1. Volume 58, Issue 3, Pages 495-506 (May 2015)
Human Chromosome Segregation Involves Multi-Layered Regulation of Separase by the Peptidyl-Prolyl-Isomerase Pin1 
Susanne Hellmuth, Scott Rata, Andreas Brown, Stefan Heidmann, Bela Novak, Olaf Stemmann 
Molecular Cell 
Volume 58, Issue 3, Pages (May 2015)
DOI: /j.molcel
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2. Molecular Cell 2015 58, 495-506DOI: (10.1016/j.molcel.2015.03.025)
Copyright © 2015 Elsevier Inc. Terms and Conditions

3. Figure 1
The Peptidyl-Prolyl Isomerase Pin1 Is a Mitosis-Specific Interactor of Vertebrate Separase
(A) Mitotic HEK293T lysate (input) was subjected to IP with anti-separase or mock IgG and analyzed by immunoblotting.
(B) Myc-separase-variants-expressing HEK293s were synchronized and subjected to Myc IP and immunoblotting. Thy., thymidine; RO, CDK1-inhibitor RO3306; ΔCLD, Cdc6-like domain deleted; H3-pS10, Ser10-phosphorylated histone H3.
(C) Cells as in (A) and transiently expressing GST-PPIase or -WW were mitotically arrested, lysed, and subjected to Myc IP.
(D) Lysates of mitotic HEK293Ts expressing Myc-tagged separase variants together with GST-WW were subjected to Myc IP.
(E) Mitotic HEK293Ts expressing Myc-separase variants were subjected to Myc IP and, where indicated, λ-phosphatase treatment. Following additional washing, bound proteins were detected by (far) western analysis.
(F) Docking of the WW domain onto phosphorylated Ser1153 recruits Pin1 to separase and facilitates the isomerization of pSer1126-Pro1127 by the PPIase domain.
See also Figure S1.
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4. Figure 2 Pin1 Is a Negative Regulator of Separase
(A) Transgenic HEK293s transfected with the indicated siRNA were induced with doxycycline (+ Dox.) to overexpress separase-WT or left uninduced and replated. Graph shows mean numbers of stained, automatically counted colonies (bars) of four experiments (dots).
(B) ZZ-Tev-separase-securin from G1/S HEK293Ts was incubated on IgG-sepharose in Pin1- or mock-depleted Xenopus egg extract supplemented with Δ90-cyclin B1 and, where indicated, GST-Pin1. Separase beads were re-isolated, washed with high salt (as indicated), and assayed for bound proteins and Scc1 cleavage.
(C) Myc-separase-WT-expressing HEK293s were transfected with siRNA against PIN1 or GL2 and a (PIN1 siRNA-resistant) plasmid encoding Flag-Pin1, treated with nocodazole and EGCG, and analyzed by Myc IP and chromosome spreads. Shown are mean values (bars) of 3–4 experiments (dots) quantifying at least 100 cells each.
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5. Figure 3
Catalytically Active Pin1 Is Required for Cdk1-Cyclin B1-Dependent Inhibition of Separase
(A) HEK293Ts expressing Myc-separase-WT, -S1126A, -P1127A, or -S1153A alone or together with Flag-PPIase-WT or -C113A were synchronized in mitosis, and lysates (input) were subjected to Myc IP and immunoblotting.
(B) Chromosome spreads of cells from (A).
(C) Multi-step, Pin1-dependent assembly of the Cdk1-cyclin B1-separase complex.
See also Figure S2.
Molecular Cell  , DOI: ( /j.molcel )
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6. Figure 4
Pin1 Isomerizes Previously Securin-Inhibited Separase in Anaphase
(A) Using anti-Myc, -securin, or -HA (hemagglutinin tag), respectively, separase, securin, or Pin1 were IPed from mitotic HEK293Ts overexpressing Myc-Tev-separase and HA-Pin1. Negative controls: Myc IP in presence of TEV-protease, IP with unspecific IgG, or HA IP from cells expressing untagged Pin1.
(B) HeLa Ks were released from Tax by ZM (t = 0 min) and analyzed at indicated time points thereafter. Where indicated, EGCG was added prior to release.
(C) Cartoon illustrating that phosphorylation energetically favors the cis-isomer while securin locks separase in trans.
(D) Model of how relieving the securin-imposed conformational constraint allows simultaneous binding of securin (fragments) and Cdk1-cyclin B1 to phosphorylated separase.
(E) Myc-Tev-separase-Δ92-securinHRV-Flag was purified by Myc IP and eluted with TEV-protease. After immobilization on anti-Flag agarose, the complex was incubated with Cdk1-Δ90-cyclin B1 and ATP, GST-Pin1, EGCG, and/or HRV-protease, as indicated, before unbound (supernatant) and bound proteins (beads) were detected by immunoblotting.
See also Figure S3.
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7. Figure 5
Pin1 Contributes to the Irreversibility of Separase Activation
(A) Securin-associated Myc-Tev-separase-WT or -S1126A on anti-Myc beads was incubated in Δ90-cyclin B1-containing Xenopus egg extract, re-isolated, and, where indicated, washed with high salt. Separases were then incubated with recombinant Δ92-securin, washed, eluted with TEV-protease, and analyzed by immunoblotting and Scc1 cleavage assay/autoradiography.
(B) Myc-Tev-separase-WT was treated and analyzed as described in (A) except for the addition of EGCG (or DMSO) prior to Δ92-securin.
(C) Tax-arrested HeLa cells were induced to express Flag-securinKDM, supplemented with EGCG where indicated, released by ZM (t = 0 min), and subjected to separase-IP at indicated times.
(D) Quantification of the securin immunoblot from (C). Black lines, open circles indicate separase-associated securinKDM-Flag relative to endogenous securin. Gray lines, black circles indicate total amount of separase-associated securin relative to separase (set to 1 at t = 0).
See also Figure S4.
Molecular Cell  , DOI: ( /j.molcel )
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8. Figure 6
Dephosphorylation of Cyclin B1 Increases Its Affinity for Separase, thereby Giving Rise to a Second Peak of Cdk1-Cyclin B1-Separase in Late Mitosis
(A) Myc-separase-WT- and Δ90-cyclin B1-WT- or -4A-expressing HEK293s were Tax-ZM treated. Association of endogenous- and Δ90-cyclin B1 and securin with separase was followed by time-resolved Myc IP.
(B) Myc-separase-WT- and cyclin B1-8A-Flag-expressing HEK293s were treated as in (A).
(C) Mathematical simulation of mitotic progression from an initial checkpoint arrest. Plotted are relative abundances of free Cdk1-cyclin B1, securin-separase, Cdk1-cyclin B1-separase, active APC/C, free separase, and cleaved cohesin for three different scenarios (all without cycloheximide): normal mitotic progression (left), presence of phosphorylation site mutant cyclin B1-8A (middle), and absence of Pin1 or replacement of endogenous separase by a S1126A or P1127A variant (right). Note that absence of isomerization results in premature sister chromatid separation only upon overexpression of separase, which is not considered here.
See also Figure S5.
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9. Figure 7 Pin1-Dependent Rapid Inactivation of Separase in Late Mitosis
(A) Separase-S1126A and -ΔCLD were purified as in Figure 5A, shifted to room temperature (t = 0), and assessed for proteolytic activity toward 35S-Scc1 at indicated times.
(B) Separase-WT was purified and treated as in (A), except that EGCG was present until elution with TEV-protease. Separase was combined with recombinant Pin1 or EGCG and then analyzed by immunoblotting and Scc1 cleavage assay/autoradiography. Note that in (A) and (B), gels were blotted and membranes cut and subjected to western analysis prior to reassembly and autoradiography.
(C) Separase-S1126A- or -ΔCLD-expressing HEK293s were Tax-ZM treated and subjected to chromatin isolation at the indicated time points. Soluble proteins (supernatant) and pelleted chromatin fractions were analyzed by immunoblotting and Coomassie staining (histones). For the α-tubulin western analysis of chromatin, supernatant was loaded into the outer lanes.
(D) HeLa cells of the Kyoto line (HeLa Ks) were EGCG or DMSO treated prior to IF. Scc1-positive chromatin was scored in telophase cells with condensed DNA (as judged by DAPI staining) and broad midbodies (as judged by the survivin signal). Scale bar = 6 μm.
Molecular Cell  , DOI: ( /j.molcel )
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