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First published online 30 November 2004
doi: 10.1242/jcs.01604

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Distinct functions of condensin I and II in mitotic chromosome assembly

Toru Hirota^1,*, Daniel Gerlich^2,*, Birgit Koch¹, Jan Ellenberg² and Jan-Michael Peters^1,

¹ Research Institute of Molecular Pathology, Dr Bohr-Gasse 7, 1030 Vienna, Austria
² European Molecular Biology Laboratory, Meyerhofstrasse 1, 69117 Heidelberg, Germany

View larger version (75K): [in a new window]   Fig. 1. Localization of condensin I and II during the cell cycle. (A-E) Analysis of NRK cells stably expressing EGFP–Flag–Kleisin-. (A) Incorporation of ectopically expressed EGFP–Flag–Kleisin- into condensin I complexes. Extracts of NRK cells expressing EGFP–Flag–Kleisin- (lane 1) or parental NRK cells (lane 2) were analyzed by SDS-PAGE and immunoblotting using antibodies to Kleisin- (upper panel), to GFP (middle panel) and to Smc4 (lower panel). The same extracts were also subjected to immunoprecipitations with antibodies to Flag-epitope (lane 3), Smc4 (lane 4), Kleisin- (lanes 5 and 7), or control IgG (lane 6) and analyzed by immunoblotting, as indicated. (B) Extracts of EGFP–Flag–Kleisin--expressing NRK cells were fractionated by 5-20% sucrose-density-gradient centrifugation. Fractions were analyzed by immunoblotting using antibodies against Kleisin-, Smc4 or a subunit of the proteasome. (C) Extracts of exponentially proliferating (log), hydroxyurea treated (HU) or nocodazole treated (Noc) NRK cells expressing EGFP–Flag–Kleisin- were separated by centrifugation into pellet (P) and supernatant (S). Endogenous and exogenous Kleisin- proteins were detected by immunoblotting using Kleisin- antibodies. EGFP–Flag–Kleisin- was expressed at approximately 10% of endogenous protein levels, estimated by quantifying the intensity of the bands. (D, E) Localization of EGFP-Kleisin- in live interphase and metaphase NRK cells. DNA was stained with Hoechst 33342 (red in overlay). (D) Mitotic time-lapse imaging of EGFP–Kleisin--expressing cells stained with Hoechst 33342 (red). Time from start of filming is indicated in minutes. Bar, 10 µm. (F) Localization of CAP-D2 and CAP-D3 by IF microscopy. Logarithmically proliferating HeLa cells were fixed with acetone-methanol solution, incubated with CAP-D2 and CAP-D3 antibodies and stained with Alexa 488 (upper panels). Merged signals of Alexa 488 (green) and DAPI (blue) are shown in the lower panels. (Left to right) Representative cells in interphase, prophase, prometaphase, metaphase, anaphase and telophase. (G) Smc2 is located on chromosomes in prophase. HeLa cells were fixed with 4% paraformaldehyde either after pre-extraction (c,d) or without preextraction (a, b), and stained with Smc2 antibodies (green). DNA was stained with DAPI (blue). Representative cells in interphase (a, c) and in prophase (b, d). Notice, staining of Smc2 on axial chromosome structures can be seen in prophase after pre-extraction, although condensed chromosomes are not seen as clearly after pre-extraction treatment.

View larger version (51K): [in a new window]   Fig. 2. Condensin I and II associate with chromosomes independently of each other. (A) Depletion of condensin subunits by RNAi in HeLa cells. Logarithmically proliferating cells were transfected with CAP-D2, CAP-D3, Smc2 siRNAs or with control reagents, and total cell extracts were analyzed by immunoblotting at the indicated times after the transfection. Because the targeted proteins were depleted between 48-72 hours after transfection, subsequent analyses were carried out in this time window. (B) HeLa cells were transfected with control, CAP-D2, CAP-D3 or Smc2 siRNAs for 48 hours. Following treatment with 50 ng/ml nocodazole for 12 hours, mitotic cells were collected and chromosome enriched fractions were prepared and analyzed by immunoblotting as indicated. Three serial dilutions were loaded for each sample. Ponceau S staining of core histones is shown as a loading control in the bottom panel. IB, immunoblot. (C) IF microscopy of condensin I and condensin II in CAP-D2-or CAP-D3-depleted cells. Fourty-eight hours after the transfection of indicated siRNAs, cells were fixed and stained with antibodies against CAP-D2 or CAP-D3 (gray in the first column, green in the second), and DNA was counterstained with DAPI (blue). Representative prometaphase cells are shown. Arrows indicate chromosome axes where CAP-D2 and CAP-D3 are enriched.

View larger version (60K): [in a new window]   Fig. 3. Condensin II is required for chromosome condensation in prophase. (A) Analysis of chromosome condensation in fixed prophase-cells depleted of condensin I or II. Logarithmically proliferating immortalized RPE cells were transfected with CAP-D2 or CAP-D3 siRNAs. Sixty hours after the transfection cells were fixed and co-incubated with antibodies against histone H3-phospho-Ser10 and either CAP-D2 or CAP-D3, and probed with Texas Red and Alexa 488, respectively. DNA was counterstained with DAPI. Cells were scored as being in prophase when the shape of their nucleus indicated that they possessed an intact nuclear envelope, and when they were positive for histone H3-phospho-Ser10. Based on DAPI staining, the extent of chromosome condensation in these cells was classified into `none', `mild', `moderate' or `strong' as exemplified in the top panels. Cells were also grouped into three different classes according to their CAP-D2 or CAP-D3 staining intensity as `no reduction', `reduced but still detectable' or `undetectable' (these different signal intensities are graphically represented by black triangles on the left). Each dot represents one prophase cell. (B) Analysis of chromosome condensation during prophase in live HeLa cells depleted of condensin I or II. To visualize chromatin, we analyzed HeLa cells that stably express H2B-EGFP. Prophase image-sequences were extracted from long-term imaging experiments and aligned on the time axis according to NEBD, as determined from the loss of a defined nuclear boundary. Imaging was 56-70 hours after siRNA transfection. Bar, 10 µm.

View larger version (42K): [in a new window]   Fig. 4. Analysis of chromosome morphology in cells depleted of condensins. (A) Metaphase plates in live HeLa cells stained with Hoechst 33342 after transfection with the indicated siRNAs. Single confocal sections of 3D-stacks at 1.6 µm slice-thickness of representative examples (n=10 for each condition) are shown. (B) Chromosome spreads from condensin-depleted cells. HeLa cells were transfected with control, CAP-D2, CAP-D3 or Smc2 siRNAs for 48 hours. Mitotic cells were collected without treatment (left and middle panels) or after 4 hours of nocodazole treatment (right panel), and incubated with either isotonic buffer (left panels) or with hypotonic buffer (middle and right panels), and fixed with Carnoy solution. Chromosome spreads made on slide-glass were stained with Giemsa. (C) Quantification of the number of spread cells whose chromosome arms had opened or remained closed after 4 hours of nocodazole treatment. 200 cells were assessed for each experiment.

View larger version (63K): [in a new window]   Fig. 5. Cohesin dissociation from chromosome arms in prometaphase requires condensin I. (A) Quantitative analysis of sister chromatid cohesion in condensin RNAi cells. Chromosome spreads from HeLa cells transfected with either CAP-D2 or CAP-D3 siRNAs were prepared as described in Fig. 4B, using hypotonic conditions. For each chromosome, 20 line scans were randomly selected on chromosome arms excluding regions where chromatids converge into centromeres (exemplified in the upper left panel). The signal intensities over the defined lines were averaged, and the distance between the peak intensities were scored (upper right panel). The resulting distance values were plotted against chromosome number in histograms (bottom panel). Data are from cells that were not treated (black bars) or treated with nocodazole for 4 hours (purple bars). (B) Analysis of cohesin in condensin depleted cells. HeLa cells, stably expressing Scc1-myc, were transfected with control or condensin siRNAs and processed for IF microscopy following a 50 ng/ml nocodazole treatment for 4 hours. Cells were incubated with anti-myc antibodies (green) and either antibodies against kinetochores (CREST sera, red) or Smc2 antibodies (red, bottom panel). DNA was counterstained with DAPI. (C) Quantitative analysis of Scc1-myc signal-intensity on chromosomes. Chromosome regions were defined by DAPI staining, and the ratio of fluorescence intensities of myc signals to CREST signals was calculated. A Student's t-test demonstrated that the difference between CAP-D2 RNAi cells and control or CAP-D3 RNAi cells was significant (P<0.01).

View larger version (25K): [in a new window]   Fig. 6. Condensin I is required for longitudinal chromosome compaction in prolonged prometaphase. (A) Identification of chromosome 1. Spread chromosomes were stained for heterochromatin with distamycin (DA) followed by DAPI staining. Arrows indicate chromosome 1 with characteristic pericentromeric heterochromatin. (B) Quantitative analysis of chromosome length after nocodazole treatment in cells depleted of condensin I or II. Forty-eight hours after transfection with CAP-D2 or CAP-D3 siRNAs, cells were either incubated with or without nocodazole for 4 hours before being processed for chromosome spreading and DA-DAPI staining. The telomere-to-centromere-to-telomere distance of chromosome 1 was measured in 30 cells for each experiment (exemplified by a dashed line in A), and the average length by which chromosomes shortened during the 4-hour nocodaozole treatment was calculated.

View larger version (71K): [in a new window]   Fig. 7. Mitotic progression in condensin-depleted cells. Mitotic progression was followed in long-term time-lapse experiments of HeLa cells expressing H2B-EGFP. (A) Representative image sequences of control cells and condensin-depleted cells. (B) The time interval between NEBD and onset of segregation was measured based on the observed chromatin pattern.

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