The spindle and kinetochore-associated (Ska) complex enhances binding of the anaphase-promoting complex/cyclosome (APC/C) to chromosomes and promotes mitotic exit

Abstract

The spindle and kinetochore-associated (Ska) protein complex is a heterotrimeric complex required for timely anaphase onset. The major phenotypes seen after small interfering RNA-mediated depletion of Ska are transient alignment defects followed by metaphase arrest that ultimately results in cohesion fatigue. We find that cells depleted of Ska3 arrest at metaphase with only partial degradation of cyclin B1 and securin. In cells arrested with microtubule drugs, Ska3-depleted cells exhibit slower mitotic exit when the spindle checkpoint is silenced by inhibition of the checkpoint kinase, Mps1, or when cells are forced to exit mitosis downstream of checkpoint silencing by inactivation of Cdk1. These results suggest that in addition to a role in fostering kinetochore-microtubule attachment and chromosome alignment, the Ska complex has functions in promoting anaphase onset. We find that both Ska3 and microtubules promote chromosome association of the anaphase-promoting complex/cyclosome (APC/C). Chromosome-bound APC/C shows significantly stronger ubiquitylation activity than cytoplasmic APC/C. Forced localization of Ska complex to kinetochores, independent of microtubules, results in enhanced accumulation of APC/C on chromosomes and accelerated cyclin B1 degradation during induced mitotic exit. We propose that a Ska-microtubule-kinetochore association promotes APC/C localization to chromosomes, thereby enhancing anaphase onset and mitotic exit.

FIGURE 1:

Depletion of Ska complex components slows alignment and arrests cells at metaphase. (A) HeLa H2B-GFP cells transfected with control siRNA or with pools of siRNA against Ska1, Ska2, and Ska3 alone or in combination at 25 nM were imaged approximately 27 h after transfection. The time taken to progress through prometaphase and metaphase was determined for every cell and plotted. A strict criterion was used to define metaphase alignment, which required that every chromosome was at the metaphase plate for at least two consecutive frames. The graph depicts the time taken to align chromosomes (blue bar), time spent at metaphase in cells that initiated anaphase (yellow bar), and time spent at metaphase in cells that initiated cohesion fatigue (red bar). The asterisk denotes a cell that exited mitosis after undergoing cohesion fatigue. Ska-depleted cells were delayed in chromosome alignment, although ultimately cells reached metaphase. The majority of Ska-depleted cells delayed or arrested at metaphase. (B) Mitotic phenotypes observed after depletion of Ska proteins. The graph denotes the percentage of cells that initiate anaphase without delay, with delay (>80 min at metaphase), or remain arrested at metaphase, eventually undergoing cohesion fatigue. The majority of Ska-depleted cells either delayed or arrested at metaphase. See also Supplemental Figure S1 and Supplemental Movies S1 and S2.

FIGURE 2:

Ska3-depleted cells are inefficient in cyclin B1 degradation and inactivate Cdk1 incompletely. (A) HeLa cells were transfected with control or Ska3 siRNA at 25 nM final concentration. At 24 h after transfection, control siRNA cultures were treated with 3.3 μM nocodazole for 8 h. Ska3-depleted cultures were washed to remove any preexisting mitotic cells and then treated with dimethyl sulfoxide (DMSO; control) or 3.3 μM nocodazole for 8 h. Mitotic cells were collected and assayed by Western blotting. Ska3-depleted cells degraded 40% cyclin B1 compared with nocodazole-arrested cells. (B) Cultures of control and Ska3-depleted cells were rinsed to remove any preexisting mitotic cells and then blocked in 3.3 μM nocodazole for 4 h. Mitotic cells were collected and then released into fresh medium without drugs. Samples were collected hourly for 6 h. Cdk1 was precipitated using p13-Suc1 beads, and associated cyclin B1 was determined by immunoblotting. Cdk1 kinase activity on histone H1 was also measured. Whole-cell lysates were blotted for Cdk1, cyclin B1, and Ska3. (The ns in the Ska3 blot indicates a nonspecific band that runs between the phosphorylated and dephosphorylated forms of Ska3.) (C) Blot quantification revealed that Cdk1-associated cyclin B1 was sixfold higher in Ska3-depleted cells vs. control cells at 6 h after nocodazole wash out (NWO). (D) Control or Ska3-depleted HeLa H2B-GFP cells were treated with DMSO (control), 3.3 μM nocodazole, or 10 μg/ml cycloheximide (CHX) and then imaged for 24 h. CHX induced mitotic exit in Ska3-depleted cells. The numbers on top of the bars in the graph indicate the number of cells that exited/total number of mitotic cells. (E) Scatterplot depiction of time taken to initiate anaphase in control (25 ± 2.8 min) and Ska3-depleted cells (222 ± 12.9 min) treated with CHX. Each dot represents one cell; long horizontal line depicts mean, and whiskers denote SEM.

FIGURE 3:

Ska3 depletion delays mitotic exit in cells treated with microtubule drugs. (A) HeLa H2B-GFP cells were transfected with control or Ska3 siRNA at 25 nM for 24 h. After washing off mitotic cells, cultures were treated with 3.3 μM nocodazole for 4 h. Reversine, 1 μM, was added, and time taken to exit mitosis in every cell was determined by imaging. Ska3-depleted cultures were delayed in mitotic exit (40 ± 1.5 min) compared with control cultures (29 ± 0.9 min). (B) Control and Ska3-depleted cells were treated as described but then released from nocodazole arrest into fresh medium for 30 min to allow spindles to form. Cells were then treated with 2 μM Taxol. Reversine, 1 μM, was added, and time taken to exit mitosis in every cell was determined by imaging. Ska3-depleted cells (42 ± 0.5 min) delayed mitotic exit compared with control cells (28 ± 0.9 min). Each dot represents one cell; the long horizontal line depicts mean, and whiskers denote SEM. (C) HeLa H2B-GFP cells were transfected with cyclin B1-mCherry and then transfected with control or Ska3 siRNA at 50 nM. Approximately 27 h posttransfection, cells were treated with 3.3 μM nocodazole. Flavopiridol, 10 μM, was added, and cyclin B1 degradation was recorded by measuring the decay of mCherry fluorescence. Ska3-depleted cells showed slower cyclin B1 degradation (p < 0.05). (D) Control and Ska3-depleted cells were treated as described but were then released from nocodazole arrest into fresh medium for 30 min to allow spindles to form. Cells were then treated with 2 μM Taxol. Then 10 μM flavopiridol was added and cyclin B1-mCherry degradation was measured. Overall, Taxol-arrested cells showed more rapid cyclin B1 degradation compared with nocodazole-arrested cells. Ska3-depleted cells showed slower cyclin B1 degradation (p < 0.005). Error bars indicate SEM, and cells were quantified from at least three independent experiments. The time taken to degrade 50% of cyclin B1 was calculated for every cell and used to determine statistical significance between control and Ska3-depleted cells. See also Supplemental Figures S2 and S3 and Supplemental Movie S3.

FIGURE 4:

Microtubules promote APC/C accumulation on chromosomes. (A) HeLa cells grown on coverslips were treated with DMSO (control) or nocodazole (3.3 μM) for 3 h. To accumulate cells in Taxol, cultures were released after 2 h from nocodazole into fresh media for 30 min to allow spindle assembly and then treated with 2 μM Taxol for 30 min. Immunofluorescence was done, and Ska3 at kinetochores was quantified. Representative images are shown in the image panel. Scale bar, 10 μm. Ska3 was maximal at metaphase kinetochores. Taxol-treated cells accumulated twice as much Ska3 at kinetochores as nocodazole-treated cells. At least five cells in each condition were measured, and mean with SEM was plotted. (B) HeLa cells were released from thymidine block into 3.3 μM nocodazole–containing media. To collect cells in Taxol, cells were released from nocodazole into media without drugs to allow spindle formation before being treated with 2 μM Taxol for 30 min. MG132-treated cells were collected by releasing cells from nocodazole into 25 μM MG132 for 1.5 h. Chromosomes were isolated from nocodazole-, Taxol-, and MG132-treated cells, and associated APC/C components (Cdc27, Cdc16) and MCC components (Mad2, BubR1, Cdc20) were determined by Western blotting. Taxol- and MG132-treated cells show ∼1.5 times more Cdc27 associated with chromosomes than nocodazole-treated cells. Taxol-treated cells also show lower levels of MCC components associated with chromosomes than do nocodazole-treated cells. (C) HeLa cells were treated with 3.3 μM nocodazole as in B, and cytoplasmic and chromosome fractions were isolated. Cell equivalent volumes were loaded on SDS–PAGE gel to compare relative concentration of APC/C in cytoplasmic and chromosome fractions. (D) The cytoplasmic fraction was diluted 15-fold, and fractions were tested for the ability to ubiquitylate myc-cyclin B1^(1-102) in vitro. The control lane consisted of ubiquitylation assay reagents lacking an APC/C source. The depletion of the nonubiquitylated myc-cyclin B1^(1-102) was quantified from three independent experiments, and mean with SEM is depicted. Although approximately twice the APC/C concentration was present in the cytoplasmic fraction compared with the chromosome fraction, the chromosome fraction exhibited stronger cyclin B1 ubiquitylation activity.

FIGURE 5:

Ska3 promotes APC/C association with chromosomes. (A) HeLa cells transfected with control or Ska3 siRNA at 25 nM for 26 h were treated with 3.3 μM nocodazole for 16 h. Chromosomes were isolated to determine the amount of associated Cdc27 and Cdc16 by Western blotting. Total amounts of APC/C were similar in whole cells, but Ska3 depletion decreased Cdc27 levels on chromosomes by ∼50%. (B) HeLa cells transfected with control or Ska3 siRNA at 25 nM were treated with 2 mM thymidine for 24 h and then released into 3.3 μM nocodazole for 12 h. Mitotic cells were collected and released from nocodazole into media containing 25 μM MG132 for 3 h. Chromosomes were isolated, and the amount of APC/C associated was determined by Western blotting. Total amounts of APC/C in whole cells were similar, but Ska3 depletion decreased Cdc27 levels on chromosomes by ∼60%. (C) HeLa cells were treated with 2 mM thymidine for 12 h to arrest in S phase. Cells were released from the thymidine block and transfected with control or Hec1 siRNA at 75 nM. At 12 h later, cells were transfected again with control or Hec1 siRNA at 75 nM and treated with 2 mM thymidine. At 24 h later, cells were released from the second thymidine block into media containing 3.3 μM nocodazole. At 12 h later, control and Hec1 siRNA–transfected cells were treated with 25 μM MG132 to prevent mitotic exit. After 2 h, mitotic cells were collected, chromosomes were isolated, and the amount of APC/C associated was determined by Western blotting. Total amounts of APC/C in whole cells were similar, but Hec1 depletion decreased Cdc27 levels on chromosomes by 80%. (D) HeLa cells grown on coverslips were treated as in C. On release from second thymidine block, control and Hec1 siRNA–transfected cells were treated with 3.3 μM nocodazole and 25 μM MG132 to prevent mitotic exit. One control coverslip was released into media containing CENP-E inhibitor GSK 923295 at 300 nM and 3.3 μM nocodazole. After 2 h, all coverslips were fixed and immunolabeled with anti-Ska3 antibody and ACA. Ska3 at kinetochores was quantified. Quantification showed that CENP-E inhibition by GSK 923295 did not affect Ska3 levels at kinetochores, whereas Hec1 depletion abolished Ska3 recruitment to kinetochores in the absence of microtubules. Kinetochores in at least five cells in each condition were quantified, and the mean with SEM is plotted. (E) HeLa cells were arrested in S phase by addition of 2 mM thymidine. At 24 h later, the cells were released into media containing 3.3 μM nocodazole and 300 nM GSK 923295. At 12 h later, mitotic cells were collected, and chromosomes were isolated to determine the amount of associated APC/C. CENP-E inhibition did not affect amount of APC/C associated with chromosomes.

FIGURE 6:

Targeting Ska complex to the kinetochore in the absence of spindle microtubules enhances cyclin B1 degradation. (A) HeLa cells grown on coverslips were transfected with Mis12-GFP, Ska1-GFP, or Mis12Ska1-GFP plasmids. Approximately 27 h posttransfection, 3.3 μM nocodazole was added for 2 h. Cells were fixed and immunolabeled with anti-Ska3 antibody and ACA. Quantification of Ska3 levels showed an ∼1.5-fold increase in kinetochore-associated Ska3 levels upon expression of Mis12Ska1-GFP plasmid. Kinetochores in at least eight cells for each condition were quantified, and the mean with SEM is plotted. Comparison of Ska3 at the kinetochore based on GFP expression levels in cells transfected with the Mis12 plasmids shows increased recruitment of Ska3 to kinetochore in cells expressing high amount of untargeted Ska1-GFP plasmid (Supplemental Figure S5A). (B) HeLa cells transfected with Mis12-GFP, Ska1-GFP, or Mis12Ska1-GFP plasmids were treated with 3.3 μM nocodazole for 15 h. Mitotic cells were collected, and chromosomes were isolated to determine the amount of associated Cdc27 and Cdc16. Blot quantification showed an approximately threefold increase in chromosomal Cdc27 in Mis12Ska1-GFP–expressing cells compared with Mis12-GFP–expressing cells. A lesser increase in chromosomal Cdc27 was also observed by overexpressing untargeted Ska1-GFP plasmid. However, Ska1-GFP plasmid was expressed at higher levels than Mis12Ska1-GFP plasmid (Supplemental Figure S5B). (C) HeLa cells were cotransfected with cyclin B1-mCherry plasmid and either Mis12-GFP, Ska1-GFP, or Mis12Ska1-GFP plasmid. Approximately 36 h after transfections, cells were treated with 3.3 μM nocodazole. Then 10 μM flavopiridol was added to induce cyclin B1 degradation. Kinetochore-targeted Mis12Ska1-GFP–expressing cells showed the strongest enhancement of cyclin B1 degradation (**p < 0.005, ****p < 0.0005 ). Although cells expressing Ska1-GFP were slightly accelerated in cyclin B1 degradation compared with Mis12-GFP–expressing cells, the two populations were not statistically significant (p = 0.26). Comparison of total GFP fluorescence levels at the initial time indicated that Ska1-GFP was expressed on average at higher levels than Mis12Ska1-GFP (Supplemental Figure S5D). Error bars indicate SEM, and cells were quantified from at least three independent experiments. The time taken to degrade 50% of cyclin B1 was calculated for every cell and used to determine statistical significance between different groups.