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First published online 25 July 2006
doi: 10.1242/jcs.03083

Journal of Cell Science 119, 3325-3336 (2006)
Published by The Company of Biologists 2006
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Cyclin-B1-mediated inhibition of excess separase is required for timely chromosome disjunction

Andrew J. Holland and Stephen S. Taylor*

Faculty of Life Sciences, Michael Smith Building, Oxford Road, University of Manchester, Manchester, M13 9PT, UK


Figure 1
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  		Fig. 1. Human separase is phosphorylated on S1126. (A) Schematic representation of human separase showing the ARM-repeat domain, the active and inactive caspase-like domains, and the regulatory region, which contains S1126 and the cyclin B1 binding domain. The catalytic cysteine C2029 and the auto-cleavage sites (AC) are shown. (B) Dot blot showing that the separase antibody recognises the phosphorylated and non-phosphorylated peptides, whereas the P-S1126 antibody only detects the phosphorylated peptide. (C) Immunoprecipitations of Myc-tagged proteins showing that the separase antibody recognises both WT and separase S:A, whereas the P-S1126 antibody only detects WT. The band marked by the arrow head represents full-length separase, the asterisk marks the N-terminal auto-cleavage product.

 

Figure 2
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  		Fig. 2. S1126 phosphorylation is mitotic-specific. (A) Anti-Myc immunoprecipitates from cells synchronised with thymidine, nocodazole or taxol, showing that separase is phosphorylated and bound to cyclin B1 only in mitotic-enriched populations. MI represents the mitotic index (%) of the population. (B) Cyclin B1 immunoprecipitations from asynchronous or nocodazole-treated HeLa cells showing that phosphorylated separase is present in the immune complex isolated from the nocodazole-treated sample.

 

Figure 3
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  		Fig. 3. S1126 phosphorylation is not required to maintain cyclin B1 binding. (A) Anti-Myc immunoprecipitates treated with {lambda} phosphatase, then washed with low-salt or high-salt buffers. Notice that, despite being dephosphorylated on S1126, separase still binds cyclin B1. (B) Anti-Myc immunoprecipitates showing that, despite being phosphorylated, the LAG and {Delta}12 mutants do not bind cyclin B1.

 

Figure 4
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  		Fig. 4. The separase S1126A mutation induces mitotic arrest. (A) Immunoblot showing tet-induced expression of Myc-tagged WT and separase S:A. Notice the increase in securin following separase induction. (B) Histograms showing accumulation of cells with DNA content >=4N cells 24 hour post-induction of separase S:A. Numbers represent mitotic index as determined by MPM-2 staining. (C) Immunoblot of tet+ cells showing equivalent expression of WT, S1126A, C2029A and the S1126A-C2029A double mutant. (D,E) Bar graphs showing the time spent in mitosis based on phase-contrast time-lapse analysis.

 

Figure 5
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  		Fig. 5. Cyclin B1 binding is required to prevent premature activation of separase. (A) Immunoprecipitations of Myc-tagged proteins from cell lines harbouring four separase transgenes blotted as indicated. (B) DNA content profiles showing G2/M defect 24 hours post-induction of separase mutants. (C) Box-plots of phase-contrast time-lapse microscopy analysis showing mitotic arrest phenotype following induction of separase mutants that do not bind cyclin B1. WT and separase S:A are shown for comparison. (D) Bar graph quantitating the number of metaphase spreads with separated chromatids 8 hours post-induction.

 

Figure 6
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  		Fig. 6. Separase S1126A induces premature loss of sister chromatid cohesion. (A) Interphase FISH images of WT and separase S:A cells 24 hour post-induction. (B) Histograms scoring number of FISH foci per cell showing that separase S:A induces aneuploidy. (C) Metaphase spreads 8 hours post-induction showing separated sister chromatids in a separase S:A cell. (D) Bar graph quantitating the number of metaphase spreads with separated chromatids in WT and separase S:A populations 8 hours post-induction. (E,F) Immunofluorescence analysis of mitotic cells expressing either WT or separase S:A, stained as indicated.

 

Figure 7
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  		Fig. 7. Separase S1126A uncouples chromatid disjunction from mitotic exit. (A) GFP histone time-lapse analysis showing a normal mitosis in a WT separase cell. (B) Prolonged mitosis in a separase S:A cell, showing rapid chromatid disjunction followed by realignment of separated chromatids. (C) Separase S:A cell showing chromatid disjunction with unaligned chromosomes (arrow heads). (D) Quantitation of normal and abnormal mitoses in WT and separase S:A populations. (E) Bar graphs showing the time from nuclear envelope breakdown to metaphase and from metaphase to sister chromatid separation. Cells expressing WT separase are sub-divided into those that performed a normal mitosis (green), or an abnormal mitosis (blue), whereas all separase S:A cells are plotted together (red). Notice that metaphase in separase S:A cells is defined as the point prior to chromatid disjunction when most or all of the chromosomes have aligned.

 

Figure 8
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  		Fig. 8. Separase overexpression sequesters securin in the cytoplasm. Immunofluorescence images showing that tet-induced expression of WT separase (A) and separase S:A (B) results in the endogenous securin accumulating in the cytoplasm of interphase cells. Notice that, whereas securin is not present in anaphase WT cells (arrow head), it is detectable in separase S:A cells that have undergone a premature loss of cohesion (arrows).

 

Figure 9
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  		Fig. 9. Securin degradation is not required for the premature loss of cohesion in separase S:A cells. Time-lapse sequences and pixel-intensity measurements showing securin-dsRed fluorescence in (A) control or (B) Separase-S:A-expressing cells. Whereas securin-dsRed is degraded prior to chromatid disjunction in the control cell, it is not degraded until mitotic exit in the separase S:A cell.

 

Figure 10
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  		Fig. 10. Increasing securin levels restores coordination of mitotic events in separase S1126A cells. (A) Time-lapse sequence of a separase S:A cell expressing dsRed showing premature loss of cohesion. (B) Time-lapse sequences of separase S:A cells expressing securin-dsRed proteins, showing rescue and cut phenotypes. (C) Quantitation of phenotypes following expression of securin-dsRed fusions in separase S:A cells.

 

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