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First published online November 3, 2003
doi: 10.1242/10.1242/jcs.00799

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Condensin-dependent localisation of topoisomerase II to an axial chromosomal structure is required for sister chromatid resolution during mitosis

¹ Instituto de Biologia Molecular e Celular, Universidade do Porto, Rua do Campo Alegre 823, 4150-180 Porto, Portugal
² Faculdade de Ciências da Saúde, Universidade Fernando Pessoa, Praça 9 de Abril, 349, 4249-004 Porto, Portugal
³ Instituto de Ciências Biomédicas de Abel Salazar, Universidade do Porto, Porto, Portugal

View larger version (53K): [in a new window]   Fig. 1. Depletion of DmSMC4 in Drosophila S2 cells by dsRNAi. (A) Depletion of DmSMC4 was monitored over time by western blot analysis. Total protein extracts from 106 cells were separated by SDS-PAGE and immunoblotted with anti-DmSMC4 or -tubulin as a loading control. Quantification of depletion was determined by normalising for -tubulin. (B) Protein extracts of different concentration of control cells were immunoblotted with anti-DmSMC4 antibody to determine the level of antibody detection. (C) Viability index of control and DmSMC4 dsRNAi-treated cells was determined by Trypan Blue staining. (D) Proliferation of control or DmSMC4 dsRNAi-treated S2 cells was monitored throughout the experiment. (E) Quantification of the mitotic index during the course of the experiment in control cells and cells treated with DmSMC4 dsRNAi was determined by counting phosphohistone H3-positive cells. (F) Anti-phosphohistone H3-positive cells were also used to quantify mitotic progression in control (C) and RNAi (R)-treated cells. Numbers of cells counted for D and E is shown in Table 1. (G) Phenotypic analysis of phosphohistone H3 positive cells in control and DmSMC4 dsRNAi-treated cells. After depletion of DmSMC4, phosphohistone H3 staining persists on chromatin bridges during telophase (arrow). Representative images of metaphase chromosomes at a higher magnification are shown in the insets. Note that sister chromatids do not resolve after DmSMC4 depletion. Scale bars: 5 µm.

View larger version (64K): [in a new window]   Fig. 2. Immunolocalisation of DmSMC4 and Barren in control and DmSMC4-depleted Drosophila S2 cells. In control cells during prometaphase and anaphase DmSMC4 and Barren co-localise to an axial structure of sister chromatids. After depletion of DmSMC4 by dsRNAi, chromosomal-associated DmSMC4 is significantly reduced after 48 hours and undetectable after 96 h. A reduction in the level of chromatin-associated DmSMC4 causes the formation of short chromosomes with unseparated sister chromatids and a highly abnormal distribution of Barren. In cells that show no DmSMC4 staining, chromosomes are short, no defined sister chromatids are observed and Barren is undetected. Scale bars: 5 µm.

View larger version (52K): [in a new window]   Fig. 3. Analysis of centromere and kinetochore function after depletion of DmSMC4. (A) Immunofluorescence analysis of CID in control cells shows discrete localisation to centromeres in prometaphase and anaphase. (B) Localisation of CID in DmSMC4-depleted cells shows centromere staining throughout mitosis. In early anaphase cells, CID staining appears to extend polewards beyond the mitotic chromatin (arrows) and in late anaphase centromeres lead migrating chromatids to the spindle poles. After incubation with colchicine, CID-stained centromeres localise over the chromosomes. (C) Control cell immunostained with anti-POLO antibodies show localisation to kinetochores and to centrosomes located at either side of the metaphase plate. (D) After depletion of DmSMC4, POLO localises to kinetochores that during anaphase can also be seen to stretch towards the poles. (E) Control cells stained for CID and tubulin show centromeres associated with microtubule bundles. A higher magnification image is shown in the left panel. (F) In DmSMC4-depleted cells centromeres stretched beyond the chromatin associated with microtubule bundles. Higher magnification images are shown in the left panels. Scale bars: 5 µm.

View larger version (42K): [in a new window]   Fig. 4. Localisation of Topo II to mitotic chromatin after depletion of DmSMC4. (A) In control cells DmSMC4 and Topo II partially co-localise to an axial structure of sister chromatids during both prometaphase or anaphase. Some accumulation of Topo II is also observed at the centromeres. Merged images show DNA (red) and Topo II (green). (B) Detailed analysis of distribution of DmSMC4 and Topo II in prometaphase chromosomes. The merged image shows DmSMC4 (green) and Topo II (red). Higher magnification view of selected regions (a and b) indicated in the merged image, show that Topo II and DmSMC4 localise mostly as an alternating string pattern with some regions in which both proteins are found. (C) After depletion of DmSMC4, Topo II does not show a defined localisation to the axial structure within sister chromatids but becomes diffusely distributed all over condensed chromatin and in chromatin bridges. Merged images show DNA (red) and Topo II (green). Scale bars: 5 µm.

View larger version (39K): [in a new window]   Fig. 5. Determination of the level of Topo II associated with chromatin in DmSMC4-depleted cells. (A) The levels of Topo II in total protein extracts from control or DmSMC4-depleted cells after 96 hours show a 19.5% reduction. (B) To determine whether this reduction corresponds to the chromosomal-associated Topo II, increasing amounts of mitotic chromatin extracts from control and DmSMC4-depleted cells immunoprecipitated with anti-phosphohistone H3 antibodies or the remaining supernatant were separated by SDS-PAGE and immunoblotted. Membranes were probed with antibodies against DmSMC4 and Topo II. Histone H3 was used as a loading control for immunoprecipitated chromatin and -tubulin for the supernatant. While DmSMC4 and Topo II are clearly found in the control extracts, in the chromatin from DmSMC4-depleted cells DmSCM4 is absent and the levels of Topo II are not significantly different from controls extracts. In the corresponding supernatant fractions, DmSMC4 and Topo II are easily detected in the control cells. However, after DmSMC4 dsRNAi treatment, DmSMC4 is not detected and Topo II is reduced.

View larger version (28K): [in a new window]   Fig. 6. In vitro activity of Topo II in control and DmSMC4-depleted extracts. (A) Determination of Topo II activity of chromatin-associated protein extracts from control cells using kDNA as substrate. For each assay 10 µg of total extracts with (+) and without (-) 10 µM of Topo II inhibitor ICRF-187 were used and the reaction was allowed to proceed for different times. The DNA was deproteinised and analysed on a 0.8% agarose gel. In control extracts the kDNA was rapidly decatenated to produce relaxed minicircles after 5 minutes of the incubation. The addition of ICRF-187 for 10 minutes completely inhibited decatenation. (B) Decatenation of kDNA was used to measure Topo II activity in control or DmSMC4-depleted extracts after 96 hours of treatment without (-) or with (+) the addition of exogenous Topo II (2U, Amersham). Different levels of protein extracts were used: 0 µg (-), 5 µg (+) or 10 µg (++). DNA was deproteinised and run on a 0.8% agarose gel containing 0.5 µg/ml ethidium bromide. Decatenation of kDNA is observed in control extracts with or without addition of exogenous Topo II. DmSMC4-depleted extracts showed no decatenation activity in the absence of exogenously added Topo II.

View larger version (40K): [in a new window]   Fig. 7. Localisation of DRAD21 in DmSMC4-depleted Drosophila S2 cells. (A) Immunolocalisation of DmSMC4 and DRAD21 in control cells. In prometaphase cells, DmSMC4 and DRAD21 do not appear to co-localise. DRAD21 is always confined to the centromeric region between the axial structure stained by DmSMC4. During anaphase while DmSMC4 localises throughout sister chromatids, DRAD21 is not detected. (B) After depletion of DmSMC4, DRAD21 is still highly abundant in prophase chromosomes but during prometaphase it is restricted to defined regions of the condensed chromosomes. In anaphase, DRAD21 is never detected either on the chromatids or the chromatin bridges. Scale bars: 5 µm. (C) Western blot of total protein extracts from control and DmSMC4-depleted cells after 96 hours show that depletion of DmSMC4 does not affect the overall levels of DRAD21 but Barren levels are reduced compared to control extracts. (D) Analysis of protein extracts from immunoprecipitated mitotic chromatin of either control or DmSMC4-depleted cells and the corresponding supernatants. In chromatin extracts from control cells DmSMC4, DRAD21 and Barren can be easily detected while in extracts from dsRNAi-treated cells neither DmSMC4 nor Barren are detected and the level of DRAD21 is reduced compared to controls. Phosphohistone H3 was used as a loading control. The corresponding supernatants are shown below. DmSMC4 is not detected by immunoblotting and the levels of DRAD21 are not different from controls. However, note that depletion of DmSMC4 causes a significant decrease in the level of Barren present in the supernatant fraction. -tubulin was used as loading control.

View larger version (57K): [in a new window]   Fig. 8. Simultaneous depletion of DmSMC4 and DRAD21 in Drosophila S2 cells. (A) Protein extracts from different concentrations of cells were immunoblotted with anti-DRAD21 antibody to determine the level of detection of the antibody. (B) Protein extracts from control or DRAD21-depleted cells were immunoblotted to detect DRAD21, DmSMC4 and -tubulin as a loading control. Note that significant depletion of DRAD21 is obtained 72 hours after treatment with RNAi while the levels of DmSMC4 remain unchanged. Cells were also treated with dsRNA for DRAD21 and DmSMC4 simultaneously and protein extracts prepared at different times and immunoblotted to reveal DRAD21, DmSMC4 and -tubulin as loading control. Significant depletion of both proteins was obtained after 96 hours of treatment therefore all phenotypic and quantification analysis was carried out at this time. (C) Cells depleted of DRAD21, stained for DRAD21 and DNA, show premature sister chromatid separation and highly condensed chromatids. (D) Cells depleted of both DmSMC4 and DRAD21, stained for DRAD21, DmSMC4 and DNA, show abnormal chromosome condensation and poorly defined sister chromatids during prometaphase and during anaphases have extensive chromatin bridges. (E) Quantification of mitotic index of control cells and cultures treated with different dsRNAs shows some accumulation of mitotic cells after depletion of DRAD21 or simultaneous depletion of DRAD21 and DmSMC4. (F) Quantification of mitotic progression after treatment of cells with different dsRNAs. Depletion of DmSMC4 does not affect mitotic progression while depletion of DRAD21 causes a significant prometaphase arrest with few cells in metaphase or anaphase. Simultaneous depletion of DmSMC4 and DRAD21 causes a significant accumulation of cells in anaphase and telophase. The number of cells used for the quantifications shown in E and F was: control n=1148, DmSMC4 RNAi n=1038, DRAD21 RNAi n=1923 and simultaneous DmSMC4/DRAD21 RNAi (double) n=1801. Scale bars: 5 µm.

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