8 Results
for author "Yue-wen Wang"
Figure 1.—cik1Δ and kar3Δ mutants exhibit HU sensitivity without notable DNA synthesis defects. (A) cik1Δ and kar3Δ mutants are sensitive to HU. Stationary phase cell cultures with indicated genotypes were 10-fold diluted and spotted onto YPD plates containing 0 or 100 mm HU. The plates were incubated at 25° for 3–4 days before being scanned. (B) cik1Δ cells show normal DNA synthesis kinetics after treatment with HU. G1-arrested WT and cik1Δ cells were released into YPD medium containing 200 mm HU for 100 min at 25°. After HU was washed off, the cells were released into YPD medium, collected at the indicated time points, and subjected to PFGE after being fixed with 70% ethanol. (C) cik1Δ and kar3Δ mutants do not show dramatic viability loss after treatment with HU. WT, cik1Δ, kar3Δ, and rad53-21 cells in midlog phase were released into YPD medium containing 200 mm HU. Cells were spread onto YPD plates at the indicated times and incubated at 25° overnight. The formation minicolony was examined under a microscope and the percentage of viable cells is shown (n > 300). (D) cik1Δ cells show normal centromere duplication. G1-arrested scc1-73 and scc1-73 cik1Δ cells with CEN5-GFP and TUB1-mCherry were released into YPD medium at 37°. The cells were collected every 30 min to examine the separation of Cen5-GFP dots by fluorescence microscopy. The percentage of large-budded cells (solid markers) and cells with two GFP foci (open markers) is shown.
Figure 2.—cik1Δ and kar3Δ mutants exhibit delayed anaphase entry after HU treatment. (A) Delayed Pds1 protein degradation in cik1Δ and kar3Δ mutants. G1-arrested PDS1-myc, cik1Δ PDS1-myc, and kar3Δ PDS1-myc cells were released into 25° YPD medium. α-factor was added back after budding to block the second round of cell cycle. Cells were collected at the indicated time points and protein samples were prepared for the analysis of Pds1 protein levels by Western blotting. Pgk1 protein levels are shown as a loading control. (B) cik1Δ and kar3Δ mutant cells fail to enter anaphase after exposure to HU. G1-arrested PDS1-myc, cik1Δ PDS1-myc, and kar3Δ PDS1-myc cells were released into YPD medium containing 200 mm HU and incubated at 25° for 120 min. HU was then washed off and the cells were released into YPD medium containing α-factor. Cells were collected at the indicated time points for the preparation of protein samples. Pds1 protein levels are shown in the bottom panel after Western blotting. The budding index is shown at the top.
Figure 3.—cik1Δ mutants fail to establish bipolar attachment after HU treatment. (A) HU treatment results in dramatically delayed chromosome segregation in cik1Δ mutants. G1-arrested MTW1-GFP and cik1Δ MTW1-GFP cells were released into YPD medium containing 200 mm HU for 2 hr at 25°. HU was then washed off and the cells were released into YPD medium. α-Factor was added to block the second round of the cell cycle. Cells were collected after release for 3 hr for fluorescence microscopy. The Mtw1-GFP signal in some representative cells is shown in the left panel. The right panel presents the percentage of large-budded cells with one Mtw1-GFP cluster before and after 3 hr release from HU arrest (n > 300). Black bars (WT); grey bars (cik1Δ).(B) cik1Δ cells exhibit abnormal spindle morphology. TUB1-GFP and cik1Δ TUB1-GFP cells in midlog phase were released into YPD medium containing 200 mm HU for 2 hr at 25°. HU was then washed off and the cells were released into YPD medium with α-factor for 3 hr. The spindle morphology before and after HU treatment is shown.
Figure 4.—Isolation of cik1HU and cik1TS mutants. (A) The growth of cik1HU and cik1TS mutants. cik1Δ cells harboring a vector and CIK1, cik1TS, and cik1HU plasmids were grown to saturation and then serial 10-fold diluted and spotted onto URA dropout plates with or without 100 mm HU. The plates were incubated at 25° or 37° as indicated for 3 days before they were scanned. (B) The diagram of mutation sites in the cik1TS and cik1HU mutants. (C) The cell cycle distribution and the spindle morphology in cik1 mutant cells. WT, cik1Δ, and cik1HU mutant cells with TUB1-mCherry were grown to midlog phase. Cells were fixed to count the budding index and to examine the spindle morphology. The left panel shows the percentage of unbudded, small, and large-budded cells. The percentage of different spindle morphologies in large-budded cells is shown in the right panel (n > 300). (D) The growth of WT, cik1HU, and cik1Δ mutants on YPD plates containing various concentrations of HU. Saturated cultures were 10-fold diluted and spotted onto YPD and HU plates. The plates were scanned after a 3-day incubation at 25°.
Figure 5.—cik1HU mutants exhibit abnormal kinetochore distribution in the presence of 20 mm HU. (A) cik1HU mutant cells show abnormal kinetochore distribution. Asynchronous WT and cik1HU cells with Tub1-mCherry and Mtw1-GFP were released into YPD medium containing 20 mm HU for 6 hr at 25°. Cells before and after HU treatment were fixed to examine the budding index and the distribution of Mtw1-GFP signals. The percentage of large-budded cells is shown in the top panel. The percentage of cells with two kinetochore clusters and a short spindle is shown in the middle. The experiment was repeated three times. The arrow (bottom panel) indicates a cik1HU mutant cell with abnormally distributed Mtw1-GFP signal. (B) cik1HU mutant cells show defects in chromosome bipolar attachment. WT and cik1HU cells with Tub1-mCherry and Cen4-GFP were treated as described in A. The percentage of large-budded cells is shown in the top panel. The percentage of cells with different Cen4-GFP localization relative to the spindle is shown in the middle (n > 300). The relative localization of Cen4-GFP to the spindle in some representative cells is shown in the bottom panel.
Figure 6.—cik1HU mutants exhibit delayed establishment of bipolar attachment after HU treatment. (A) Comparison of cell cycle progression in synchronized WT, cik1Δ, and cik1HU mutants. G1-arrested PDS1-myc, cik1Δ PDS1-myc, and cik1HU PDS1-myc cells were released into 25° YPD medium. Cells were collected at the indicated time points and protein samples were prepared for the analysis of Pds1 protein levels by Western blotting. Pgk1 protein levels are shown as a loading control. The budding index is shown in the top panel. (B) cik1HU mutants show delayed anaphase entry after exposure to HU. G1-arrested PDS1-myc, cik1Δ PDS1-myc, and cik1HU PDS1-myc cells were released into YPD medium containing 200 mm HU and incubated at 25° for 120 min. HU was then washed off and the cells were released into YPD medium containing α-factor. The cells were treated as described in A. The budding index and the Pds1 protein levels are shown. (C and D) The cell cycle progression of WT and cik1HU mutant cells after HU treatment. G1-arrested WT and cik1HU cells with Cen4-GFP and Tub1-mCherry were released into 200 mm HU YPD and incubated at 25° for 120 min. HU was then washed off and the cells were released into YPD medium containing α-factor. Cells were collected at the indicated time points for budding index and fluorescence microscopy. The percentage of large-budded cells is shown in C, and the percentage of cells with elongated spindles is shown in D. (E) cik1HU mutant cells exhibit defects in establishing chromosome bipolar attachment. The relative localization of Cen4-GFP to the spindle in the cells collected above was analyzed by fluorescence microscopy. The percentage of cells within different categories is shown in the top panel (n > 300), and the representative cells are shown in the bottom panel. The arrow indicates a cell with Cen4-GFP away from the spindle.