Sds22 and Repo-Man stabilize chromosome segregation by counteracting Aurora B on anaphase kinetochores

Repo-Man and Sds22 counteract Aurora B phosphorylation of Dsn1 and thus regulate the kinetochore–microtubule interface during anaphase.


Introduction
Aurora B is the catalytic subunit of the chromosomal passenger complex, which regulates mitotic spindle assembly and cyto kinesis (Ruchaud et al., 2007). During mitotic spindle as sembly, centromerelocalized Aurora B destabilizes kinetochoremicrotubule interactions that are under low mechanical tension (Tanaka et al., 2002;Hauf et al., 2003;Lampson et al., 2004;Lampson and Cheeseman, 2011) by phosphorylating several components of the outer kinetochore, including NDC80/HEC1, KNL1, and Dsn1 (Welburn et al., 2010;DeLuca et al., 2011). The Aurora B-mediated error correction is important for bipolar attach ment of all chromosomes, which satisfies the spindle assembly checkpoint to initiate anaphase (Musacchio and Salmon, 2007).
Loss of sister chromatid cohesion at anaphase onset reduces the mechanical tension on kinetochores, yet, kinetochores need to remain attached to microtubules until complete segregation. Removal of Aurora B from centromeres at anaphase onset, regu lated by a drop in Cdk1 activity (Pereira and Schiebel, 2003;Gruneberg et al., 2004;Hümmer and Mayer, 2009;Oliveira et al., 2010), the p97 system (Ramadan et al., 2007), and the E3 ubiqui tin ligase Cul3 (Sumara et al., 2007), may explain why anaphase kinetochores are not sensed and detached despite low tension. Supporting this idea, elevated Aurora B levels on anaphase chro mosomes lead to untimely spindle checkpoint reengagement (Mirchenko and Uhlmann, 2010;VázquezNovelle and Petronczki, 2010), and premature removal of cohesin from metaphase chro mosomes in Drosophila melanogaster embryos results in ir regular chromosome segregation (Oliveira et al., 2010). This suggests that Aurora B removal from anaphase chromosomes is necessary for unperturbed segregation. However, Aurora B may still reach distant targets on anaphase chromatin through a spa tial gradient of activity emerging from the central spindle (Fuller et al., 2008).
The regulation of anaphase kinetochores likely involves yet unknown phosphatases. PP1 phosphatase counteracts Aurora B on chromosome arms (Murnion et al., 2001;Sugiyama et al., 2002;Wang et al., 2008), and PP1 also targets to kinetochores D uring mitotic spindle assembly, Aurora B kinase is part of an error correction mechanism that detaches microtubules from kinetochores that are under low mechanical tension. During anaphase, how ever, kinetochore-microtubule attachments must be main tained despite a drop of tension after removal of sister chromatid cohesion. Consistent with this requirement, Aurora B relocates away from chromosomes to the central spindle at the metaphase-anaphase transition. By ribo nucleic acid interference screening using a phosphorylation biosensor, we identified two PP1targeting subunits, Sds22 and RepoMan, which counteracted Aurora B-dependent phosphorylation of the outer kinetochore component Dsn1 during anaphase. Sds22 or RepoMan depletion induced transient pauses during poleward chromosome movement and a high incidence of chromosome missegregation. Thus, our study identifies PP1targeting subunits that regu late the microtubule-kinetochore interface during anaphase for faithful chromosome segregation.
Sds22 and Repo-Man stabilize chromosome segregation by counteracting Aurora B on anaphase kinetochores phosphorylation ( Fig. 1 F and Table S1), yet, they were not further considered in our study, which aims at the identifica tion of phosphatases counteracting Aurora B.
To validate the phenotypes of the topranking candidate hits, we first measured siRNA target protein levels (Fig. S1). Three different siRNAs depleted RepoMan 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 timelapse imag ing showed that depletion of RepoMan or Sds22 by any of the different siRNA oligonucleotides delayed dephosphorylation of the biosensor (n ≥ 12 cells; Fig. 2 [A-C] 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. Codeple tion of Sds22 and RepoMan further delayed dephosphorylation of the biosensor (Fig. 2 E).
Confocal timelapse imaging of cells in which the PP2A catalytic (PPP2CA), scaffold (PPP2R1A), or regulatory (PPP2R2A/B55) subunits were depleted individually or alto gether (Fig. S1, M-R), however, could not confirm their require ment for timely Aurora B biosensor dephosphorylation (n ≥ 18 cells; Fig. S2 D). We suspect that the falsepositive scoring in our primary screen may have been caused by misclassification of mitotic stages owing to perturbed chromatin morphology . 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 de pletion levels achieved in our RNAi experiments.
The delayed biosensor dephosphorylation in Sds22 or RepoMan 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 RepoMan nor Sds22 RNAi affected Aurora B localization, in contrast to an Mklp2 RNAi-positive control (Fig. 3, A and B; Gruneberg et al., 2004;Hümmer and Mayer, 2009). Immunofluorescence staining by phosphospe cific 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, by binding to CENPE (Kim et al., 2010), KNL1 , and Sds22 . During spindle assembly, PP1 sta bilizes microtubule binding at kinetochores in mammalian tissue culture cells Posch et al., 2010), as does the bud ding yeast homolog Glc7 on reconstituted kinetochores (Sassoon et al., 1999). During anaphase, the regulatory subunit RepoMan recruits PP1 to chromatin , which reverts Haspin and Aurora B-dependent phosphorylations on histone 3 (Qian et al., 2011;Vagnarelli et al., 2011). Whether these or any other phosphatases regulate the kinetochoremicrotubule interface during anaphase is not known. Here, we identify and characterize two PP1targeting subunits, RepoMan and Sds22, which regulate the kinetochore-microtubule interface during anaphase to ensure faithful chromosome segregation.

Results and discussion
To screen for phosphatases counteracting Aurora B during ana phase, 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 chromatintargeted version of the bio sensor (Fuller et al., 2008) and classified mitotic stages based on chromatin morphology using inhouse-developed, supervised machinelearning methods (CellCognition; Fig. 1, A and B; Held et al., 2010). The fully automated assay was validated by RNAi depletion of Aurora B (Fig. S1, A and B), which efficiently inhib ited biosensor phosphorylation in mitotic cells ( Fig. 1, C-E).
Three different siRNA oligonucleotides targeting each of a genomewide set of 225 annotated human phosphatases, includ ing catalytic, regulatory, and scaffold subunits , were transfected individually into the reporter cells. Can didate 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 ( Fig. 1 F and Table S1). The five topscoring target genes encode Repo Man (also termed CDCA2), Sds22 (also termed PPP1R7), PPP2CA, PPP2R1A, and PPP2R2A (also termed B55).
RepoMan is a PP1targeting subunit, which had previously been shown to counteract Aurora B on anaphase chromatin 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 . Three other PP1targeting 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 het erotrimeric PP2A complex that reverts Cdk1 substrate phos phorylations during mitotic exit (Mochida et al., 2009;Schmitz et al., 2010). Several siRNAs caused decreased biosensor Sds22 and Repo-Man stabilize chromosome segregation • Wurzenberger et al.

Elevated Aurora B levels on anaphase chromatin in Mklp2
RNAi cells correlated with delayed dephosphorylation of the chromatintargeted biosensor (Fig. 2 D). The delay was further increased by codepleting Sds22 or RepoMan (Fig. 2 E), yet, biosensor dephosphorylation reached similar levels as in con trol cells 15 min after anaphase onset, despite the persistence 2002; Honda et al., 2003;Sessa et al., 2005;Salimian et al., 2011) showed that Sds22 or RepoMan RNAi did not affect the levels of active Aurora B on anaphase chromosomes (Figs. 3 [C-E] and S2 [E-G]). Thus, RepoMan and Sds22 contribute to anaphase dephosphorylation of chromosomal Aurora B sites independent of Aurora B translocation or inactivation.  (Fuller et al., 2008) were excited with 426-440-nm light. Pseudocolored YPet/CyPet emission ratios indicate phosphorylation of the biosensor (low ratios indicate high phosphorylation). Mitotic stages were classified by supervised machine learning , as indicated by colored contours. Nontargeting control siRNA (siControl) was transfected 42 h before imaging. Bar, 50 µm.  Sds22 and RepoMan may dephosphorylate chromosomal Aurora B substrates that had been phosphorylated before re location of the kinase to the central spindle or counteract a sus tained Aurora B activity emerging from the central spindle via a longrange spatial gradient (Fuller et al., 2008) or from re sidual Aurora B on anaphase chromatin. Aurora B inhibition by of high levels of Aurora B on chromatin at these late time points (compare Fig. 3 [A and B] with Fig. 2 E). The phosphatase ac tivity that eventually dephosphorylates the biosensor in this ex periment may emerge from residual amounts of the respective RNAi target proteins, from functional redundancy between Sds22 and RepoMan, or from unknown other phosphatases. ZM1 (Girdler et al., 2006) briefly after anaphase onset signifi cantly accelerated biosensor dephosphorylation in RepoMan and Sds22 RNAi cells (Fig. 2 F), indicating that Aurora B continues to regulate substrate phosphorylation on chromo somes after anaphase onset. This could be either by direct substrate phosphorylation on chromosomes or indirectly by regu lating counteracting phosphatases.
Sds22 or RepoMan RNAi had minor effects on dephos phorylation of a cytoplasmic Aurora B phosphorylation FRET biosensor (Fig. 2 G;Fuller et al., 2008). This suggests that Sds22 and RepoMan act locally on chromosomes, which is in accordance with their reported localization to kinetochores and chromatin, respectively Posch et al., 2010).
To investigate the relevance of timely Aurora B substrate dephosphorylation, we recorded 3D confocal livecell videos from HeLa cells stably expressing H2BmCherry. RepoMan 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 twosided binomial test; Fig. 4, A and B). To test for ontarget specificity of the phenotype, we transfected siRNAs specific for human RepoMan or Sds22 in HeLa cell lines stably expressing the respective siRNAresistant, EGFPtagged mouse counterparts (Fig. S3, A-D). Depletion of endogenous human RepoMan 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 missegre gation results from ontarget depletion of RepoMan or Sds22.
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 RepoMan (3.1 ± 0.5% in control RNAi cells, 2.8 ± 0.7% in RepoMan 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 chromo some 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 RepoMan RNAi cells; n > 400 cells), yet, this fraction is much smaller than the fraction of cells with anaphase segregation errors (Fig. 4, A and B).
To gain more insight into the chromosome missegrega tion phenotype, we tracked kinetochores in 3D videos of live HeLa cells stably expressing EGFP-CENPA (Jaqaman et al., 2010). By measuring sister kinetochore distances, we determined segregation dynamics independent of spindle rotation and trans lation (Fig. 4, C and D). RepoMan or Sds22 depletion had only a weak effect on the overall mean segregation speed (Fig. S3 G). (C and E) Kymographs of sister kinetochore pairs (yellow arrow highlights interkinetochore distance). Tracked sister kinetochores are highlighted by colored circles; green represents linear segregation, and red represents paused segregation (net movement of <0.1 µm in three subsequent frames). t = 0 min at chromosome segregation onset (dashed lines). Bars, 5 µm. (D, F, G, and H) 3D interkinetochore distances of sister kinetochore pairs from metaphase until full segregation. Paused segregation (net movement of <100 nm in three subsequent frames) is highlighted in red. t = 0 min at chromosome segregation onset (dashed lines). (I) Incidence of sister kinetochore pairs per cell with paused segregation after initially normal segregation during poleward movement 0-5 min after anaphase onset. n ≥ 9 cells (10 kinetochore pairs per cell) per condition. Mean ± SEM. **, P < 0.001, using a two-sided binomial test.
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 Sds22depleted metaphase cells . In contrast, Dsn1 Ser100 phosphorylation was significantly elevated during prometaphase and metaphase in Sds22 RNAi cells (Fig. 5 C). Even though the mechanism underlying differ ential regulation of Aurora B substrates by Sds22 is unclear, we also observed that depletion of Sds22, but not of RepoMan, delayed progression from mitotic entry until anaphase, as previ ously reported (Fig. S3 L; Posch et al., 2010).
Our data indicate that the PP1targeting subunits RepoMan 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 align ment (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 highresolution tracking of segregating chromosomes shows that oscillatory chromosome dynamics observed in meta phase (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 RepoMan and Sds22 RNAi cells could arise from defects in microtubule However, a large fraction of individual kinetochores tempo rarily paused segregation or even transiently moved backward in cells depleted of Sds22 or RepoMan (Fig. 4, C-I). The seg regation 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 chromo somes, 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 phos phorylation, we maintained high levels of Aurora B on anaphase chromatin using Mklp2 RNAi (Figs. 2 D and 3 [A and B]; Gruneberg et al., 2004;Hümmer and Mayer, 2009 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 affin ity is regulated by differential phosphorylation of Aurora B sites on outer kinetochore components (Welburn et al., 2010;DeLuca et al., 2011). Ser100phosphorylated Dsn1 (Welburn et al., 2010) was indeed elevated on anaphase kinetochores of RepoMan or Sds22 RNAi cells (Fig. 5, A and C), whereas total Dsn1 levels were unaffected (Fig. 5, B and D).

Inhibitor treatment
For inhibition of Aurora B in quantitative immunofluorescences, ZM1 was added to a final concentration of 2 µM 1 h before fixation. For anaphasespecific inhibition of Aurora B during time-lapse imaging, ZM1 (Tocris Bioscience) was prediluted to 20 µM stock solution in prewarmed imaging medium and added directly after chromosome segregation to a final concentration of 2 µM.
Automated imaging for RNAi screening was performed on a screening microscope (ImageXpress Micro; Molecular Devices) equipped with a reflection-based laser autofocus, a CoolSNAP HQ camera (Roper Scientific), and CFP-YFP FRET filters for sensitized emission ratio imaging (excitation filter of 426-440 nm and emission filters of 467-492 nm [CFP] or 529-556 nm [YFP]) using a 10× 0.5 NA Fluor-S dry objective (Nikon) and controlled by in-house-developed MetaMorph macros . Cells were maintained at 37°C in a humidified atmosphere of 5% CO 2 .
Scanning confocal microscopy was performed on a customized microscope (LSM 510 Axiovert; Carl Zeiss) equipped with piezo focus drive (piezosystem jena GmbH) and custom-designed filters (Chroma Technology Corp.) using a 63× 1.4 NA oil Plan Apochromat objective or a 40× 1.3 NA oil differential interference contrast EC Plan Neofluar objective (Carl Zeiss) and controlled by the LSM 510 AIM software. Cells were maintained at 37°C in a humidified atmosphere of 5% CO 2 by an EMBL incubation chamber.
Live-cell imaging of EGFP-CENP-A cells for tracking purposes was performed on a multiplexed system (Olympus IX71 DeltaVision; Applied Precision) equipped with a camera (CoolSNAP HQ2) and temperature incubation, using a 100× 1.4 NA oil UPlanSApo objective (Olympus) and the softWoRx acquisition software. 20 z sections with a z step of 0.5 µm were imaged at a temporal resolution of 15 s. Cells were imaged for a maximum of 5 min before chromosome segregation to reduce phototoxic side effects.
Chromosome missegregation leads to aneuploidy, a potentially carcinogenic state (Ganem et al., 2007). Previous studies on the mechanisms ensuring faithful chromosome seg regation mainly focused on the spindle checkpoint and error correction machinery (Musacchio and Salmon, 2007;Lampson and Cheeseman, 2011). Less well characterized but equally im portant 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.

Cell lines and plasmids
The HeLa Kyoto cell line was obtained from S. Narumiya (Kyoto University, Kyoto, Japan) and cultured in DME (Gibco) supplemented with 10% (vol/vol) FBS (PAA Laboratories) and 1% (vol/vol) penicillin-streptomycin (Invitrogen). Monoclonal reporter cell lines were generated as previously described ). All reporter cell lines used in this study showed similar proliferation rates as HeLa Kyoto wildtype cells.
The chromatin-targeted FRET biosensor (Fuller et al., 2008) was N-terminally fused to full-length core histone 2B (H2B) and was subcloned into the pIRESpuro2 vector (Takara Bio Inc.). The cytoplasmic FRET biosensor was generated by removing the N-terminal H2B sequence.
Murine bacterial artificial chromosomes (BACs; RP23-417I6 [AURKB], RP23-316O17 [Sds22/PPP1R7], and RP24-290N12 Repo-Man/CDCA2) harboring the genes of interest were obtained from the BACPAC Resources Center. Mouse Sds22 protein has 94% amino acid sequence identity with the human homolog, and mouse Repo-Man protein has 53% amino acid sequence identity with the human homolog. The N-terminal NFLAP (EGFP-Neo) or LAP (EGFP-IRES-Neo) cassette was PCR amplified using primers that carry 50 nucleotides of homology to the N terminus (Repo-Man) or C terminus (Aurora B and Sds22) of the target genes. Recombineering and stable transfection of the modified BAC was performed as previously described (Poser et al., 2008). In brief, both a plasmid carrying two recombinases and the purified tagging cassette were introduced into the Escherichia coli strain containing the BAC vector using electroporation. Precise incorporation of the tagging cassette was confirmed by PCR and sequencing. GFP-tagged BACs were isolated from bacteria and transfected into HeLa Kyoto cells using Effectene (QIAGEN). Pools of HeLa cells stably expressing the GFP-tagged transgenes were selected by cultivation in selection media containing 400 µg/ml geneticin (G418; Invitrogen) and were analyzed by Western blot and immunofluorescence using an anti-GFP antibody (Roche) to verify correct protein size and localization of the tagged protein.
The cell pools stably expressing tagged mouse Sds22 or Repo-Man proliferated at normal rate and did not show elevated levels of apoptosis. The incidence of chromosome missegregation was similar to control cells in mouse Sds22-expressing cell pools, yet, it was increased to 20% in cell pools stably expressing mouse Repo-Man. Cell pools stably expressing BAC transgenes of Aurora B, Sds22, and Repo-Man were cotransfected with H2B-mCherry and selected for monoclonal cell lines stably coexpressing both genes of interest.

siRNA transfection
The human siRNA phosphatase library was as published in Schmitz et al. (2010). siRNAs targeting PP1 catalytic subunits, Repo-Man, Sds22, and Mklp2 were obtained from QIAGEN and are listed in Table 1. The nontargeting control siRNA used was AllStars (QIAGEN). siRNA duplexes were frequency of sister kinetochores that displayed paused segregation was scored for 10 kinetochore pairs per cell.
Phospho-Thr232 Aurora B, total Aurora B, phospho-Ser893/894 INCENP, and total INCENP levels on chromatin were measured in mean projections of optical z stacks. Regions of interest were defined by thresholding on the Hoechst channel using ImageJ. Fluorescence intensities were background subtracted and normalized to prometaphase control RNAi cells.
Dsn1 and phospho-Ser100 Dsn1 levels were measured in regions of interest of constant size around single kinetochores (16 kinetochores per cell) in mean projections of z stacks using ImageJ. Fluorescence intensities on the Dsn1, phospho-Dsn1, and CREST channel were background subtracted, and Dsn1/CREST or pSer100 Dsn1/CREST ratios were normalized to control RNAi prometaphase cells. Staining by pSer100 Dsn1 revealed prominent cytoplasmic foci in all RNAi conditions, which were not apparent in stainings for total Dsn1 and therefore likely represent an unspecific cross-reaction of the antibody. We did not measure fluorescence close to cytoplasmic foci.
Quantitative real-time PCR Cells were transfected with siRNAs in 12-well plates (Thermo Fisher Scientific) 40 h before RNA isolation by phenol-chloroform extraction using TRIZOL (Invitrogen), according to the manufacturer's instructions. cDNA was synthesized by reverse transcription using random hexamer primers (Microsynth) and M-MuLV reverse transcription (Thermo Fisher Scientific). Real-time PCR was performed on a LightCycler 480 (Roche) using the Light-Cycler 480 SYBR green I Master Mix (Roche). Efficiency of gene amplification was determined by a standard curve, and expression levels were quantified relative to glyceraldehyde 3-phosphate dehydrogenase levels. Primers were designed using Autoprime software. For amplification of human and mouse Repo-Man or Sds22, specificity of primer sequences was validated by sequence alignment using the BLASTN algorithm. All primer pairs are listed in Table 2.
Online supplemental material Fig. S1 shows validation of target gene depletion. Fig. S2 shows biosensor phosphorylation kinetics for various experimental conditions and immunofluorescence staining for INCENP phosphorylation. Fig. S3 shows on-target validation of siRNAs by phenotype rescue and spindle rotation Image analysis Automated image analysis for FRET biosensor-based screening was performed using the in-house-developed CellCognition software . To compensate for inhomogeneous illumination, images were flatfield corrected by background images acquired in empty wells. Nuclei were segmented by adaptive thresholding, and texture and shape features were calculated for each object, as described in Held et al. (2010). Samples for nine user-defined morphology classes (interphase, prophase, prometaphase, metaphase, early anaphase, late anaphase, telophase, binucleate cells, and apoptotic cells) were annotated manually to train a support vector machine classifier. Supervised classification was then applied to high-throughput screening data. Mean fluorescence intensities were measured in the nuclei, and background-subtracted YPet/CyPet emission ratios were normalized to the mean of all interphase cells of one experimental replicate. Z scores of individual siRNA conditions in late anaphase were calculated as the deviation of mean YPet/CyPet ratios of one condition to the overall mean of all data points of one experimental replicate, normalized to the SD of all data points. Z scores for hit ranking were calculated as the mean of the three siRNAs targeting each gene and both experimental replicates. siRNA conditions with n < 15 late anaphase cells were excluded from further analysis.
Automated timing measurements of mitotic progression from nuclear envelope breakdown to chromosome segregation were performed using the CellCognition software, as previously described Schmitz et al., 2010). Nuclei were tracked over time and classified based on their chromatin morphology by supervised machine learning. Nuclear envelope breakdown was detected by a decrease in the ratio of the mean nuclear versus cytoplasmic IBB-EGFP fluorescence.
Confocal time-lapse videos of the FRET biosensor were analyzed using ImageJ (National Institutes of Health). Regions of interest were determined by thresholding on the YFP channel, and fluorescence intensities were extracted from mean intensity projections. Background-subtracted YPet/CyPet emission ratios were normalized to late metaphase (1-5 min before onset of chromosome segregation) and late anaphase (6-8 min after onset of chromosome segregation) of control RNAi cells and plotted over time aligned on anaphase onset.
Chromosomal localization of Aurora B-EGFP was analyzed similarly in mean projections of confocal z stacks. Nuclear regions were defined by thresholding on the H2B-mCherry channel, and Aurora B-EGFP mean fluorescence on chromatin was normalized to H2B-mCherry mean fluorescence to compensate for chromosome condensation. Relative Aurora B levels were then calculated individually in each cell relative to mean levels in metaphase to compensate for variable expression levels of the two markers in different cells.
Kinetochore tracking in anaphase was performed using Imaris software (Bitplane). 3D kinetochore positions were automatically determined with subpixel resolution using the Spots function of Imaris. Sister kinetochores were then identified and tracked manually from 2 min before anaphase onset until up to 20 min after anaphase onset. 3D interkinetochore distances were calculated and plotted over time. Mean segregation speed was determined by linear regression of the segregation curve from 0 to 4 min after onset of chromosome segregation. Pausing events were defined as a net movement of <0.1 µm between three subsequent frames, and the