Figure 1.
Kinetochore-microtubule interaction in HU-treated cells.
(A) Examination of kinetochore integrity and kinetochore-microtubule interaction during S-phase. G1-arrested cells with untagged (U) or myc-tagged ASK1 and NNF1 were released into YPD medium containing 200 mM HU for 2 hrs at 30°C and then released into 30°C YPD medium. Cells were collected at indicated time points for ChIP assay. DNA from total (input) and immunoprecipitates (IP) were used for PCR reaction with primers specific to CEN1, CEN3, and ACT1. H and R stand for HU and Release, respectively. The quantified association of Ask1 and Nnf1 with CEN3 is shown on the right panel. (B) Centromere localization in HU-arrested yeast cells. G1-arrested TUB1-mCherry CEN4-GFP cells were released into YPD medium containing 200 mM HU. After incubation at 30°C for 2 hrs, cells were collected for the examination of fluorescence signals. The percentage of cells with different Cen4-GFP distribution is shown on the right.
Figure 2.
Premature spindle elongation and abnormal kinetochore distribution in HU-arrested ask1-3 mutant cells.
(A) ask1-3 and dam1-DDD mutants show HU sensitivity. Cells with the indicated genotypes were grown to saturation. After 10-fold dilution, the cells were spotted onto YPD plates with and without 100 mM HU. The plates were incubated at 25°C for 4 days before they were scanned. (B) HU treatment results in dramatic anaphase entry delay in ask1-3 cells. G1-arrested WT and ask1-3 cells were released into YPD medium with and without 200 mM HU at 25°C. HU was washed off after 2 hr incubation and the cells were then resuspended in 25°C YPD medium. α-factor was added to block the second round of cell cycle. Cells were collected at the indicated time points for DAPI staining. (C) The spindle length in WT and ask1-3 mutants after HU treatment. G1-arrested cells (TUB1-GFP and ask1-3 TUB1-GFP) were released into 25°C YPD medium containing 200 mM of HU for 2 hrs. The cells were then collected, fixed with formaldehyde, and subjected to fluorescence microscopy. The spindle morphology is shown in the left panel and the percentage of cells with different spindle lengths is shown on the right. More than 100 cells were measured for spindle length for each strain. (D) Premature spindle elongation and abnormal kinetochore localization in HU-arrested ask1-3 cells. G1-arrested TUB1-mCherry MTW1-3GFP and ask1-3 TUB1-mCherry MTW1-3GFP were released into medium containing 200 mM HU and incubated for 4 hrs at 25°C. Cells were collected and fixed for fluorescence microscopy. Mtw1-GFP localization and spindle morphology in the representative cells are shown in the top panel; the percentage of cells with elongated spindles or two separated kinetochore clusters is shown in the bottom panel.
Figure 3.
The abnormal kinetochore localization in HU-arrested ask1-3 cells is not a result of bipolar attachment.
G1-arrested NUF2-mCherry CEN4-GFP and ask1-3 NUF2-mCherry CEN4-GFP were released into YPD medium containing 200 mM HU for 4 hrs at 25°C. Cells were collected and fixed for fluorescence microscopy. The localization of Nuf2-mCherry and Cen4-GFP falls into two categories (A and B). (A) A representative cell with a single Nuf2-mCherry cluster and Cen4-GFP dot. (B) A representative cell with two Nuf2 clusters but one Cen4-GFP dot. (C) The percentage of cells with phenotypes shown in A and B.
Figure 4.
The anaphase entry delay in ask1-3 mutant cells after HU treatment.
(A) ask1-3 cells exhibit delayed chromosome segregation after HU treatment. G1-arrested NUF2-mCherry CEN4-GFP and ask1-3 NUF2-mCherry CEN4-GFP cells were released into YPD medium containing 200 mM HU and incubated at 25°C. Two hours later, HU was washed off and the cells were released into YPD medium. α-factor was added back to block the second round of cell cycle. Cells were collected for the examination of fluorescence signals. The localization of Nuf2 and Cen4 at the indicated times is shown in the top panel. The percentage of budded cells with one Cen4-GFP dot is shown in the bottom panel. (B) ask1-3 cells exhibit spindle checkpoint-dependent anaphase entry delay after HU treatment. G1-arrested PDS1-myc, ask1-3 PDS1-myc, and ask1-3 mad1Δ PDS1-myc cells were released into YPD medium containing 200 mM HU for 2 hrs. After HU was washed off, the cells were released into YPD medium and incubated at 25°C. The cells were collected at the indicated time points for protein preparation and DAPI staining. Pgk1 protein levels are shown as a loading control. (C) Deletion of tension checkpoint SGO1 partially suppresses the anaphase entry delay in ask1-3 mutants after HU treatment. Cells with indicated genotypes were treated as described in B. Here shows the percentage of large budded cells with a single Cen4-GFP dot after HU wash off.
Figure 5.
Deletion of CIN8 partially suppresses the phenotypes of ask1-3 mutants.
(A) Deletion of CIN8 partially suppresses the HU sensitivity of ask1-3 mutants. Cells with indicated genotypes were grown to saturation and then 10-fold diluted and spotted onto YPD plates with and without 100 mM HU. The plates were incubated at 25°C for 4 days before being scanned. (B) cin8Δ mutation suppresses spindle elongation in HU-treated ask1-3 mutants. Asynchronous cells with indicated genotypes were grown to mid-log phase and HU was then added to the cell cultures to a final concentration of 200 mM. After 2 hr incubation at 25°C, cells were fixed for fluorescence microscopy. The spindle morphology in some representative cells is shown in the top panel and the percentage of cells with different spindle length and the average spindle length are shown in the bottom panel. (C) cin8Δ mutation partially suppresses the abnormal kinetochore distribution phenotype in HU-treated ask1-3 mutant cells. G1-arrested WT, ask1-3 and cin8Δ ask1-3 cells with MTW1-GFP were released into YPD medium containing 200 mM HU and incubated at 25°C for 4 hrs. Cells were collected and fixed for the examination of Mtw1-GFP signal. Shown here is the percentage of cells with two clear GFP clusters. (D) Deletion of CIN8 partially suppresses the anaphase entry delay in ask1-3 mutants after HU treatment. G1-arrested WT, cin8Δ, ask1-3 and cin8Δ ask1-3 cells were released into YPD medium containing 200 mM HU for 2 hrs. After HU was washed off, the cells were released into 25°C YPD medium and collected at the indicated time points for DAPI staining. The percentage of budded cells with undivided nuclei is shown.
Figure 6.
Overexpression of ASE1 or CIN8 results in spindle elongation and abnormal kinetochore distribution in the presence of HU.
(A) Overexpression of ASE1 or CIN8 leads to spindle elongation in HU-arrested cells. G1-arrested TUB1-GFP cells with a vector, PGAL-ASE1 or PGAL-CIN8 plasmid were released into galactose medium containing 200 mM HU and incubated at 30°C for 2.5 hrs. The cells were then collected and fixed for fluorescence microscopy. The spindle morphology in some representative cells is shown. (B) The percentage of cells with different spindle lengths (<1.5 µM; 1.5–2.5 µM; >2.5 µM). The spindle length of more than 100 cells was measured for each sample. (C) Overexpression of ASE1 and CIN8 results in scattered kinetochore distribution along the spindle in HU-arrested cells. NUF2-mCherry CEN4-GFP cells with a vector, PGAL-ASE1, or PGAL-CIN8 plasmid were grown to mid-log phase in raffinose medium. Cells were synchronized in G1 phase with α factor and then released into galactose medium containing 200 HU and incubated at 30°C. Cells were collected after 4 hr incubation for the examination of fluorescence signals. (D) Overexpression of ASE1 or CIN8 induces abnormal kinetochore distribution in HU- but not in cdc13-1-arrested cells. G1-arrested cdc13-1 NUF2-mCherry cells with a vector or PGAL-ASE1, PGAL-CIN8 plasmid were released into galactose medium and incubated at 34°C. Nuf2 distribution was examined after 4 hr incubation. Shown here is the percentage of budded cells with scattered Nuf2 signals in HU-arrested cells (experiment C) and in cdc13-1-arrested cells.
Figure 7.
Overexpression of ASE1 or CIN8 in HU-arrested cells causes anaphase entry delay.
(A) Overexpression of ASE1 or CIN8 in HU-arrested cells causes delayed anaphase entry after HU is washed off. G1-arrested CEN4-GFP cells with a vector, PGAL-ASE1 or PGAL-CIN8 plasmid were released into galactose medium containing 200 mM HU for 3 hrs at 30°C. HU was washed off and the cells were released into glucose medium and incubated at 30°C. α-factor was added to block the second round of cell cycle. Cells were collected at the indicated times and fixed for fluorescence microscopy. The top panel shows cells collected at 2.5 hr after HU release. The percentage of budded cells with a single Cen4-GFP is shown in the bottom panel. (B) Overexpression of ASE1 and CIN8 leads to spindle checkpoint-dependent anaphase entry delay. G1-synchronized WT and mad1Δ cells with a vector, PGAL-ASE1 or PGAL-CIN8 plasmid, were released into galactose medium containing 200 mM HU for 3 hrs at 30°C. The cells were released into 30°C glucose medium containing α-factor. Cells were collected at the indicated times for DAPI staining. The percentage of budded cells with undivided nuclei is shown.