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First published online 26 April 2005
doi: 10.1242/jcs.02355

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Functional contribution of Pds5 to cohesin-mediated cohesion in human cells and Xenopus egg extracts

Ana Losada^*, Tomoki Yokochi and Tatsuya Hirano

Cold Spring Harbor Laboratory, 1 Bungtown Road, Cold Spring Harbor, NY 11724, USA

View larger version (11K): [in a new window]   Fig. 1. Schematic representation of the primary structures of Pds5A and Pds5B from human and Xenopus. The yellow bars indicate the `core' region conserved between Pds5A and Pds5B, which contains multiple HEAT repeats (shown in magenta). The C-termini of Pds5A (blue line) and Pds5B (red line) display little similarity. Consensus sites for Cdk phosphorylation are clustered in the C-terminal domain of Pds5B (indicated by thin, vertical red lines) but not of Pds5A. The shadowed N-terminal region of Xenopus Pds5B has not yet been cloned.

View larger version (47K): [in a new window]   Fig. 2. Pds5A and Pds5B associate with cohesin in HeLa cell nuclear extracts. (A) Immunoblot of HeLa cell nuclear extracts with antibodies against Pds5A (lane 1), Pds5B (lane 2) and the cohesin subunit Smc3 (lane 3). (B) The same antibodies were used in immunoprecipitations carried out on HeLa cell nuclear extracts in the absence (lanes 1, 3 and 5) or presence (lanes 2 and 4) of the corresponding antigen peptide (0.5 mg/ml). The immunoprecipitates were fractionated in a 7.5% SDS-polyacrylamide gel and stained with silver. The positions of cohesin subunits, Pds5A and Pds5B are marked by arrowheads, a white circle and a black circle respectively, in lanes 1 and 3. The two polypeptides of 105 and 110 kDa present in the Pds5B precipitate (asterisks in lane 3) are likely to be non-specific because neither of them was detectable when a different antibody against Pds5B was used. (C) Immunoblot analysis of the immunoprecipitates obtained with non-immune rabbit IgG as control (lane 1), anti-Pds5A (lane 2), anti-Pds5B (lane 3) or anti-Smc3 (lane 4). (D) The immunoprecipitates obtained with anti-Smc3 were washed with a buffer containing increasing concentrations of KCl (0.2 M, 0.4 M and 0.8 M, lanes 1-3) before being analyzed by immunoblotting.

View larger version (64K): [in a new window]   Fig. 3. Association of Pds5A with chromatin requires functional cohesin in HeLa cells. (A) HeLa cells were transfected with siRNAs specific to the cohesin subunit Scc1, Pds5A or Pds5B or mock-transfected with buffer as control. To estimate the extent of depletion, aliquots of siRNA-treated cell extracts were analyzed by immunoblotting (lanes 5-7) alongside increasing amounts of a control cell extract (expressed as percentage, lanes 1-4). The levels of Orc2 were analyzed as a loading control. (B,C) HeLa cells transfected as in A were pre-extracted with detergent before fixation and stained with DAPI and anti-Smc3 (B) or anti-Pds5A (C). All images were acquired over the same exposure time. Bar, 50 µm.

View larger version (60K): [in a new window]   Fig. 4. Cohesion defects in cells with reduced levels of cohesin or Pds5 proteins. (A) HeLa cells grown on coverslips that had been transfected with no siRNA (control) or with siRNAs specific to Scc1, Pds5A or Pds5B were incubated in 60 mM KCl for 30 minutes, fixed and stained with DAPI, anti-condensin (CAP-E/Smc2) and anti-aurora B. In the `merge' panels, CAP-E and aurora B signals appear in red and green, respectively. Representative examples of a normal metaphase cell (control), and of the different types of abnormalities found among the cells treated with siRNA (type I, type II and type III). (B) Quantification of the defects described in A. The total number of metaphase cells scored was 283 (control), 450 (Scc1 siRNA), 260 (Pds5A siRNA), and 243 (Pds5B siRNA). (C) Examples of metaphase chromosome spreads prepared from siRNA-transfected cells and co-stained with DAPI and anti-CAP-E. The top panels show control chromosomes and the bottom panels show chromosomes with unpaired chromatids (type I). The frequency of this latter phenotype is also shown. The total number of metaphase cells scored was 269 (control), 279 (Scc1 siRNA), 227 (Pds5A siRNA), and 225 (Pds5B siRNA). Bar, 5 µm (A,C); 2 µm (insets in A).

View larger version (45K): [in a new window]   Fig. 5. Chromosome alignment and segregation defects in HeLa cells with reduced levels of cohesin or Pds5 proteins. HeLa cells that had been mock-transfected (control) or transfected with siRNAs specific for Scc1, Pds5A or Pds5B were fixed and labeled with antibodies against -tubulin (green) and CENP-E (red). DNA was stained with DAPI (blue). Representative examples of a normal metaphase cell (a), and of the different types of abnormalities found among the cells treated with siRNA (b-e). The frequency of the defects among the population of prometaphase/metaphase cells in the different cultures is shown in the graph below the images. The number of cells scored was 284 (control), 461 (Scc1 siRNA), 269 (Pds5A siRNA) and 242 (Pds5B siRNA). Bar, 5 µm.

View larger version (66K): [in a new window]   Fig. 6. Characterization of Pds5A and Pds5B in Xenopus egg extracts. (A) Pds5A (lane 2) and Pds5B (lane 3) were immunoprecipitated from Xenopus HSS with the corresponding antibodies. Cohesin complexes were immunoprecipitated in parallel reactions with anti-Smc3 (lane 4), and non-immune rabbit IgG was used as control (lane 1). The immunoprecipitates were analyzed by immunoblotting. (B) Sperm chromatin was incubated in interphase LSS to assemble interphase nuclei and then half a volume of CSF (mitotic) LSS was added to trigger entry into mitosis. Chromatin fractions were isolated at the times indicated (lanes 3-13), and analyzed by immunoblotting. A sample from a mock assembly reaction without sperm chromatin (lane 2) and an aliquot of the interphase extract were also included (lane 1). (C) Interphase chromatin was assembled in interphase LSS that had been immunodepleted with non-immune IgG (lanes 1 and 4; mock-dep), anti-Pds5B (lanes 2 and 5; Pds5B-dep) or a mixture of anti-Smc1 and anti-Smc3 (lanes 3 and 6; cohesin-dep). Aliquots of each extract (lanes 1-3) and the corresponding chromatin fractions (lanes 4-6) were analyzed by immunoblotting.

View larger version (67K): [in a new window]   Fig. 7. Morphological analysis of chromosomes assembled in the absence of Pds5A and Pds5B. (A) Representative images of individual chromosomes assembled in mock-depleted (left), cohesin-depleted (center) and Pds5-depleted (both Pds5A and Pds5B) extracts (right) and stained with DAPI, anti-CAP-G (condensin I), and anti-CAP-H2 (condensin II). The signals of condensin I and II appear in green and red, respectively, in the merged images shown in the bottom panels. The arrowheads indicate the enrichment of CAP-H2 at the centromeric region. (B) Mitotic chromosomes were assembled in extracts that had been mock-depleted (top) or depleted of Pds5A and Pds5B (bottom) as in A, and co-stained with DAPI, anti-SA1 (cohesin) and anti-CAP-E (condensin). A mass of entangled chromosomes is shown. (C) Mitotic chromosomes assembled in extracts that had been mock-depleted (left), depleted of cohesin (middle) or depleted of both Pds5A and Pds5B (right) were co-stained with anti-INCENP (green), anti-CENP-E (red) and DAPI (blue). Merged images of DAPI and CENP-E (whole chromosome) or CENP-E and INCENP (inset) are shown. To better compare the organization of the pericentromeric region of these chromosomes, we show a chromosome from the cohesin-depleted sample with a less severe cohesion defect than the chromosome presented in Fig. 6A, center. The defect is nonetheless obvious, as judged by the increased separation between the sister chromatids in the upper part of the chromosome as well as between the sister kinetochores. Bar, 5 µm.

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