Figure 4
Phospho-deficient dam1-3A mutants show chromosome mis-segregation after nocodazole exposure. G1-arrested WT, dam1-3A and dam1-3D cells with CEN4-GFP TUB1-mCherry were released into YPD containing 20 µg/ml nocodazole at 30 °C for 2hr. Then, nocodazole was washed off and the cells were released into YPD at 30 °C (Time 0). α-factor was restored to block next cell cycle. Cells were collected every 20 min for the examination of budding index and fluorescent signals. (A) The percentage of large-budded cells and cells with a short spindle (SP) after nocodazole release. (B) The distribution of CEN4–GFP and spindle morphology in representative cells. The arrows indicate cells with co-segregated CEN4–GFP. (C) The percentage of cells with an elongated spindle as well as co-segregated CEN4–GFP at 120 and 140 min (n ≥ 100). (D) The viability loss of WT, dam1-3A and dam1-3D mutants after nocodazole treatment. The cells arrested in G1 and cells after nocodazole (Noc) exposure for 2 hrs were spread onto YPD plates to count the plating efficiency after overnight growth at 25 °C (n ≥ 200). (E) The number of CEN4-GFP dots in G1 cells after nocodazole exposure. After nocodazole exposure, the cells were released and allowed to enter next G1 phase. The percentage of G1 cells with 0, 1, or 2 CEN4-GFP dots was counted (n > 100). (F). Representative images show the CEN4-GFP signals in WT, dam1-3A and dam1-3D cells in G1 phase. The arrow indicates a G1 cell with 2 CEN4-GFP dots.
