Figure 3
Dam1 phosphorylation is required for the establishment of chromosome bipolar attachment after nocodazole treatment. (A) G1-arrested cdc13-1 and cdc13-1 dam1-3A cells with MTW1-GFP TUB1-mCherry were released into YPD medium containing 20 µg/ml nocodazole at 34 °C for 2hr. Nocodazole was washed off, and then cells were released into YPD at 34 °C. Cells were collected every 10 min and fixed for the examination of fluorescent signals. Confocal microscopy was used for the projection of maximum-intensity images. The Mtw1-GFP fluorescence density of each kinetochore cluster was determined, and the average ratio of the intensity between the two kinetochore clusters in each cell was shown (n > 20). (B) Representative images for the Mtw1-GFP signal acquired by confocal microscopy after nocodazole was washed off for 10 and 20 min. Scale bar: 5 µm. (C) cdc13-1, cdc13-1 dam1-3A and cdc13-1 dam1-3D cells with CEN4-GFP TUB1-mCherry were treated as described in A. The percentage of cells with CEN4-GFP localized at the center region of the spindle is shown. (D) The relative localization of CEN4-GFP (green) to the spindle (red) in some representative cells are shown. Scale bar: 5 µm.
