Aurora-B/AIM-1 Regulates the Dynamic Behavior of HP1 at the G₂–M Transition

Supplemental Material

This article contains the following supporting material:

• Figure 1 - Mitotic phosphorylation of histone H3 correlates with the dissociation of HP1α from heterochromatin, and Aurora-B/AIM-1 colocalization with HP1α at the heterochromatin region.
  G1/S phase CHO cells expressing EGFP-HP1 α were obtained from a double-thymidine block. G2 phase cells were obtained through release of cells synchronized by a double-thymidine block into fresh medium for a period of 8-10 h. M phase cells were obtained through release from a thymidine/nocodazole block. After removal of nocodazole, cells were further cultured for 10, 20, 30, and 40 min in fresh medium prior to fixation. Fixed cells were labeled with DAPI (DNA), anti-H3 phos10 (H3P) (A), or anti-AIM-1 (Aur-B/AIM-1) (B) antibodies. Prometaphase cells were extracted with Triton X-100, fixed with formaldehyde. Scale bar: 10 μm.

• Movie 1 - Transient overexpression of kinase-negative Aurora-B/AIM-1 KR inhibited the dissociation of HP1α from the chromosome arms, and led to failed chromosome segregation and cytokinesis.
  CHO cells expressing EGFP-HP1α were transfected with kinase-negative Aurora-B/AIM-1 KR as shown in Movie 1. After transfection, the cells were synchronized by thymidine treatment and released. After 10 h, the cells were subjected to time-lapse video microscopy. HP1α failed to dissociate from the chromosome arms, and the chromosomes failed to align on the metaphase plate (arrows). Eventually, both chromosome segregation and cytokinesis failed, leading to multinucleated cells.

• Movie 2 - HP1α dissociation from heterochromatin occurred normally at the onset of mitosis.
  CHO cells expressing EGFP-HP1α were synchronized by thymidine treatment and subsequent release. After 10 h cells were recorded with time-lapse video microscopy. When a cell entered into mitosis, HP1α began to dissociate from the chromosome and eventually dispersed into the cytoplasm.