hSgo2 localization and expression patterns. (A) HeLa cells released from a double thymidine block were costained with Bub1 (b, f, j, n, r, z) and hSgo2 (c, g, k, o, s, 1). Shown are cells from G2 through anaphase A. Insets show enlarged images of kinetochore pairs. Color images show the merged signals of Bub1 (green) and hSgo2 (red) staining. (B) Western blots of lysates prepared from cells released from a mitotic block. Lane 1, asynchronous cells; lanes 2–7, cells at 0, 30, 60, 90, 120, and 150 min after nocodazole washout; lane 8, cells released into medium with MG132 and harvested at 150 min. Bub3 is used as a loading control. (C) Costaining of hSgo2, MCAK, and ACA in prophase (Pro) and prometaphase (Prometa) HeLa cells. (D) Intensity profiles of hSgo2 (red), MCAK (green), and ACA (blue) of a representative kinetochore (C, arrow) in prophase (left) and prometaphase (right). Intensity values were obtained from the separate channels and plotted as a function of distance (inset, white dotted line). 25 kinetochores with discrete signals in all three channels were measured.

hSgo2 localization is affected by kinetochore tension. (A) Cells treated with a low-dose nocodazole to suppress microtubule dynamics, and thus reduce tension between bipolar attached kinetochores, were costained with Bub1 (red), hSgo2 (green), and DAPI (blue). Insets depict enlarged images of single kinetochore pairs highlighted by the dashed boxes. 1, unattached kinetochore pair; 2 and 3, bipolar attached kinetochores of aligned chromosomes. (B) Control metaphase cells. 1 and 2, bipolar attached kinetochores under full tension. Distances were measured from the centers of Bub1 and hSgo2 intensity profiles. Scale, 1 pixel = 0.117 μm. All images were captured and processed identically.

Characterization of cells depleted of hSgo2 by siRNA. (A) Efficiency of depletion of hSgo2 by different siRNAs. Lysates were harvested 48 h after transfection and probed for hSgo2 and CENP-F, which served as a loading control. Lanes 1–3, 50, 10, and 5 μg of lysates from cells transfected with control siRNA; lanes 4–7, 50 μg of lysates from cells transfected with pooled, 1, 2, 3, and 4 hSgo2 siRNAs. The hSgo2 signal intensity from cells transfected with oligos 2, 3, and the pooled siRNAs was less than or equal to the signal obtained from control samples containing 10-fold less protein (>90% depletion). (B) Cells transfected with hSgo1 exhibit attachment (top left) and cohesion defects (bottom left). Cells depleted of hSgo2 by the pooled siRNA (middle) can achieve metaphase alignment, as with controls (right). (C) Select frames from videos of HeLa (GFP/H2B) cells transfected with the different siRNAs. Two videos of hSgo2-depleted cells are shown to document different spindle orientations relative to the slide. (D) Fraction of cells from time-lapse studies that exhibited mitotic errors in anaphase. Orange, morphologically normal anaphase (nondisjunction would not be scored); green, lagging chromosomes; blue, died in mitosis. (E) HeLa (GFP/H2B) cells transfected with control, hSgo2, and MCAK siRNAs were examined by time-lapse videomicroscopy. The time of anaphase onset was determined as the time from NEBD to chromosome separation. Anaphase times for cells (n > 50 for each sample) were determined in each experiment (n = 6) and plotted as the frequency of all mitotic cells at each of the recorded times. Black, control siRNA; pink, hSgo2 siRNA smartpool; green, hSgo2 siRNA 3; red, MCAK siRNA. Error bars represent the SEM from six independent experiments.

hSgo2 specifies localization of MCAK to the inner centromere. (A) Prometaphase cells after transfection with control and hSgo2 siRNAs were costained for hSgo2, MCAK, and ACA. Chromosomes were stained with DAPI. (B) Comparison of the intensity ratios of MCAK (left) and MCAK normalized to ACA (right) between control and hSgo2-depleted cells (n > 30). (left) The depletion efficiency of MCAK siRNA. (right) Ratios of the intensity of MCAK to ACA at kinetochores (n > 30) in cells treated with nocodazole. (C) Mitotic cells harvested after transfection with control and hSgo2 siRNAs were probed with anti–CENP-F, anti-hSgo2, and anti-MCAK antibodies. The slower migrating MCAK is hyperphosphorylated (p-MCAK). (D) Cells transfected with control, MCAK, and hSgo2 siRNAs were treated with a high dose of nocodazole to depolymerize microtubules, and then costained for ACA, hSgo2, and MCAK.

Kinetochores depleted of hSgo2 exhibit attachment defects. (A) Metaphase cells transfected with control, hSgo2, and MCAK siRNAs were chilled as previously described (Lampson and Kapoor, 2005) before fixing and staining for ACA and tubulin. Images of whole cells are from maximum projections. Insets are images from a single optical slice. (B) Control (top row) and hSgo2 siRNA-transfected cells (middle and bottom rows) were costained with hSgo2 and Mad1 to monitor microtubule attachment status at aligned (middle row) and unaligned (bottom row) kinetochores. ACA was used to identify kinetochores. (C) To measure the interkinetochore distance, sister kinetochores were identified by pairs of Bub1 foci that flanked Aurora B (not depicted). Interkinetochore distances (n > 40) of aligned chromosomes in control metaphase- (1), hSgo2- (2), and MCAK-depleted (3) cells and in cells treated with a low dose of nocodazole (4). Interkinetochore distances of unattached kinetochores at low dose (5), high dose nocodazole (6), and in hSgo2-depleted cells (7). Black bars represent the mean.

hSgo2 localization depends on hBuB1 and Aurora B. Cells were transfected with control, hBuB1, hBuBR1, and AuroraB siRNAs and stained with the indicated antibodies. Samples were also costained with hSgo2, MCAK, or ACA. Exposure times were identical between control and siRNA samples.

Kinetochore ultrastructure is altered in cells depleted of hSgo2 and Aurora B. EM images of thin sections of mitotic cells that were transfected with control (A), hSgo2 (B and C), and Aurora B (D) siRNAs. Insets show enlarged views of kinetochores indicated by the white and black arrows. (A) Normal kinetochores (n = 32) with discrete inner (small white arrow) and outer plates (large white arrow). (B) hSgo2-depleted kinetochores (n = 12) reveal an outer plate, but lack a discernable inner plate, and the subjacent chromatin appears undercondensed (bracket). (C) Example of a C-shaped kinetochore with a prominent fibrous corona. (D) Cells depleted of Aurora B (n = 24) exhibit C-shaped kinetochores.