Figure 3.

The H2A-I112A mutation causes mono-polar attachment, particularly after nocodazole treatment. (A) A schematic representation of the tet operator (tetO)/TetR-GFP system with kinetochore-microtubule bi-polar (left) and mono-polar (right) attachment in budding yeast. GFP-fused TetR binds tetO sequences integrated into the pericentromeric regions of chromosome 5 (CEN5-GFP). Centromere stretching in metaphase is observed when cells are arrested by depletion of Cdc20, a mitotic APC/C activator (Tanaka et al, 2000). (B) Time course for the observation of chromosome missegregation. Cells were released from the mitotic arrest caused by depletion of Cdc20 with (+Noc) or without (NT, no treatment) additional nocodazole treatment for 2.5 h at 25 °C. Images of dividing cells were taken by time-lapse imaging. (C) Images shown are representative photographs merged with CEN5-GFP in cells in anaphase. The scale bar represents 1 μm. (D) The rates of chromosome missegregation in H2A-WT and -I112A cells. Chromosome missegregation was evaluated by the behaviour of CEN5-GFP dots in dividing cells. The rates are represented as the mean±the standard deviation. (E) Time course for the observation of kinetochore-microtubule attachment in metaphase after nocodazole treatment. Cells were arrested by Cdc20 depletion with or without nocodazole (+Noc or NT) for 2 h at 25 °C and released from the nocodazole block. After additional metaphase arrest by Cdc20 depletion for ∼2 h, images of metaphase-arrested cells were taken by time-lapse imaging. (F) Images are representative photographs merged with CEN5-GFP and CFP-Tub1 in H2A-WT and -I112A cells in metaphase. The cyan signal of CFP-Tub1 has been converted to red. The scale bar represents 1 μm. (G) The rate of mono-polar attachment in H2A-WT and -I112A cells. Kinetochore-microtubule attachment was evaluated by the movement of GFP-labelled CEN5 in metaphase-arrested cells. The rates were represented as mean with standard deviation.