Phosphatase-regulated recruitment of the spindle- and kinetochore-associated (Ska) complex to kinetochores

Abstract

Kinetochores move chromosomes on dynamic spindle microtubules and regulate signaling of the spindle checkpoint. The spindle- and kinetochore-associated (Ska) complex, a hexamer composed of two copies of Ska1, Ska2 and Ska3, has been implicated in both roles. Phosphorylation of kinetochore components by the well-studied mitotic kinases Cdk1, Aurora B, Plk1, Mps1, and Bub1 regulate chromosome movement and checkpoint signaling. Roles for the opposing phosphatases are more poorly defined. Recently, we showed that the C terminus of Ska1 recruits protein phosphatase 1 (PP1) to kinetochores. Here we show that PP1 and protein phosphatase 2A (PP2A) both promote accumulation of Ska at kinetochores. Depletion of PP1 or PP2A by siRNA reduces Ska binding at kinetochores, impairs alignment of chromosomes to the spindle midplane, and causes metaphase delay or arrest, phenotypes that are also seen after depletion of Ska. Artificial tethering of PP1 to the outer kinetochore protein Nuf2 promotes Ska recruitment to kinetochores, and it reduces but does not fully rescue chromosome alignment and metaphase arrest defects seen after Ska depletion. We propose that Ska has multiple functions in promoting mitotic progression and that kinetochore-associated phosphatases function in a positive feedback cycle to reinforce Ska complex accumulation at kinetochores.

Keywords: Anaphase-promoting complex/cyclosome; Cell cycle; Microtubules; Mitosis; Spindle checkpoint.

Fig. 1.

Phosphatases PP1 and PP2A promote Ska recruitment and normal progression through mitosis. (A) HeLa cells grown on coverslips were transfected with control, PP1γ or PP2A Aα siRNA. 45 h after transfection, cells were treated with 3.3 μM nocodazole for 3 h and then prepared for immunofluorescence. Ska3 at kinetochores was quantified. PP1γ or PP2A Aα depletion reduced Ska3 at kinetochore. (B) Bar graph depicting mean fluorescence intensity of Ska3 at kinetochores normalized to anti-centromere antibody (ACA). (C) HeLa cells grown on coverslips were transfected with control, Plk1 or BubR1 siRNA. 45 h after transfection cells were treated for 3 h with 3.3 μM nocodazole and prepared for immunofluorescence. Ska3 at kinetochores was quantified. (D) Plk1 or BubR1 depletion reduced Ska3 at kinetochores. (E) HeLa H2B-GFP cells were transfected with PP1γ or PP2A Aα siRNA individually or in combination at 50 nM final concentration. Approximately 30 h post transfection, mitotic progression was followed by video microscopy. Bar graph depicts mean chromosome alignment times. PP1 depletion causes a slight delay in alignment. PP2A Aα depletion shows a stronger delay while combined depletion of PP1γ and PP2A Aα shows the most robust delay. (F) Bar graph depiction of time taken to initiate anaphase in individual cells. PP1γ or PP2A Aα depletions either individually or in combination delay mitotic progression. The combined depletions are more penetrant suggesting a compensatory or redundant role for the phosphatases when depleted individually. (G) Bar graph showing percentage of siRNA-treated cells arrested in metaphase and undergoing cohesion fatigue after depleting PP1γ, PP2A Aα or both. Graphs B, D-F show mean±s.e.m., unpaired Student's t-test, two-tailed distribution.

Fig. 2.

Kinase and phosphatase inhibitors regulate Ska recruitment to kinetochores and APC/C recruitment to mitotic chromosomes. (A) HeLa cells grown on glass coverslips were treated with 3.3 µM nocodazole and 25 μM MG132 for 2 h. Coverslips were individually treated with DMSO (control), 1μM Reversine (Rev, Mps1 inhibitor), 25 µM ZM447439 (ZM, Aurora B inhibitor) or 0.5 μM Okadaic acid (OA, phosphatase inhibitor) for an additional 1 h. Cells were pre-extracted, fixed and labeled with anti-Ska3 and ACA antibodies, then processed for immunofluorescence. (B) Quantification shows that Aurora B or Mps1 inhibition increased Ska3 at kinetochores while phosphatase inhibition decreased Ska3 at kinetochores. (C) HeLa cells, treated as in A, were collected. A portion was lysed directly into sample buffer (whole cells) and the rest used to prepare isolated mitotic chromosomes (chromosomes). Western blots were labeled with antibodies to histone H3 and Cdc27, a component of the APC/C. In whole cell lysate, most Cdc27 is hyperphosphorylated and shows slower mobility. That bound to mitotic chromosomes shows lower phosphorylation and higher electrophoretic mobility. (D) Quantification of western blots shows that like Ska, Cdc27 levels increase in chromosomes treated with Aurora B or Mps1 inhibitors and decrease in OA-treated cells. Graphs B and D show mean±s.e.m., unpaired Student's t-test, two-tailed distribution.

Fig. 3.

Expression of a Nuf2PP1 fusion in the presence of intact spindle microtubules promotes Ska recruitment to kinetochores and APC/C recruitment to chromosomes. (A) HeLa cells grown on coverslips were transfected with Nuf2-mCherry, PP1-mCherry or Nuf2PP1-mCherry. 36 h after transfection 3.3 μM nocodazole was added to cells for 3 h and cells were prepared for immunofluorescence. (B) PP1 localization to kinetochores was quantified. Nuf2PP1 expression increased PP1 at kinetochores by 50% compared to controls. (C) HeLa cells transfected with Nuf2-mCherry, PP1-mCherry or Nuf2PP1-mCherry were released from 330 nM nocodazole into MG132 for 2 h. Cells were prepared for immunofluorescence and PP1 at kinetochores was measured. (D) HeLa cells were treated as in A and B, then prepared for immunofluorescence. In cells arrested in nocodazole, additional PP1 at kinetochores did not measurably increase recruitment of Ska3. (E) HeLa cells were treated as in C and Ska3 at kinetochores was measured. In cells released from nocodazole and allowed to progress to metaphase in MG132, additional PP1 at kinetochores increased recruitment of Ska3. (F) Quantification of Ska3 levels in metaphase arrested cells expressing Nuf2-mCherry, PP1-mCherry and Nuf2PP1-mCherry. (G) Cells were treated as in A and C and then used to prepare whole cell lysate (Total) and mitotic chromosomes (Chromosome). These samples were then blotted for APC2, Histone H3 and for mCherry to reveal expression levels of the transgenes. In nocodazole, targeting PP1 to kinetochores did not significantly increase the amount of chromosome-associated APC/C. Chromosome bound APC/C is similar in cells expressing Nuf2-mCherry, PP1-mCherry, or Nuf2PP1-mCherry. (H) Cells were treated as in C and then used to prepare whole cell lysate (Total) and mitotic chromosomes (Chromosome). In metaphase cells, targeting PP1 to kinetochores increases the level of chromosome-associated APC/C. Quantification of this result is depicted in Fig. S2C. Graphs B, C and F show mean±s.e.m., unpaired Student's t-test, two-tailed distribution.

Fig. 4.

Expression of Nuf2PP1 fusion partially rescues mitotic defects caused by Ska depletion. (A) Tracking of fates for individual HeLa cells stably expressing histone H2B-GFP and transfected with Nuf2-mCherry, PP1-mCherry or Nuf2PP1-mCherry. 12 h after transfection, cells were treated with control or Ska3 siRNA. Time-lapse imaging was initiated 24 h after siRNA transfection. The mCherry constructs did not affect cells transfected with control siRNA so only the Nuf2-mCherry control is shown here. The other controls are summarized in Fig. S2D. The time intervals to align chromosomes, initiate anaphase or undergo metaphase arrest/cohesion fatigue were measured. Expression of Nuf2-PP1 partially rescued alignment times, metaphase delays, and cohesion fatigue induced by Ska3 depletion. (B) Time intervals for chromosome alignment in control siRNA or Ska3 siRNA for Nuf2-mCherry, PP1-mCherry or Nuf2PP1-mCherry expressing cells. Expression of Nuf2PP1-mCherry partially reduced alignment in Ska3-depleted cells. Horizontal lines indicate mean and whiskers indicate standard error. P values were determined using the Mann–Whitney test. (C) The percentages of cells that arrested at metaphase for the duration of imaging and/or underwent cohesion fatigue in control or Ska3-depleted cells expressing Nuf2-mCherry, PP1-mCherry or Nuf2PP1-mCherry. In Ska3-depleted cells, expression of Nuf2PP1 increased the proportion of cells that underwent anaphase onset compared to expression of Nuf2 or PP1.