(A) Schematic overview of cell synchronisation and transfection procedure.
(B) Characterization of Sgo1 antibodies. Affinity-purified Sgo1 antibodies raised against two different regions of Sgo1 recognize the same 72-kDa protein by Western blotting. Synchronised HeLa cells were transfected with water (Mock) or Sgo1 siRNA. Cells were harvested 7 and 11 h after release from thymidine block, and chromatin fractions were resolved by SDS-PAGE and probed with antibodies 94 and 95. A major band at 72 kDa was detected by both antibodies, which disappeared or was greatly reduced upon Sgo1 knockdown. The blot was reprobed with HP-1β antibody as a loading control.
(C) Localisation of Sgo1. Cells were costained for Sgo1 with antibody 94 (shown in green), and with CREST antiserum (shown in red). DNA was counterstained with DAPI. Five different stages of mitosis are shown: (a) prophase, (b) prometaphase, (c) metaphase, (d) anaphase, and (e) telophase. Bar = 10 μm. (f) A single pair of CREST labelled kinetochores are enlarged, showing two adjacent Sgo1 foci as commonly observed for late stage metaphases. Bar = 1 μm.
(D) In situ immunofluorescence of (a) mock-treated or (b) Sgo1 siRNA-transfected HeLa cells, demonstrating the disappearance of the Sgo1 signal following siRNA treatment. The latter shows the typical in situ appearance of an Sgo1-depletion arrested cell (see Figures 3 and 6). Sgo1 was stained with antibody 94 (shown in green), and CREST is shown in red. DNA was counterstained with DAPI.

Chromosome spreads were stained for Sgo1 with antibody 94 (shown in green), and counterstained with (A) CREST antiserum (bar = 10 μm), and (B) antibody to Aurora B, (both shown in red). DNA was stained with DAPI (shown in blue). Enlarged views show a single Sgo1 focus located between two sister CREST dots (A), and overlapping with Aurora B focus (B). Bar = 1 μm [applies to images b–d in (A) and (B)].

Synchronised HeLa cells were transfected with Sgo1 siRNA or dH₂O, harvested at 2-h intervals following release and examined by chromosome spreading and Giemsa staining. 100 cells were scored for mitotic index, and 100 mitotic cells were classified into seven categories based on chromosome configuration as exemplified in (B) for each time point.
(A) The frequency of each category of mitotic cell is given as a percentage of total cell numbers, such that the sum of each column represents the mitotic index.
(B) Representative pictures of seven categories of mitotic cells. Chromosome spreads: (a) prophase; (b) metaphase/metaphase-like; (c) anaphase; and (d) telophase. (e) Early phase of precocious sister disjunction. At this stage, sisters are beginning to separate and some or all presumptive sister pairs are still discernible. Arrowheads indicate chromosomes whose centromeric cohesion seems to be lost. (f) Later phases of sister chromatid separation. Sister pairs at this stage are no longer discernible, remnants of the metaphase plate are still visible, and sisters have not yet hypercondensed. (g) Scattered single chromatids. Sisters are completely separated and distributed randomly in relation to one another, individual chromatids are hypercondensed, giving a “curly” appearance. Note that chromatid separation in normal anaphases (c) are different from precocious sister chromatid separation (e–g), in that disjoined and paired chromatids coexist in the same cell.
(C) A portion of the cells harvested for the analysis in Figure 3A and 3B were ethanol-fixed and their DNA content was analysed by flow cytometry.

(A) Synchronised HeLa cells expressing EGFP-tagged histone H2B were transfected with Sgo1 siRNA or dH₂O as in Figure 1A, and analysed with time-lapse confocal microscopy. Stacks of 12 different z-plane images were obtained every 5 min and projected images for several time points are shown. Note that several chromosomes failed to congress to the metaphase plate, which was followed by progressive sister chromatid separation over time. Typically, a pair of disjoining sister chromatids dissociated simultaneously from the metaphase plate towards opposite poles.
(B) Summary of live cell analysis. Cells prepared as in (A) were examined by time-lapse immunofluorescence microscopy. Mitotic cells were aligned on the time axis according to NEBD as determined from the loss of a defined nuclear boundary. Time from NEBD to anaphase onset/sister chromatid separation (white portion of bars) and from this point to chromosome decondensation or apoptosis (grey portion of bars) was measured.
(C) To follow centromere dynamics, cells expressing EGFP-CENP-A were either mock-treated or Sgo1 siRNA transfected. Images were obtained by time-lapse confocal microscopy. Note that in cells depleted of Sgo1, dots of paired GFP-CENP-A can be found in earlier unaligned chromosomes (arrows). Single dots progressively fell apart in later phases, resulting in collapse of metaphase plate (images in lower row).

(A) Synchronised HeLa cells that inducibly express Scc1-myc were transfected with control or Sgo1 siRNA and processed for immunofluorescence microscopy with or without 4-h treatment with nocodazole. Mitotic cells were spun down on glass slides and analysed for cohesin with antibodies to myc epitope (right photomicrographs). Cells were costained with P-H3 (left photomicrographs, red) to identify cells from prophase to metaphase. DNA was counterstained with DAPI (left photomicrographs, blue). Note that with Sgo1 RNAi, Scc1-myc was undetectable in the majority of P-H3 positive cells (arrows). Cells with myc staining are indicated as staining controls (asterisks). Bar = 10 μm.
(B) Quantification of Scc1-myc staining. Approximately 200 cells with positive staining for P-H3 were assessed for Scc1-myc staining, which was classified as positive, reduced, or negative, as indicated.
(C) Lack of centromere enrichment of cohesin in Sgo1-depleted cells. Mitotic cells were prepared as in (A), and the centromeric enrichment of Scc1-myc was analysed by immunofluorescence microscopy. Merged pictures of Scc1-myc (green) and CREST antigen (red) are shown. Approximately 200 cells were scored for each experiment. Bar = 5 μm.
(D) Centromeric cohesin is not maintained in Sgo1-depleted cells. Mitotic cells were prepared and processed for immunofluorescence as in (C). Representative cells with various levels of Scc1-myc are shown. Note that centromeric staining of Scc1-myc emerges as the bulk of cohesin dissociates from chromosomes in controls (upper panels). However, in Sgo1 RNAi cells, centromeric enrichment is hardly seen at any stage of arm cohesin dissociation (lower photomicrographs). Bar = 10 μm.

(A) Sgo1 depletion prevents destruction of cyclin B1. Synchronised HeLa cells mock-treated or transfected with Sgo1 siRNA were processed for immunofluorescence microscopy using cyclin B1 antibodies (green) and CREST sera (red) 11 h after release from the second thymidine block. DNA was visualized by DAPI staining (blue). In mock-treated cells, cyclin B1 staining in prophase (a) and in prometaphase (b) disappears as cells segregate their chromosomes upon anaphase entry (c). Sgo1-depleted cells which segregate chromosomes asynchronously and arrest in a mitotic state retain preanaphase levels of cyclin B1 (d–e). Bar = 10 μm.
(B) Mitotic cells shown in (A) were classified into five categories based on DAPI-labelled chromosome configuration, and scored for presence or absence of cyclin B1 staining. Black bars represent the frequency of each category, and red bars represent the percentage of cells that fall into each category and are positive for cyclin B1 staining.
(C) Recruitment of Mad2 in cells depleted of Sgo1. Cells prepared as in (A) were stained with cyclin B1 antibody, and counterstained with Mad2 antibody (red) and DAPI to visualize DNA (blue). (a) Prometaphase mock-transfected cells show Mad2 staining at kinetochores, which is lost as the metaphase plate assembles and disappears before cells enter anaphase (unpublished data). (b) Sgo1-transfected cells which have prematurely segregated sisters remain positive for Mad2 kinetochore staining. Bar = 10 μm.
(D) Aurora B remains at centromeres in Sgo1-depleted cells. Cells prepared as in (A) were stained with Aurora B antibody (green) and counterstained with CREST antiserum (red), and DAPI to visualize DNA (blue). In prometaphase (a) mock-transfected cells, Aurora B is found at centromeres before relocalizing to the central spindle as cells enter anaphase (b). In Sgo1 transfected cells that show precocious sister separation, Aurora B remains localized at centromeres (c).

(A and B) Synchronised HeLa cells were mock-treated or transfected with the indicated combination of Sgo1 and Plk1 siRNA and harvested at the time shown following release from thymidine block. Chromosomes were then spread on glass slides and examined by Giemsa staining. As in Figure 3, the percentage of mitotic cells were calculated out of 200 cells, and mitotic chromosome spreads were then further classified into one of 10 categories (n = 200 mitotic spreads for each time point). (A) The frequency of each category of mitotic cell (see examples in Figures 3B and 7B) is given as a percentage of total cell numbers, such that the sum of each column represents the mitotic index. (B) In addition to the seven categories in Figure 3A and 3B, the following categories (illustrated in Figure 7B) were scored: (a) sister centromeres separated, arms still cohesed; (b) scattered chromatids, same as Figure 3B, part g; (c) sisters are separated and randomly distributed in relation to one another, but are not hypercondensed; and (d) sister chromatids are cohesed along their length, and rod-shaped chromosomes are hypercondensed, as is characteristically seen with Plk1 knockdown [20]. For simplicity, the categories illustrated in Figure 3B, part e and f, and Figure 7B, part a–c, were combined into a single category representing premature sister separation (shown in red). In smaller pictures, bar = 10 μm; in enlarged portions, bar = 2 μm.
(C and D) Synchronised HeLa cells were mock-treated or transfected as indicated. At 6 h after release from the second thymidine block, cells were treated with nocodazole with or without Aurora B inhibitor Hesperadin as indicated, and harvested at hourly intervals up to 3 h thereafter. Mitotic index and chromosome spreads were assessed as in (A). (C) The frequency of each category of mitotic cell (illustrated in Figures 3B and 7D) is given as a percentage of total cell numbers, such that the sum of each column represents the mitotic index. (D) The seven categories scored for Figure 3A and 3B were also scored for this experiment. (a) Scattered chromatids as in Figure 3B, part g; (b) early phase of precocious sister disjunction as in Figure 3B, part e. In addition the following categories (illustrated in Figure 7D) were also scored: (c) Chromosomes have begun to decondense prior to separation, and sister resolution is defective, characteristic of Hesperadin treatment. (d) Centromeres cohesed, and arms opened and hypercondensed, characteristic of nocodazole treatment.

(A) Total cell extracts prepared from HeLa cells that, upon doxycycline treatment, inducibly express either a wild-type (SA2-wt) or nonphosphorylatable (SA2–12xA) myc-tagged version of Scc3-SA2 were resolved by SDS-PAGE and probed with anti-SA2 antibody. The lower band represents endogenous Scc3-SA2, the upper band, myc-tagged Scc3-SA2. The first lane contains total cell extract prepared from untagged HeLa cells.
(B) Expression of nonphosphorylatable Scc3-SA2 suppresses sister separation caused by Sgo1 depletion. Cycling HeLa cells carrying inducible myc-tagged versions of Scc3-SA2 were treated with (or without) doxycycline at 2 μg/ml for 72 h prior to and during transfection to induce expression, as indicated. Cells were harvested 18 h and 24 h post transfection, chromosomes were spread on glass slides and Giemsa-stained. As in Figure 7, the percentage of mitotic cells were calculated out of 200 cells, and mitotic chromosome spreads were then further classified into one of five categories (n = 200 mitotic spreads for each time point). The frequency of each category of mitotic cell (see examples in Figures 3B, 7B, and 7D) is given as a percentage of total cell numbers, such that the sum of each column represents the mitotic index.
(C) Nonphosphorylatable Scc3-SA2-mediated suppression is still observed when combined with nocodazole arrest. The indicated treatments were repeated as outlined in (B) in the presence of nocodazole which was added to cultures 4 h posttransfection, i.e., 14 and 20 h prior to harvesting of cells. Samples were processed as in (B), with the addition of a sixth mitotic category.
(D) Live cell analysis of Sgo1 depletion of nonphosphorylatable Scc3-SA2 (SA2–12xA) expressing cells. To follow chromosome behaviour, cells stably expressing EGFP-H2B and inducibly expressing SA2–12xA were used. Expression of SA2–12xA was induced 72 h prior to transfection as in (B). Cells were transfected with Sgo1 siRNA as in Figure 1A and examined by time-lapse fluorescence microscopy. Significant number of cells exit mitosis by expressing nonphosphorylatable Scc3-SA2.
(E) Nonphosphorylatable SA2 (SA2–12xA) is found at centromeres even in the absence of Sgo1. HeLa cells containing either the myc-tagged wild-type (a) or SA2–12xA (b) inducible transgene were either uninduced or induced as in (B) 72 h before transfection. Transfection of Sgo1 siRNA was performed prior to the second thymidine block as in Figure 1A. At 8.5 h after the release from early S phase, mitotic cells were spun down on glass slides and analysed for cohesin localisation by immunofluorescence microscopy using antibodies to the myc epitope (shown in green). Cells were costained with CREST antiserum to label kinetochores (shown in red) DNA was counterstained with DAPI (shown in blue).
(F) Quantification of SA2-myc staining. Samples similar to those described in (E) were stained with myc and P-H3 antibodies (the latter to identify cells from prophase to metaphase). Approximately 200 P-H3-positive cells were assessed for SA2-myc staining, and the percentage of cells that were both P-H3- and SA2-myc-positive was plotted. We believe that the apparent drop in the number of SA2–12xA-myc positive cells observed with depletion of Sgo1 relative to mock transfection is a statistical artefact caused by the mitotic arrest and accumulation of those cells that did not express SA2–12xA-myc (∼30%) but that were depleted of Sgo1.

(A) During an unperturbed mitosis (Wild Type), arm cohesin (red circles) is removed in a kinase-dependent manner during prophase/prometaphase. Sgo1 protects centromeric cohesin (brown circles) until the metaphase-anaphase transition. Once all chromosomes have successfully bi-oriented on the metaphase plate, Mad2 inhibition of the APC/C is relieved, allowing separase activation. Separase in turn removes cohesin remaining at centromeres through cleavage of the α kleisin Scc1 subunit, allowing the cell to enter anaphase. In the absence of Sgo1 (Sgo1 Depletion), cohesin is removed from the chromosome arms and at the centromere during prophase/prometaphase before chromosomes have properly bi-oriented and been attached to their full complement of spindles. Thus, Mad2 activity continues to maintain the spindle checkpoint, causing cells to arrest for a prolonged period in a prometaphase-like state with separated sister chromatids. Expression of nonphosphorylatable Scc3-SA2 (Sgo1 Depletion + Cohesin containing SA2–12xA) prevents the prophase removal of cohesin both from arms and centromeres, thus allowing cells to proceed through an apparently normal anaphase even in the absence of Sgo1 activity.
(B) The phosphorylation state of cohesin may be the result of a dynamic balance in which Sgo1 somehow functions to antagonize the activity of mitotic kinases, including Plk1 and Aurora B and, potentially, other as-yet unidentified kinases (Kinase X, Y). In this model, when the direction of the reaction is artificially sent towards the hyperphosphorylated state (following Sgo1 knockdown), cohesin efficiently dissociates from chromatin; when the opposite state is favoured (e.g., as a result of Plk1 depletion) cohesin remains tightly associated with chromatin.