Bod1 is required for the efficient resolution of syntelic attachments. (A) The mitotic profile of control and Bod1-depleted cells was determined 72 h after transfection of siRNA. (B) GFP–CENP-B–transfected HeLa cells were cotransfected with control or Bod1 siRNA, and time-lapse microscopy was performed 72 h after transfection. Projections of selected time points are shown. (C) Control or Bod1 siRNA HeLa cells were treated with monastrol for 3 h, washed, and released into media containing MG132 for 1 h before fixing. The mitotic profile before and after monastrol release is shown. (D) Syntelic attachments in Bod1 siRNA cells 1 h after release from monastrol showing microtubules (green), anti-Hec1 (red), and ACA (blue). Insets are magnified images of boxed areas. Error bars represent SD. Bars, 5 μm.

MCAK is not efficiently phosphorylated in Bod1^siRNA cells. (A) Aurora B is not delocalized in Bod1-depleted cells. Phospho-Ser10-histone H3 staining in control and Bod1 siRNA cells indicating Aurora B activity. (B–E) Cells were transfected with control or Bod1 siRNA. After 72 h, cells were treated with monastrol for 3 h and released into media containing MG132 for 1 h before fixing. (B and C) Cells were stained for total MCAK population, and levels at kinetochores were quantified. Boxed areas are magnified below the main images. (D and E) Cells were stained for phospho-Ser92-MCAK, and levels at aligned and unaligned kinetochores were quantified. Dashed lines indicate orientation of the metaphase plate. Error bars represent SD. Bars, 5 μm.

Depletion of Bod1 by siRNA causes major chromosome alignment defects. (A) Immunoblot using an anti-Bod1 antibody showing the effective depletion of Bod1 72 h after transfection of siRNA duplexes. Tubulin was used as a loading control. (B) One-step RT-PCR analysis of control and Bod1 siRNA RNA lysates showing the specific knockdown of Bod1 in relation to other Fam44 family members. (C) FACS analysis of control and Bod1-depleted cells. (D) Taxol or nocodazole was added to control or Bod1 siRNA cells 48 h after transfection, and the percentage of mitotic cells was determined 18 h later. (E) HeLa cells were transfected with control siRNA or siRNA against Bod1 and processed for immunofluorescence 72 h later. Microtubules are shown in red, chromosomes are shown in blue, and ACA is shown in green. The inset is a magnified image of the boxed area. The arrow shows lateral attachment, and the arrowhead shows end-on attachment. (F) Control or Bod1 siRNA–transfected HeLa cells stained with Bub1 and ACA. (G) The spindle length of control cells and Bod1 siRNA cells. Error bars show SD. (H) Anti-Eg5 and anti–Aurora A staining of control and Bod1 siRNA–transfected HeLa cells. Bars, 5 μm.

Microtubule and kinetochore dynamics in Bod1-depleted cells. (A) Cold stable microtubule assay of control and Bod1-depleted cells. Microtubules are shown in red, and ACA is shown in green. (B–D) Cells were imaged by transiently transfecting either control or Bod1 siRNA, mCherry-tubulin (red), and GFP–CENP-B (green). Imaging was started 72 h after transfection. (B) Projections of selected time points from a cell transfected with control siRNA are shown. Time from the onset of imaging is shown in the top left corner. (C) Cells transfected with Bod1 siRNA failed to align many of their chromosomes. Arrowheads highlight unaligned kinetochores becoming bioriented. Circles highlight pairs of sister kinetochores that remain behind the spindle pole. (D) Bod1-depleted cell showing unaligned sister kinetochores (boxed area). (right) Oscillation of sister kinetochores over 4 min. Black and white panels show saturated images of tubulin staining to highlight microtubules. Arrows show single oscillating kinetochore pairs; arrowheads show oscillating microtubules. (E) The distance between the sister kinetochores highlighted in D and the centrosome were plotted over time. The black bar highlights the measurements taken from images in D. (F) Selected time points from a Bod1-depleted cell showing CENP-B–GFP. (G) The distance between kinetochores in the unaligned sisters (green line; “un” arrowheads in F) and aligned sisters (aligned 1 and aligned 2, blue lines; a1 and a2 arrowheads in F) depicted in F were measured over time. The distance between three selected sister kinetochore pairs from control cells were also measured over time (red lines). Bars, 10 μm.

Bod1 is a novel protein localizing to centrosomes and kinetochores. (A and B) Selected maximum intensity projections from time-lapse images of HeLa cells show gene-specific mitotic phenotypes. Cells were transfected with the pU6YH plasmid expressing scrambled shRNA (A) or shRNA targeting Bod1 (B). Images were taken 60 h after transfection over a period of 60–120 min. Numbers indicate time (minutes) from the establishment of a metaphase plate. (C) HeLa cells transfected with Bod1-GFP were fixed after 48 h with PFA. (top) Localization of Bod1-GFP at various points in the cell cycle. (bottom) A merged image with microtubules (red), chromosomes (blue), and Bod1-GFP (green). Arrowheads indicate concentrations of Bod1-GFP at microtubule ends. Insets are magnified images of boxed areas. (D) Chromosome spreads of nocodazole-arrested HeLa cells stained for endogenous Bod1 (green), ACA (red), and DNA (blue). (E and F) The localization of Bod1-GFP was determined with respect to Aurora B (E) and ACA (F). Kinetochore localization of Bod1-GFP is indicated by arrowheads. (G) Hydrodynamic analysis of nocodazole-arrested HeLa lysates by glycerol gradient centrifugation (left) and size exclusion chromatography (right) showing that Bod1 is present in two different complexes. Asterisks mark cross-reacting bands. Bars, (A and B), 10 μm; (C), 5 μm.