7 Results
for author "Yue-wen Wang"
Figure 1bub1-∆K mutants exhibit premature SAC silencing in response to CIK1-CC-induced syntelic attachment. (A) Overexpression of CIK1-CC is toxic to bub1-∆K cells. Saturated WT and mutant cells with a vector (V) or a PGALCIK1-CC plasmid (CC) were 10-fold serial diluted, spotted onto glucose and galactose plates, and incubated at 30° for 2 days. (B) The viability loss of bub1-∆K cells after CIK1-CC overexpression. WT and bub1-∆K cells with a vector or a PGALCIK1-CC plasmid were grown to log phase in raffinose medium and then released into galactose medium at 30°. Cells were collected at 0, 3, and 6 hr and spread onto YPD plates to determine the plating efficiency after overnight growth at 25° (n ≥ 200). (C) The expression of truncated Bub1. BUB1-13-myc and bub1-∆K-13myc cells in midlog phase were treated with 20 µg/ml of nocodazole for 120 min at 30°. The cells lysates were prepared for the examination of myc-tagged Bub1 and Bub1-∆K. (D) CIK1-CC overexpression causes chromosome missegregation in bub1-∆K mutant cells. A vector or a PGALCIK1-CC plasmid was introduced into WT and bub1-∆K cells with CEN4-GFP TUB1-mCherry. The transformants were first arrested in G1 phase in raffinose medium and then released into galactose medium at 30°. α-factor was restored after budding to block the second round of cell cycle. Cells were collected at the indicated time points for the examination of fluorescence signals. The percentage of CEN4-GFP missegregation is shown in the center panel; the distribution of CEN4-GFP and spindle morphology in some representative cells is shown on the right. The budding index is shown in the left panel. Bar, 5 µm. (E) bub1-∆K mutation alleviates the delay of Pds1 degradation induced by CIK1-CC overexpression. G1-arrested PDS1-18myc and bub1-∆K PDS1-18myc cells with a vector or a PGALCIK1-CC plasmid were released into galactose medium and incubated at 30°. α-factor was restored after budding. Cells were collected at the indicated time points and protein samples were prepared for Western blotting. The budding index and Pds1 levels are shown. Pgk1 protein levels are used as a loading control.
Figure 2bub1-∆K and sgo1Δ mutants suppress the anaphase entry delay in dam1-3D cells. (A) The delayed Pds1 degradation in dam1-3D cells is suppressed by bub1-∆K. G1-arrested WT and mutant cells with Pds1-18myc were released into YPD medium at 30°. α-factor was added after budding. Cell lysates were prepared at the indicated times for western blotting with anti-myc antibody. The budding index and Pds1 protein levels are shown. Pgk1, loading control. (B) The delay of Mad1 dephosphorylation in dam1-3D cells is suppressed by bub1-∆K. G1-arrested WT and mutant cells with Mad1-3HA were released into YPD medium at 30°. Cell lysates were prepared at the indicated time points for Western blotting with anti-HA antibody. The Mad1 modification is show in the left panel and the budding index is shown in the right panel. (C) The delay of Mad1 dephosphorylation in dam1-3D cells is suppressed by sgo1Δ. The cells were treated as described above. The Mad1 modification and the budding index are shown.
Figure 3The abolishment of Bub1-dependent H2A phosphorylation leads to premature SAC silencing. (A) h2a-S121A mutant cells are sensitive to CIK1-CC overexpression. WT and h2a-S121A cells with a vector (V) or a PGALCIK1-CC (CC) plasmid were serial 10-fold diluted and then plated onto glucose and galactose plates for further incubation at 30° for 2 days. (B) h2a-S121A mutants lose viability after CIK1-CC overexpression. Cells with the indicated genotypes in raffinose medium were released into galactose medium at 30°. Cells were collected at 0, 2, 4, and 6 hr and spread onto YPD plates to examine the plating efficiency after overnight growth at 25° (n ≥ 200). (C) h2a-S121A mutation abolishes the anaphase entry delay in dam1-3D mutants. Cells with the indicated genotypes were arrested in G1 phase with α-factor and then released into cell cycle in YPD. Cells were collected over time to examine the Pds1 protein levels. Budding index and Pds1 protein levels are shown. Pgk1, loading control. (D) h2a-S121A mutant cells show efficient metaphase arrest in response to nocodazole treatment. G1-arrested PDS1-18myc and h2a-S121A PDS1-18myc cells were released into YPD medium containing 20 μg/ml of nocodazole and incubated at 30°. Pds1 protein levels were determined after Western blotting. The Pds1 levels and budding index are shown. Pgk1, loading control.
Figure 4rts1Δ mutants are sensitive to CIK1-CC overexpression. (A) Overexpression of CIK1-CC is toxic to rts1Δ cells. WT and rts1Δ cells with a vector (V) or a PGALCIK1-CC (CC) plasmid were 10-fold serial diluted and spotted onto glucose and galactose plates for further incubation at 30° for 2 days. (B) rts1∆ cells lose viability after CIK1-CC overexpression. WT and rts1∆ cells with a vector or a PGALCIK1-CC plasmid were grown to log phase in raffinose medium at 30° and then galactose was added into the medium. Cells were collected at 0, 3, and 6 hr and spread onto YPD plates to assess the plating efficiency (n ≥ 200). (C) CIK1-CC overexpression leads to chromosome missegregation in rts1Δ cells. CEN4-GFP TUB1-mCherry and rts1Δ CEN4-GFP TUB1-mCherry cells with a vector or a PGALCIK1-CC plasmid were arrested in G1 phase in raffinose medium and then released into galactose medium at 30°. Cells were collected at indicated time points and fixed for the examination of fluorescence signals. The percentage of cells with missegregated sister CEN4-GFPs among all anaphase cells is shown on the top left (n > 100). The localization of CEN4-GFP as well as spindle morphology in some representative cells is shown at the bottom. The budding index is shown on the right. Bar, 5 µm.
Figure 5rts1Δ mutation suppresses the anaphase entry delay in dam1-3D cells without compromising SAC activation. (A) The delay of Pds1 degradation in dam1-3D cells is suppressed by rts1Δ. G1-arrested WT and mutant cells with Pds1-18myc were released into YPD medium at 30°. α-factor was added back after budding. Cell lysates were prepared at the indicated time points for Western blotting with anti-myc antibody. The Pds1 levels and budding index are shown. Pgk1, loading control. (B) rts1∆ cells show intact SAC function. G1-arrested PDS1-18myc and rts1Δ PDS1-18myc cells were released into YPD medium containing 20 μg/ml of nocodazole and incubated at 30°. Cells were collected every 20 min for the budding index and the determination of Pds1 protein levels. Pgk1, loading control. The Pds1 levels and the budding index are shown.
Figure 6The binding of Sgo1 to H2A and PP2A is essential to prevent premature SAC silencing. (A) Overexpression of CIK1-CC is toxic to sgo1 mutants with abolished binding to either H2A or PP2A. Saturated cells with a vector (V) or a PGALCIK1-CC plasmid (CC) were 10-fold serial diluted and spotted onto glucose and galactose plates, which were incubated at 30° for 2 days. (B) The viability loss of SGO1, sgo1Δ, sgo1-N51I, and sgo1-4A cells after CIK1-CC overexpression. Log-phase cells in raffinose were released into galactose medium at 30°. Cells were collected at 0, 3, and 6 hr and spread onto YPD plates to determine the plating efficiency (n ≥ 200). (C–E) CIK1-CC overexpression causes chromosome missegregation in sgo1 mutants. A vector or a PGALCIK1-CC plasmid was introduced into WT, sgo1Δ, sgo1-N51I, and sgo1-4A mutants with CEN4-GFP TUB1-mCherry. G1-arrested cells were released into galactose medium at 30°. α-factor was restored after budding. Cells were collected at the indicated time points for the examination of fluorescence signals. (C) The budding index . (D) The percentage of anaphase cells that show cosegregated CEN4-GFP. (E) The distribution of CEN4-GFP and spindle morphology in some representative cells. Bar, 5 µm.