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 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 (Held et al., 2010), as indicated by colored contours. Nontargeting control siRNA (siControl) was transfected 42 h before imaging. Bar, 50 µm. (B) Enlarged images of mitotic cells. Bar, 10 µm. (C) Cells imaged, as in A, 42 h after transfection of Aurora B–targeting siRNA (siAurora B). Bar, 50 µm. (D and E) Statistical analysis of Aurora B biosensor phosphorylation. YPet/CyPet emission ratios were calculated for individual cells, and all measurements were then normalized (norm.) to the median of interphase control cells. Median (line), quartiles (boxes), and 10–90% data range (error bars) indicate YPet/CyPet ratios at different mitotic stages. (D) Biosensor phosphorylation of control cells transfected with nontargeting siRNA. (E) Aurora B RNAi cells. (F) Screen of an siRNA library targeting a genome-wide set of human phosphatases. FRET biosensor phosphorylation in late anaphase cells was assayed as in A–E. Data points represent mean z scores of YPet/CyPet emission ratios in late anaphase for three different siRNAs targeting per gene and two independent experimental replicates. Low z scores indicate hyperphosphorylation of the FRET biosensor.

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) Quantification of Aurora B–EGFP levels on chromatin by ratios of mean Aurora B–EGFP fluorescence divided by mean H2B-mCherry fluorescence. t = 0 min at the onset of chromosome segregation (dashed line). Fluorescence ratios of individual cells were normalized to metaphase values. n ≥ 18 cells. (C) Immunofluorescence staining with antibodies against Aurora B and Aurora B phosphorylated at Thr232 (pT232). DNA was stained with Hoechst 33342. Representative example images of prometaphase, metaphase, and anaphase cells 42 h after transfection with control, Repo-Man, or Sds22 RNAi are shown. Bar, 10 µm. (D and E) Quantification of Thr232-phosphorylated Aurora B (D) or total Aurora B (E) on chromatin in immunofluorescence images, as shown in C. Fluorescence intensities were normalized to control siRNA–transfected prometaphase cells. The dashed lines separate measurements of phospho–Aurora B levels on chromatin for the different siRNA conditions (left) from ZM1 treatment (inhibitor of Aurora B kinase, used as a positive control). Mean ± SEM. n = 7 (D) or 4 (E) independent experiments (>120 [D] or >70 [E] cells per experimental condition).

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 et al., 2006; B). Representative example images of cells costained for pSer100 Dsn1 (A) or Dsn1 (B), kinetochores (CREST), and DNA (Hoechst 33342). Bars, 10 µm. (C and D) Quantification of phosphorylated Dsn1 or total Dsn1 on kinetochores. pSer100 Dsn1/CREST or Dsn1/CREST fluorescence intensity ratios were normalized to control siRNA–transfected prometaphase cells. Mean ± SEM. n = 3 experiments (>30 cells per experimental condition; 16 kinetochores per cell). **, P < 0.001, using a two-tailed Student’s t test.

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 siRNA (siControl), or siRNAs targeting Repo-Man or Sds22. YPet/CyPet emission ratios are displayed in pseudocolor. Time is shown in min/s. Bars, 10 µm. (B–F) Quantification of YPet/CyPet emission ratios from cells, as shown in A, normalized (norm.) to mean YPet/CyPet ratios of control RNAi cells in metaphase. Curves and bars display mean ± SEM. Videos were aligned to the onset of chromosome segregation (t = 0 min; dashed lines). (B and C) Phosphorylation of the chromatin-targeted biosensor after transfection of different siRNAs targeting Repo-Man or Sds22. n ≥ 12 cells. (D and E) Phosphorylation of the chromatin-targeted biosensor after transfection of siRNAs targeting Mklp2, Repo-Man, or Sds22. n ≥ 10 cells. (F) Phosphorylation of the chromatin-targeted biosensor after transfection of siRNAs targeting Repo-Man or Sds22. ZM1 was added to a final concentration of 2 µM immediately after the onset of chromosome segregation. n ≥ 14 cells. (G) Phosphorylation kinetics of a cytoplasmic Aurora B biosensor (Fuller et al., 2008). Normalized YPet/CyPet emission ratios of live HeLa cells stably coexpressing the cytoplasmic biosensor and H2B-mCherry. Mean ± SEM. n ≥ 18 cells. t = 0 min at chromosome segregation onset (dashed line).

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 cells stably expressing H2B-mCherry (n > 500 cells; seven independent experiments; **, P < 0.001, using a two-sided binomial test). Bars, 10 µm. (C–I) 3D tracking of kinetochores in HeLa cells stably expressing EGFP–CENP-A (Jaqaman et al., 2010) reveals pauses in poleward segregation. (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.