1. Volume 9, Issue 3, Pages 515-525 (March 2002)
The Dissociation of Cohesin from Chromosomes in Prophase Is Regulated by Polo-like Kinase 
Izabela Sumara, Elisabeth Vorlaufer, P.Todd Stukenberg, Olaf Kelm, Norbert Redemann, Erich A. Nigg, Jan-Michael Peters 
Molecular Cell 
Volume 9, Issue 3, Pages (March 2002)
DOI: /S (02)
Copyright © 2002 Cell Press Terms and Conditions

2. Figure 1
Fractionation of a Mitosis-Specific Cohesin-Dissociation Activity
(A) A S100 fraction from mitotic Xenopus extract containing cyclin B-Δ90 (S100m) was fractionated by gel filtration chromatography. Each column fraction was incubated for the indicated periods of time with chromatin from HeLa cells stably expressing SCC1-myc. Subsequently, chromatin was removed by centrifugation and the amount of cohesin dissociation was analyzed by SDS-PAGE and immunoblotting the supernatant fractions with Myc antibodies.
(B) S100 fractions from interphase (S100i) Xenopus extracts were fractionated and analyzed for their ability to dissociate cohesin from chromatin as in (A). The corresponding fractions from S100m were analyzed side by side.
(C) Cohesin was immunoprecipitated with SA1 antibodies from interphase (i) and CSF (m) Xenopus extracts and analyzed by SDS-PAGE and immunoblotting with antibodies to phosphorylated threonine (P-Thr), SA1, and SCC1.
(D) Gel filtration chromatography fractions from (A) were analyzed by immunoblotting with antibodies to the indicated protein kinases. The peak fractions in which thyroglobulin (698 kDa), catalase (206 kDa), BSA (67 kDa), and chymotrypsinogen (25 kDa) eluted in a parallel fractionation experiment are indicated.
Molecular Cell 2002 9, DOI: ( /S (02) )
Copyright © 2002 Cell Press Terms and Conditions

3. Figure 2
Polo-like Kinase Is Required for Cohesin Dissociation from Chromatin, but Aurora A and B Are Not
(A) Aurora A and Aurora B immunoblots showing Xenopus interphase extracts and CSF-arrested extract before and after immunodepletion with either control antibodies or antibodies to Aurora A or B (Ab Depleted).
(B) Interphase extracts depleted as in (A) were incubated with sperm nuclei for 30 min to allow chromatin assembly. Subsequently, half volumes of CSF extracts depleted as in (A) were added to trigger entry into M phase. At the indicated time points after addition of CSF extracts, chromatin was reisolated and analyzed by immunoblotting with antibodies to subunits of cohesin (SCC1, SA1) or condensin (SMC2). MCM3 was analyzed as a loading control. P-H3, histone H3 phosphorylated on serine 10.
(C) Interphase and CSF extracts depleted with control or PLX1 antibodies were analyzed as in (B). In vitro translation mixture containing S35-labeled CDC25C was added together with the CSF extracts. In two cases, PLX1-depleted extracts were supplemented with 10 ng/μl of either recombinant wild-type PLX1 kinase (+ WT His-PLX1) or of a catalytically inactive PLX1 mutant (+ N172A His-PLX1) at the same time when the CSF extracts were added. Extract samples (four top panels) or reisolated chromatin (five bottom panels) were collected at the indicated time points and analyzed by SDS-PAGE and either phosphorimaging (S35CDC25C and phosphorylated histone H1, P32-H1) or immunoblotting with antibodies to the indicated proteins (all other panels). The cell cycle state of the extracts was analyzed by monitoring the phosphorylation of S35-labeled CDC25C and histones H1 and H3. Proteasomes were analyzed as a loading control. In some cases, phosphorylated H3 and SMC4 bound to chromatin can already be observed at the zero time point. This may be due to the fact that different samples have to be kept on ice for different periods of time until they are processed by centrifugation and mixing with SDS sample buffer. Progression into M phase may continue to occur slowly in the samples kept on ice.
Molecular Cell 2002 9, DOI: ( /S (02) )
Copyright © 2002 Cell Press Terms and Conditions

4. Figure 3
Immunofluorescence Microscopy of Chromatin Assembled in PLX1-Depleted Extracts
(A) Xenopus interphase extracts depleted with control or PLX1 antibodies were incubated with sperm nuclei for 120 min to allow chromatin assembly, formation of nuclei, and DNA replication. In vitro translation mixture containing S35-labeled CDC25C was added to monitor the cell cycle state of the extracts. Subsequently, control or PLX1-depleted CSF extracts were added to trigger entry into M phase. In a control experiment, PLX1-depleted extract was supplemented with 10 ng/μl recombinant wild-type PLX1 (+ WT His-PLX1) at the same time when CSF extract was added. At the indicated time points, extract samples were analyzed by SDS-PAGE and either phosphorimaging (S35CDC25C) or immunoblotting with antibodies to phosphorylated histone H3 (P-H3) and PLX1.
(B) Chromatin assembled as in (A) was isolated on cover slips by centrifugation through 40% glycerol and analyzed by indirect immunofluorescence microscopy. Chromatin was isolated from interphase extracts (I) at 120 min before addition of CSF extracts (upper panels) and from M phase extracts (M) 120 min after addition of CSF extracts (lower panels). DNA was stained with DAPI, cohesin with antibodies to SCC1, and condensin with antibodies to SMC2.
(C) Higher magnification of chromosomes isolated from M phase extracts as in (B). Note that individual chromosomes composed of two sister chromatids can be observed in control depleted extracts, whereas no individual chromosomes can be seen in PLX1-depleted extracts.
Molecular Cell 2002 9, DOI: ( /S (02) )
Copyright © 2002 Cell Press Terms and Conditions

5. Figure 4
Polo-like Kinase Is Sufficient to Induce Cohesin Dissociation in Interphase
(A) Xenopus sperm nuclei were incubated for 30 min in interphase extracts from cycloheximide-treated Xenopus eggs to allow chromatin assembly. Then either DMSO, or 1 μM OA or 10 ng/μl recombinant wild-type PLX1 (WT-PLX1) or hyperactive mutant (T201D-PLX1) was added. At different time points, extracts samples (top panels) were analyzed by SDS-PAGE and phosphorimaging (S35CDC25C). Chromatin was reisolated by sucrose cushion centrifugation and analyzed by immunoblotting with antibodies to the indicated proteins (three bottom panels).
(B) Experiment as in (A), except that 10 ng/μl or 20 ng/μl recombinant wild-type PLX1 was used.
Molecular Cell 2002 9, DOI: ( /S (02) )
Copyright © 2002 Cell Press Terms and Conditions

6. Figure 5
Polo-like Kinase Is Required and Sufficient for Cohesin Phosphorylation
(A) Cohesin was purified from HeLa cells (cohesini) arrested in S phase by HU and incubated in either Xenopus interphase extracts (Xti), CSF extracts (CSF), CSF extracts depleted with control antibodies (CSFΔContr), or CSF extracts depleted with PLX1 antibodies (CSFΔPLX1). Subsequently, cohesin was reisolated from the extracts and analyzed side by side with cohesin purified from HeLa cells arrested by nocodazole (cohesinm) by SDS-PAGE and immunoblotting with antibodies to phosphorylated threonine (P-Thr). The same filters were reprobed with SA2 and SCC1 antibodies.
(B) The cell cycle state of the extracts from (A) was determined either by phosphorimaging (S35CDC25C) or immunoblotting with antibodies to CDC27 at different time points.
(C) Xenopus interphase extracts (Xti) were depleted either with control antibodies (Xtm Δ Contr), PLX1 antibodies (Xtm Δ PLX1), or with PLX1 antibodies, and supplemented with recombinant human PLK1 kinase (Xtm Δ PLX1 + PLK1). Xenopus interphase extracts (Xti) and mitotic extracts (obtained by addition of nondegradable cyclin B Δ 90 and 1 μM of OA, Xtm) were analyzed by immunoprecipitation with antibody to Xenopus SA1. Cohesin immunoprecipitates were analyzed by immunoblotting with antibody to phosphorylated threonine (P-Thr). The same filters were reprobed with SA2 and SCC1 antibodies.
(D) The cell cycle state of the extracts used in (C) was determined either by phosphorimaging (S35CDC25C) or immunoblotting with antibodies to CDC27.
(E) Cohesin was purified from logarithmically growing HeLa cells and from cells arrested by nocodazole (cohesinm). Cohesini was incubated with recombinant PLK1 kinase, and reactions were terminated at the indicated time points and analyzed by silver staining and phosphorimaging (P32-exposure). The positions of cohesin subunits are indicated on the left. SA2 subunit is indicated by an arrowhead.
Molecular Cell 2002 9, DOI: ( /S (02) )
Copyright © 2002 Cell Press Terms and Conditions

7. Figure 6
Cohesin Phosphorylation by PLK1 Reduces Cohesin's Ability to Bind Chromatin
(A) Cohesin was purified from logarithmically growing HeLa cells (cohesini, lanes 1 and 3) and from cells arrested by nocodazole (cohesinm, lanes 2 and 4). Cohesin eluates were incubated in PLX1-depleted interphase extracts from cycloheximide-treated Xenopus eggs containing sperm nuclei with and without addition of 1 μM of OA. Subsequently, chromatin was reisolated and analyzed by SDS-PAGE and immunoblotting with antibodies to MCM3 and Myc (top). Inputs of cohesini (lane 1, bottom) and cohesinm (lane 2, bottom) were analyzed by SDS-PAGE and immunoblotting with antibody to phosphorylated threonine (P-Thr) and cohesin antibodies as indicated.
(B) Purified cohesin from HeLa cells arrested by nocodazole (cohesinm, lanes 1, 3, 4, and 5) was treated with λ-PPase (lane 2) and incubated in the extract as in (A). As a control, the indicated amounts of λ-PPase were added directly to the extract (lanes 3–5). Chromatin was reisolated and analyzed by SDS-PAGE and immunoblotting with antibodies to MCM3 and Myc (top). Inputs of cohesinm and λ-PPase-treated cohesinm were analyzed by SDS-PAGE and immunoblotting with antibody to phosphorylated threonine (P-Thr) and cohesins as indicated.
(C) Cohesin purified from HeLa cells arrested by HU (cohesini, lanes 1 and 3) was phosphorylated with recombinant GST-PLK1 (lane 2) and incubated in the extracts as above. As a control, a higher amount of GST-PLK1 recombinant kinase was added directly to the extract (lane 3). Reisolated chromatin and inputs of purified cohesin complexes were analyzed as in (A) and (B).
Molecular Cell 2002 9, DOI: ( /S (02) )
Copyright © 2002 Cell Press Terms and Conditions