Analysis of Scc1-deficient cells defines a key metaphase role of vertebrate cohesin in linking sister kinetochores

Abstract

Cleavage of the cohesin subunit Scc1p/Mcd1p/Rad21 permits sister chromatid separation and is considered to trigger anaphase onset. It has also been suggested that the cohesin complex is essential for chromosome condensation and for assembling fully functional kinetochores. Here, we used vertebrate cells conditionally deficient in Scc1 to probe cohesin function in mitosis. Cells lacking cohesin arrest in prometaphase, with many chromosomes failing to align at a metaphase plate and high levels of the spindle assembly checkpoint protein, BubR1, at all kinetochores. We show that the structural integrity of chromosomes is normal in the absence of Scc1. Furthermore, specific inhibition of topoisomerase II, which is required for decatenation of replicated chromosomes, can bypass the cohesin requirement for metaphase chromosome alignment and spindle checkpoint silencing. Since the kinetochore effects of Scc1 deficiency can be compensated for by topoisomerase II inhibition, we conclude that Scc1 is not absolutely required for kinetochore assembly or function, and that its principal role in allowing the onset of anaphase is the establishment of sufficient inter-sister tension to allow biorientation.

Figure 1

Scc1-deficient chromosomes have a stable higher-order structure. (A) Assay for mitotic chromosome structural integrity (Hudson et al, 2003). Note that this is a morphological assay and might not detect the possible loss or alteration of associated proteins during the refolding cycles. (B) Even though the sister chromatids (arrows) show an abnormal degree of premature separation, cohesin (Scc1)-depleted chromosomes have normal structural integrity. (C) Quantitation of the results presented in panel (B). Examples of normally refolded and aberrantly refolded chromosomes are shown at the right. Scc1-depleted chromosomes recover normally.

Figure 2

Successful alignment of Scc1-deficient chromosomes at a metaphase plate following treatment with topo II inhibitors. (A) A metaphase cell was defined as a cell with its chromosomes all aligned on the plate, as shown at the top, whereas cells with chromosomes scattered about the plate (arrows, lower panels) were defined as prometaphase. Cells on polylysine-treated slides were fixed and incubated with antibodies to α-tubulin (red) and CENP-C (green), along with DAPI (grey in left panels). Scale bar, 10 μm. (B) Histogram showing the relative percentage of metaphase Scc1^OFF cells following the treatment of synchronized cells with the topo II inhibitors ICRF-159 or ICRF-187 for 1 h before analysis. Cells in which the spindle axis was not parallel to the slide were excluded from this analysis. Data shown are the mean±standard deviation of at least three experiments in which at least 25 cells with appropriately aligned spindles were scored blind. Levels of alignment under all conditions differed significantly from those observed in Scc1^OFF conditions (χ²-test; P<0.01). A control experiment on 25 Scc1^ON metaphases showed 85 and 84% aligned at metaphase after treatment with ICRF-159 and ICRF-187, respectively.

Figure 3

Decrease in kinetochore-associated BubR1 in Scc1-deficient cells following topo II inhibitor treatment. (A, B) All Scc1^OFF cells in early mitosis have levels of BubR1 accumulation at kinetochores characteristic of prometaphase for both aligned and nonaligned chromosomes. (C, D) Treatment of Scc1^OFF cells with the topoisomerase inhibitors ICRF-159 (C) and ICRF-187 (D) promotes the alignment of chromosomes at a metaphase plate and results in a downregulation of BubR1 levels at the kinetochores. Synchronized, Scc1-deficient cells were treated as for Fig 2. Staining is shown with antibodies to α-tubulin (red) and BubR1 (green), along with DAPI (grey in the right panels). (E) Treatment of Scc1^OFF cells with ICRF-159 promotes the bipolar attachment of sister kinetochores to opposite spindle poles. Synchronized, Scc1-deficient cells were treated as for Fig 2. Staining is shown with antibodies to α-tubulin (green) and CENP-C (red), along with DAPI (blue). Scale bars, 5 μm.