To screen for phosphatases counteracting Aurora B during anaphase, we used a fluorescence resonance energy transfer (FRET) biosensor for Aurora B substrate phosphorylation (Fuller et al., 2008
). A conformational change in the biosensor upon Aurora B phosphorylation reduces FRET between a CFP for energy transfer (CyPet) donor and a YFP for energy transfer (YPet) acceptor (Fuller et al., 2008
). To quantify phosphorylation levels of the biosensor at specific stages of mitosis, we recorded YPet and CyPet emission images of unsynchronized live HeLa Kyoto cells stably expressing a chromatin-targeted version of the biosensor (Fuller et al., 2008
) and classified mitotic stages based on chromatin morphology using in-house–developed, supervised machine-learning methods (CellCognition; ; Held et al., 2010
). The fully automated assay was validated by RNAi depletion of Aurora B (Fig. S1, A and B
), which efficiently inhibited biosensor phosphorylation in mitotic cells ().
Figure 1. FRET biosensor–based RNAi screen for Aurora B–counteracting phosphatases. (A) Live-cell assay for Aurora B phosphorylation. Live HeLa Kyoto cells stably expressing a histone 2B (H2B)–targeted Aurora B FRET biosensor (Fuller et (more ...)
Three different siRNA oligonucleotides targeting each of a genome-wide set of 225 annotated human phosphatases, including catalytic, regulatory, and scaffold subunits (Schmitz et al., 2010
), were transfected individually into the reporter cells. Candidate hits that caused biosensor hyperphosphorylation in late anaphase cells 42 h after transfection were ranked based on mean z scores calculated per target gene from all respective siRNA oligonucleotides and two experimental replicates ( and Table S1
). The five top-scoring target genes encode Repo-Man (also termed CDCA2), Sds22 (also termed PPP1R7), PPP2CA, PPP2R1A, and PPP2R2A (also termed B55α).
Repo-Man is a PP1-targeting subunit, which had previously been shown to counteract Aurora B on anaphase chromatin (Trinkle-Mulcahy et al., 2006
; Qian et al., 2011
; Vagnarelli et al., 2011
). Sds22 had previously been shown to target PP1 to kinetochores during preanaphase stages to regulate Aurora B (Posch et al., 2010
). Three other PP1-targeting subunits, PPP1R3C, PPP1R12A (also termed MYPT1), and PPP1R2 (also termed inhibitor 2), also scored relatively high in the RNAi screen (Table S1), yet, they were not further analyzed in this study. PPP2CA, PPP2R1A, and PPP2R2A form a heterotrimeric PP2A complex that reverts Cdk1 substrate phosphorylations during mitotic exit (Mochida et al., 2009
; Schmitz et al., 2010
). Several siRNAs caused decreased biosensor phosphorylation ( and Table S1), yet, they were not further considered in our study, which aims at the identification of phosphatases counteracting Aurora B.
To validate the phenotypes of the top-ranking candidate hits, we first measured siRNA target protein levels (Fig. S1). Three different siRNAs depleted Repo-Man to 7–13% (Fig. S1, C and D), whereas Sds22 was only reduced to ~50% by three different siRNAs (Fig. S1, E and F). Confocal time-lapse imaging showed that depletion of Repo-Man or Sds22 by any of the different siRNA oligonucleotides delayed dephosphorylation of the biosensor (n
≥ 12 cells; and Videos 1–3
). Low variability between individual cells (Fig. S2, A and B
) suggests that residual protein levels (Fig. S1) are distributed relatively homogenously within the cell population. Codepletion of Sds22 and Repo-Man further delayed dephosphorylation of the biosensor ().
Figure 2. Repo-Man and Sds22 are required for timely dephosphorylation of the chromatin-targeted FRET biosensor. (A) Confocal time-lapse images of HeLa Kyoto cells expressing the H2B-targeted Aurora B FRET biosensor, 42 h after transfection of a nontargeting control (more ...)
RNAi depletion of PP1 catalytic subunits PP1α or PP1γ (Fig. S1, G–L) also caused delayed biosensor dephosphorylation (n
≥ 7 cells; Fig. S2 C), consistent with the proposed function of Repo-Man and Sds22 as PP1-targeting subunits (Ohkura and Yanagida, 1991
; Stone et al., 1993
; MacKelvie et al., 1995
; Renouf et al., 1995
; Dinischiotu et al., 1997
; Trinkle-Mulcahy et al., 2006
). Even though depletion of PP1β alone had little effect, its codepletion with the two other PP1 catalytic subunits (Fig. S1, G–L) further increased the delay in biosensor dephosphorylation (Fig. S2 C), suggesting functional redundancy between PP1 catalytic subunits that may conceal strong phenotypes after individual subunit depletion. In conclusion, PP1 and its targeting subunits Repo-Man and Sds22 contribute to timely Aurora B substrate dephosphorylation on anaphase chromatin.
Confocal time-lapse imaging of cells in which the PP2A catalytic (PPP2CA), scaffold (PPP2R1A), or regulatory (PPP2R2A/B55α) subunits were depleted individually or altogether (Fig. S1, M–R), however, could not confirm their requirement for timely Aurora B biosensor dephosphorylation (n
≥ 18 cells; Fig. S2 D). We suspect that the false-positive scoring in our primary screen may have been caused by misclassification of mitotic stages owing to perturbed chromatin morphology (Schmitz et al., 2010
). Because all of the other PP2A regulatory subunits scored within the SD of the screening dataset, we conclude that PP2A is not rate limiting for Aurora B substrate dephosphorylation on anaphase chromatin at the protein depletion levels achieved in our RNAi experiments.
The delayed biosensor dephosphorylation in Sds22 or Repo-Man RNAi cells may be caused by incomplete Aurora B removal from chromatin or by inefficient dephosphorylation of its substrates. Confocal imaging of HeLa cells stably expressing Aurora B–EGFP from its endogenous promotor (Poser et al., 2008
) showed that neither Repo-Man nor Sds22 RNAi affected Aurora B localization, in contrast to an Mklp2 RNAi–positive control (; Gruneberg et al., 2004
; Hümmer and Mayer, 2009
). Immunofluorescence staining by phosphospecific antibodies against the autophosphorylation site Thr232 on Aurora B (an essential activation site; Yasui et al., 2004
; Sessa et al., 2005
; Ruchaud et al., 2007
) and INCENP phosphorylated on Ser893/Ser894 (Aurora B–dependent phosphorylation sites essential for full Aurora B activation; Bishop and Schumacher, 2002
; Honda et al., 2003
; Sessa et al., 2005
; Salimian et al., 2011
) showed that Sds22 or Repo-Man RNAi did not affect the levels of active Aurora B on anaphase chromosomes ( and S2 [E–G]). Thus, Repo-Man and Sds22 contribute to anaphase dephosphorylation of chromosomal Aurora B sites independent of Aurora B translocation or inactivation.
Figure 3. Repo-Man and Sds22 function downstream of Aurora B. (A) Confocal time-lapse imaging of HeLa Kyoto cells stably expressing Aurora B–EGFP and H2B-mCherry. Time is shown in min/s. t = 0 min at chromosome segregation onset. Bars, 10 µm. (B) (more ...)
Elevated Aurora B levels on anaphase chromatin in Mklp2 RNAi cells correlated with delayed dephosphorylation of the chromatin-targeted biosensor (). The delay was further increased by codepleting Sds22 or Repo-Man (), yet, biosensor dephosphorylation reached similar levels as in control cells ~15 min after anaphase onset, despite the persistence of high levels of Aurora B on chromatin at these late time points (compare with ). The phosphatase activity that eventually dephosphorylates the biosensor in this experiment may emerge from residual amounts of the respective RNAi target proteins, from functional redundancy between Sds22 and Repo-Man, or from unknown other phosphatases.
Sds22 and Repo-Man may dephosphorylate chromosomal Aurora B substrates that had been phosphorylated before relocation of the kinase to the central spindle or counteract a sustained Aurora B activity emerging from the central spindle via a long-range spatial gradient (Fuller et al., 2008
) or from residual Aurora B on anaphase chromatin. Aurora B inhibition by ZM1 (Girdler et al., 2006
) briefly after anaphase onset significantly accelerated biosensor dephosphorylation in Repo-Man and Sds22 RNAi cells (), indicating that Aurora B continues to regulate substrate phosphorylation on chromosomes after anaphase onset. This could be either by direct substrate phosphorylation on chromosomes or indirectly by regulating counteracting phosphatases.
Sds22 or Repo-Man RNAi had minor effects on dephosphorylation of a cytoplasmic Aurora B phosphorylation FRET biosensor (; Fuller et al., 2008
). This suggests that Sds22 and Repo-Man act locally on chromosomes, which is in accordance with their reported localization to kinetochores and chromatin, respectively (Trinkle-Mulcahy et al., 2006
; Posch et al., 2010
To investigate the relevance of timely Aurora B substrate dephosphorylation, we recorded 3D confocal live-cell videos from HeLa cells stably expressing H2B-mCherry. Repo-Man or Sds22 depletion significantly increased the frequency of anaphase bridges and lagging chromosomes (n
> 500 cells in seven independent experiments; P < 0.001, using a two-sided binomial test; ). To test for on-target specificity of the phenotype, we transfected siRNAs specific for human Repo-Man or Sds22 in HeLa cell lines stably expressing the respective siRNA-resistant, EGFP-tagged mouse counterparts (Fig. S3, A–D
). Depletion of endogenous human Repo-Man or Sds22 in the cell lines expressing the corresponding mouse genes did not significantly increase the frequency of chromosome missegregation (Fig. S3, E and F). Thus, chromosome missegregation results from on-target depletion of Repo-Man or Sds22.
Figure 4. Repo-Man and Sds22 stabilize anaphase chromosome segregation. (A and B) Depletion of Repo-Man or Sds22 increases the incidence of anaphase bridges (A) and lagging chromosomes (B). Segregation defects were detected in confocal videos of live HeLa Kyoto (more ...)
Chromosome missegregation may result from defects before anaphase onset, like chromosome misattachment during early mitosis or cytokinesis failure in preceding cell divisions. The fraction of binucleated cells was not increased in cells depleted of Sds22 or Repo-Man (3.1 ± 0.5% in control RNAi cells, 2.8 ± 0.7% in Repo-Man RNAi cells, and 3.1 ± 0.3% in Sds22 RNAi cells; n > 1,900 cells per condition), indicating effective cytokinesis. We detected a small increase in chromosome misalignment in cells depleted of Sds22 (5.4 ± 1.8% compared with 4.5 ± 0.7% in control RNAi or 4.2 ± 1.0% in Repo-Man RNAi cells; n > 400 cells), yet, this fraction is much smaller than the fraction of cells with anaphase segregation errors ().
To gain more insight into the chromosome missegregation phenotype, we tracked kinetochores in 3D videos of live HeLa cells stably expressing EGFP–CENP-A (Jaqaman et al., 2010
). By measuring sister kinetochore distances, we determined segregation dynamics independent of spindle rotation and translation (). Repo-Man or Sds22 depletion had only a weak effect on the overall mean segregation speed (Fig. S3 G). However, a large fraction of individual kinetochores temporarily paused segregation or even transiently moved backward in cells depleted of Sds22 or Repo-Man (). The segregation pauses occurred at variable timing after anaphase onset, did not correlate with increased spindle rotation (Fig. S3, H–K), and were not simultaneously observed on adjacent chromosomes, indicating that irregular segregation results from a defect at individual chromosomes rather than an overall perturbation of the spindle.
To investigate whether the observed segregation defects may be a consequence of misregulated Aurora B substrate phosphorylation, we maintained high levels of Aurora B on anaphase chromatin using Mklp2 RNAi ( and ; Gruneberg et al., 2004
; Hümmer and Mayer, 2009
). Indeed, Mklp2 RNAi also induced transient pauses in poleward segregation ( and S3 K), consistent with a requirement for timely Aurora B substrate dephosphorylation for steady chromosome segregation.
Pauses in chromosome segregation may be caused by defective chromosome structure, incomplete resolution of sister chromatid arms, or by destabilized kinetochore–microtubule interactions. In metaphase, the kinetochore–microtubule affinity is regulated by differential phosphorylation of Aurora B sites on outer kinetochore components (Welburn et al., 2010
; DeLuca et al., 2011
). Ser100-phosphorylated Dsn1 (Welburn et al., 2010
) was indeed elevated on anaphase kinetochores of Repo-Man or Sds22 RNAi cells (), whereas total Dsn1 levels were unaffected ().
Figure 5. Repo-Man and Sds22 are required for timely dephosphorylation of the microtubule attachment factor Dsn1. (A and B) Immunofluorescence staining with an antibody against Dsn1 phosphorylated at Ser100 (pS100; Welburn et al., 2010; A) or total Dsn1 (Kline (more ...)
Misregulated phosphorylation of the kinetochore–microtubule interface may explain the segregation defects observed in Repo-Man and Sds22 RNAi cells. However, other defects like incompletely resolved sister chromatids may also contribute to chromosome segregation pauses. A previous study reported decreased phosphorylation levels of several Aurora B substrates in Sds22-depleted metaphase cells (Posch et al., 2010
). In contrast, Dsn1 Ser100 phosphorylation was significantly elevated during prometaphase and metaphase in Sds22 RNAi cells (). Even though the mechanism underlying differential regulation of Aurora B substrates by Sds22 is unclear, we also observed that depletion of Sds22, but not of Repo-Man, delayed progression from mitotic entry until anaphase, as previously reported (Fig. S3 L; Posch et al., 2010
Our data indicate that the PP1-targeting subunits Repo-Man and Sds22 contribute to faithful chromosome segregation. Repo-Man and Sds22 counteract a sustained activity of Aurora B on anaphase chromatin and on kinetochores after translocation of the chromosomal passenger complex to the central spindle. Thus, a balance between opposing kinase and phosphatase activities shapes the spatial gradient of Aurora B–dependent phosphorylation on anaphase chromatin (Fuller et al., 2008
). This is in agreement with previous studies reporting that PP1 counteracts Aurora B in vitro and during chromosome alignment (Murnion et al., 2001
; Sugiyama et al., 2002
; Wang et al., 2008
; Kim et al., 2010
; Liu et al., 2010
; Posch et al., 2010
Our high-resolution tracking of segregating chromosomes shows that oscillatory chromosome dynamics observed in metaphase (Jaqaman et al., 2010
) switch to a steady linear movement during anaphase. Therefore, the drop in mechanical tension at anaphase onset does not destabilize microtubule attachments in cells, as observed with in vitro reconstituted kinetochores (Akiyoshi et al., 2010
). Transient segregation pauses in Repo-Man and Sds22 RNAi cells could arise from defects in microtubule flux or depolymerization (Civelekoglu-Scholey and Scholey, 2010
; Walczak et al., 2010
). A perturbed chromosome architecture, as reported for Repo-Man–deficient cells (Vagnarelli et al., 2006
), could further contribute to segregation errors.
Chromosome missegregation leads to aneuploidy, a potentially carcinogenic state (Ganem et al., 2007
). Previous studies on the mechanisms ensuring faithful chromosome segregation mainly focused on the spindle checkpoint and error correction machinery (Musacchio and Salmon, 2007
; Lampson and Cheeseman, 2011
). Less well characterized but equally important for faithful chromosome segregation is an unperturbed anaphase. Our study provides new insight into the regulation of anaphase chromosome segregation and reveals the diversity of phosphatases coordinating the different events of mitotic exit.