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First published online November 3, 2003
doi: 10.1242/10.1242/jcs.00797

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RNAi analysis reveals an unexpected role for topoisomerase II in chromosome arm congression to a metaphase plate

Wellcome Trust Centre for Cell Biology, Institute for Cell and Molecular Biology, Kings Buildings, University of Edinburgh, Mayfield Road, Edinburgh EH9 3JR, Scotland, UK

View larger version (88K): [in a new window]   Fig. 1. Efficient depletion of Topo II in Drosophila S2 cells using RNAi. (A) Immunoblots: (upper) Topo II levels begin to fall by 48 hours after the addition of dsRNA and become undetectable by 72 hours (1x106 cells loaded per lane); (lower) loading control (anti-tubulin). –, control RNAi; +, Topo II RNAi treated. (B) The anti-Topo II antibody used can detect Topo II in 5x104 cells, but not 1x104 cells. (C-F) Immunofluorescence analysis showing Topo II depletion at the 72nd hour after treatment; (C,E) controls, (D,F) treated cells, (C,D) prometaphase, (E,F) anaphase. In all merged images, DAPI is blue, Cid is red and Topo II is green. Scale bar: 5 µm.

View larger version (22K): [in a new window]   Fig. 2. (A,B) Scoring of mitotic cells in the different phases of mitosis reveals only slight differences between cells following control RNAi treatment (using dsRNA corresponding to a human intronic sequence; A) and Topo II RNAi (B). The Topo II RNAi causes a significant increase in the fraction of anaphase cells seen at later times. (C) The percentage of cells in each stage of the cell cycle at various times after treatment. There is no significant difference in any of the mitotic phases when control and Topo II RNAi-treated cells (selected because the spindle is viewed from the side and the metaphase plate can be unambiguously identified) are scored at 72 hours post addition of dsRNA. (D) Mitotic index. There is no significant difference in mitotic index between control and Topo II RNAi-treated cultures over the course of the experiment. (E) Cell growth curves of control and Topo II RNAi-treated cultures. These data are from three independent experiments. In each experiment, more than 2000 cells were scored at every time point. The mitotic index is determined by observing the DNA and spindle staining.

View larger version (72K): [in a new window]   Fig. 3. Topo II depletion causes abnormalities in chromosome structure, but does not affect histone H3 phosphorylation on serine10. All images are from cultures at 72 hours after addition of Topo II dsRNA. (A-A'") Control RNAi-treated metaphase cell. (B-B'") Prometaphase cell with abnormal chromosome morphology viewed parallel to spindle axis. (C-C'") Two metaphase cells, one of which has a highly elongated chromosome arm extending to one spindle pole. Both are viewed nearly perpendicular to the spindle axis. (A-C) DAPI staining for DNA; (A'-C') anti-tubulin shows the mitotic spindle; (A",B") histone H3 phosphorylated on serine10 is stained with a specific antibody; (A'"-C'") merged images (DAPI is blue, tubulin is red, histone H3 is green. Scale bar: 5 µm.

View larger version (61K): [in a new window]   Fig. 4. The condensin subunit Barren associates normally with mitotic chromosomes in Topo II-depleted cells. (A,B) Control RNAi-treated normal metaphase and anaphase cells. (C,D) Topo II RNAi-treated metaphase cells with a highly elongated chromosome arm extending to one spindle pole. (E) Topo II RNAi-treated anaphase cell with the bulk of the chromatin stretched out between the separating kinetochores. (A-E) DAPI staining for DNA. (A'-E') anti-tubulin shows the mitotic spindle; (A"-E") staining for Barren shows the position of the condensin complex; (A'"-E'") merged images (DAPI is blue, tubulin is red, Barren is green). Scale bar: 5 µm.

View larger version (32K): [in a new window]   Fig. 5. Many metaphases in Topo II-depleted cells have an unusual phenotype in which one or more chromosome arm(s) becomes highly elongated and stretches towards the spindle pole. (A) Control RNAi-treated normal metaphase cell. (B) Topo II RNAi-treated metaphase cell with a highly elongated chromosome arm extending to one spindle pole. (C) Topo II RNAi-treated metaphase cell with J-shaped chromosome arms extending to spindle poles. (D) Statistical analysis of the protruding arm phenotype. (A-C) DAPI staining for DNA; (A'-C') anti-tubulin shows the mitotic spindle; (A"-C") staining for Cid/CENP-A shows the position of kinetochores; (A'"-C'") merged images (DAPI is blue, tubulin is red, Cid is green). Scale bar: 5 µm.

View larger version (39K): [in a new window]   Fig. 6. Identification of the chromosomal component in the protruding arm phenotype. Analysis by FISH at 72 hours after the addition of Topo II dsRNA using the following probes. (A) Euchromatic chromosome 3 probe (BACH47K04); (B) Euchromatic chromosome X probe (BACH47E07). In A and B, arrowheads indicate FISH signal in protruding arm. (C) Euchromatic chromosome 2 probe (BACHN09); (D) Heterochromatic chromosome X probe (359 satellite); large arrowhead points to extended FISH signal in protruding arm; small arrowhead points to condensed FISH signal in metaphase plate. In all merged images, the probe is green and the DNA is red. Scale bar: 5 µm.

View larger version (89K): [in a new window]   Fig. 7. The chromosomal passenger protein INCENP is localised normally in Topo II-depleted cells with the exception of a few highly abnormal anaphase cells. (A) Control RNAi-treated normal prometaphase cell. (B) Control RNAi-treated normal telophase cell. (C) Topo II RNAi-treated prometaphase cell with abnormal chromosomal morphology. (D) Topo II RNAi-treated late prometaphase cell with abnormal chromosomal morphology. (E) Topo II RNAi-treated abnormal cell with INCENP stretched along the chromosome arms. (F) Topo II RNAi-treated anaphase cell with massive chromatin bridging, but INCENP located normally on the central spindle. (G) Topo II RNAi-treated late prometaphase cell with abnormal chromosomal morphology: Aurora-B localises normally on the centromeres. (A-G) DAPI staining for DNA; (A'-G') anti-tubulin shows the mitotic spindle; (A"-F") staining for INCENP; (G") staining for Cid shows the positions of the kinetochores; (G'") staining for the chromosomal passenger Aurora B. In A'"-F'" DAPI is blue, tubulin is red, INCENP is green, and in G'''' DAPI is blue, Cid is red, Aurora-B is green. Scale bar: 5 µm.

View larger version (26K): [in a new window]   Fig. 8. Anaphase in Topo II-depleted cells is characterised by the presence of massive chromatin bridges, however sister centromeres usually manage to disjoin and move towards the spindle poles. (A) Control RNAi-treated normal anaphase cell. (B) Topo II RNAi-treated anaphase cell with the bulk of the chromatin stretched out between the separating kinetochores. (A-B) DAPI staining for DNA; (A'-B') anti-tubulin shows the mitotic spindle; (A"-B") staining for Cid/CENP-A shows the position of kinetochores; (A'"-B'") merged images (DAPI is blue, tubulin is red, Cid is green). Scale bar: 5 µm. (C) Statistical analysis of the lagging chromosome phenotype.

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