale bar: 5 µm. (C) Hec1-S55 phosphorylation in RPE-1 cells arrested in metaphase depleted of Aurora A using the AID system. Cells treated as in Fig. S3 A with or without AZD1152 and/or MLN8237 were fixed and stained with anti-Hec1 (blue, shown as green) and anti–phospho-Hec1-S55 (red) antibodies. The mClover signal (green, shown as monochrome) was acquired by fluorescence microscopy. 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). (D) Quantification of the Hec1-S55 phosphorylation signal in RPE-1 cells arrested in metaphase depleted of Aurora A using the AID system. Relative intensity of the phospho-Hec1-S55 signal in cells treated as in C 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 205 kinetochores from five cells for each condition. P values were obtained using the Steel–Dwass multiple comparisons test. (E) Quantification of distance between spindle pole and Hec1 signal on kinetochores depending on the Hec1-S55 phosphorylation. A schematic of measurement is shown at the top, and the result from 227 kinetochores from five cells is shown as box and dot plots as in Fig. 2 B. P value was obtained using the Mann–Whitney U test. (F and G) Phosphorylation of Hec1-S55 (F) or S69 (G) along the metaphase plate. Upper panels: Metaphase RPE-1 cells expressing Aurora A–mAID-mClover treated as in Fig. S3 A in the absence of IAA were fixed and stained with anti-GFP (green), anti–phospho-Hec1-S55 (F), or S69 (G; blue) and anti-centromere (ACA; red) antibodies. Scale bars: 5 µm. Lower graphs: Intensities of Aurora A–mAID-mClover (green), phospho-Hec1-S55 (F), or S69 (G; blue) and ACA (red) signals in the cells shown in the upper panels were plotted against relative distance from the spindle equator, where the distance from the spindle equator to a spindle pole was set as 5. (H) Replacement of endogenous TPX2 with WT TPX2 (WT), TPX2-F307E F334E F335E (3E), or the N-terminal fragment (aa 1–43) of TPX2 (N). RPE-1 cells containing mAID-mClover-TPX2 and expressing mCherry-TPX2 (WT, 3E, or N) were treated as in Fig. S3 A, then fixed and stained with anti-mCherry (red) or anti–Aurora A (blue). The mClover signal (green) was acquired by fluorescence microscopy. Cells arrested in metaphase are shown. Scale bar: 5 µm. (I) Hec1-S55 phosphorylation in RPE-1 cells arrested in metaphase expressing TPX2-WT, 3E, or N. Cells explained in H were treated as in Fig. S3 A, 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). (J) Quantification of the Hec1-S55 phosphorylation in RPE-1 cells arrested in metaphase expressing TPX2-WT, 3E, or N. Relative intensity of the phospho-Hec1-S55 signal in cells treated as in I 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 188 kinetochores from five cells for each condition. P values were obtained using the Steel–Dwass multiple comparisons test. AURKA, Aurora kinase A; Neo, neomycin resistance gene; Dox, doxycycline.

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