Histone H3S10 phosphorylation in mouse preimplantation embryos. (A) Time-lapse images of an embryo injected with 100 µg/ml Fab311-488 and 5 µg/ml H2B-mRFP mRNA (maximum projection of 51 z stacks). Arrows indicate the concentration of Fab311 in the centromere-rich regions. See Video 4. (B) Maternal chromosomes are selectively detected by Fab313. Fab313-488 (red) and H2B-mRFP (cyan) images in first and second mitosis are shown. The two-cell image is contrast enhanced. See Video 5. (C) Pups developed from Fab313-488–injected embryos with their recipient mother. (D) Development of embryos injected with fluorescent Fab. Embryos generated by in vitro fertilization were injected with various concentrations (conc.) of Fab together with H2B-mRFP mRNA, imaged up to the four to eight-cell stage, and transferred to the oviduct of day 0.5 pseudopregnant mothers. The rates of normal development were similar to controls (Yamagata et al., 2009). ^aImaging minus (−) means the embryos were injected with Fab but not imaged. Bars, 10 µm.

Visualization of aurora B with H3S10ph in living cells and the effects of kinase and phosphatase inhibitors. (A) HeLa cells loaded with FabAuB-488 and Fab311-555. Maximum projections of three z-stack images at 1.5-µm intervals are shown. Arrows and closed arrowheads indicate FabAuB concentrations in heterochromatin, the midzone, and the midbody. Open arrowheads indicate Fab311 foci before the accumulation on condensed chromosomes (asterisks). See Video 8. (B) A fibroblast loaded with FabAuB-488 (green) and Fab311-555 (magenta). Deconvolved and maximum-projected images of five z-stack images at 0.75-µm intervals taken every 2 min are shown. Full time series images of the boxed areas are shown. (top) White arrowheads indicate FabAuB and Fab311 foci. (bottom) The positions of Fab311 foci with the highest intensities are indicated (arrowheads). See Video 9. (C) BrdU incorporation in cells exhibiting aurora B and H3S10ph. Cells were incubated in BrdU for 10 min to 5 h, fixed, and immunolabeled for aurora B, H3S10ph, and BrdU. (left) When treated with BrdU for 10 min, aurora B–positive cells often exhibit BrdU signals, which are unassociated (arrows; HeLa) or associated (arrowheads; fibroblasts) with H3S10ph. (right) BrdU-positive cells were counted among those positive for aurora B, H3S10ph, or condensed chromosomes (n > 20); cells in early mitotic phase (i.e., prophase to prometaphase; M) were counted in HeLa because of their prolonged mitosis (> 1 h vs. ∼30 min in fibroblasts). The means ± SD from three independent experiments are shown. (D) A fibroblast treated with ZM. (E) A fibroblast treated with TMC followed by ZM. (F) A HeLa cell treated with TMC. Bars, 5 µm.

Fluorescently labeled Fab binds to phosphorylated histone H3S10 during mitosis in HeLa cells. (A) Specificity of mAbs evaluated by ELISA using the indicated peptides. The peptides that reacted with individual mAbs are indicated in the graphs. (B) Immunofluorescence. Fixed cells were stained with Fab311-488 (Alexa Fluor 488–labeled Fab from CMA311; red), Fab313-555 (Alexa Fluor 555–labeled Fab from CMA313; green), and anti–CENP-C (blue). Images of a prophase cell are shown with magnified views of boxed area. See Fig. S1 for other cells at different phases of the cell cycle. (C) Time-lapse images of a cell loaded with Fab311-488. Arrows indicate concentrations of Fab311-488. See Video 1. (D) Period of mitosis in Fab311-488–loaded and control cells. By collecting three z-stack images every 3 min, the period from prophase to anaphase was measured (n = 27) using phase-contrast images. No significant difference was seen; P = 0.66 (Student’s t test). (E) The mobility of Fab311-488 by FRAP. After bleaching a 2-µm spot (white circle), images were collected for 12 s every 0.13 s (left) or for 120 s every 0.4 s (right). Means ± SD are shown (n = 12). The red line shows the fitted curve using single exponential association kinetics. Residence time (k⁻¹; k = association coefficient) of the transiently bound fraction is calculated as 44 s. (F) H3S10ph detected in live and fixed samples. Cells loaded with Fab311-488 were imaged every 3 min. When foci were detected in two consecutive frames (01:00 and 01:03), cells were fixed and immunolabeled with Fab311-555. Foci observed during live imaging (arrows) were also detected after fixation and immunolabeling. Bars: (B, C, and F) 5 µm; (E) 1 µm.

Aberrant chromosome segregation and interphase phosphorylation monitored using fluorescent Fab. (A) Anaphase HeLa cells showing aberrant chromosome segregation. Fixed cells were stained with Fab311-488 (red), anti–aurora B (green), and DAPI (blue). Arrows indicate a missegregated chromosome (left) and a chromosome bridge (right). (B) Missegregated chromosomes are readily detectable in living cells using Fab311-555. HeLa cells expressing histone H2B–GFP were loaded with Fab311-555, and images were taken every 3 min. A missegregated chromosome (arrows) is easily visible by Fab311, whereas contrast enhancement is required for detection by H2B-GFP. (C) Frequency of chromosome missegregation and period of Fab311-488 foci appearance before mitosis measured using time-lapse imaging. (left) The chromosome missegregation rate. (right) The period of Fab311-488 foci appearance before mitosis. (D) Time-lapse images of a fibroblast loaded with Fab311-488. H3S10ph focus (arrow) appears >5 h before mitosis, and others appear (arrowheads) later. See Video 7. Bars, 5 µm.

Effects of ZM treatment during the interphase. Cells were loaded with GST-NLS-GFP and Fab311-555 and treated with ZM for ∼20 min. (A) Response of Fab311 to ZM addition and removal in hTERT-RPE1. See Video 10. (B) An hTERT-RPE1 cell that exhibited chromosome missegregation after transient exposure to ZM. (C) Effects of transient exposure to ZM on the formation of Fab311 foci and chromosome segregation. ^aLoaded cells were incubated in the absence (−) or presence (+) of ZM. ^bThe period when Fab311 foci appeared before the nuclear membrane broke down (judged by the release of GST-NLS-GFP from the nucleus into the cytoplasm). Mean ± SD, range, and number of cells analyzed (n) are indicated. ^cThe percentage of cells showing aberrant chromosome segregation. Bars, 5 µm.