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First published online May 28, 2005
doi: 10.1242/10.1242/jcs.02392

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Drosophila CAP-D2 is required for condensin complex stability and resolution of sister chromatids

¹ Wellcome Trust Centre for Cell Biology, Institute of Cell and Molecular Biology, University of Edinburgh, Michael Swann Building, King's Buildings, Mayfield Road, Edinburgh, EH9 3JR, UK
² Instituto de Histologia, Faculdade de Medicina, Universidade de Lisboa, Av. Prof. Egas Moniz, 1649-028 Lisboa, Portugal
³ St Georges Hospital Medical School, Cranmer Terrace, London, SW17 0RE, UK

View larger version (66K): [in a new window]   Fig. 1. Identification of a condensin complex in Drosophila and characterisation of CAP-D2 during interphase. (A) Identification of a condensin complex in 0- to 5-hour embryo extracts following immunoprecipitation with an antibody recognizing Drosophila CAP-D2. Three 0.5 M NaCl washes and three 2% SDS elutions are shown for preimmune and immune precipitations. The samples were analysed by immunoblotting with SMC2, SMC4, CAP-D2 and CAP-H/Barren antibodies. A fraction of SMC2 and SMC4 eluted in the washes, but most was eluted with 2% SDS, while CAP-D2 and CAP-H/Barren only eluted with 2% SDS. (B-D) S2 cultured cells were cytospun onto poly-L-lysine slides, then fixed and stained with various antibodies as described. (B) CAP-D2 localises in the nucleus during interphase. G1 cells (using -tubulin as a marker) show nuclear staining for CAP-D2 with a diffuse cytoplasmic staining. Cells in G1 phase (single centrosome, arrow in upper panel) have less nuclear CAP-D2 than cells with two centrosomes (cells in S or G2 phase, arrow in lower panel). -tubulin (red), CAP-D2 (green) and DNA (blue). (C) S phase cells (detected after BrdU incorporation) show strong nuclear staining for CAP-D2, but at differing levels. BrdU (green), CAP-D2 (red), and DNA (blue). (D) Cells in late G2/early prophase (arrow) show an intense staining for CAP-D2. Cyclin B (nuclear during late G2/early prophase) was used as a marker for this stage. Cyclin B (green), CAP-D2 (red) and DNA (blue).

View larger version (83K): [in a new window]   Fig. 2. Characterisation of CAP-D2 during mitosis. (A) CAP-D2 localises to a central axis in chromatids and shows accumulation at the centromeres from prometaphase until telophase. P-H3 was used as a mitotic marker. During late anaphase and telophase, when chromosomes begin to decondense, CAP-D2 still associates with chromosomes. CAP-D2 (red), P-H3 (green), and DNA (blue). (B) CAP-D2 accumulates at centromeres during metaphase. CID was used as a centromere marker. CAP-D2 (red), CID (green) and DNA (blue). (C) CAP-D2 is still strong in the daughter nuclei of a cell undergoing cytokinesis (arrow). CAP-D2 (red), -tubulin (green) and DNA (blue).

View larger version (55K): [in a new window]   Fig. 3. CAP-D2 dsRNA-mediated interference in Drosophila S2 cells results in abnormal chromosome morphology and segregation. (A) Immunoblot showing the depletion of CAP-D2 after treatment with dsRNA. 5 x105 cells were loaded per lane. Lanes: C, control dsRNA; –, no dsRNA; D, CAP-D2 dsRNA. CAP-D2 levels start to decrease as early as 48 hours and the protein is undetectable at 96 and 120 hours. -tubulin was used as a loading control. (B,C) Immunofluorescence analysis of control and CAP-D2 RNAi cells. Cells were cytospun onto poly-L-lysine slides 65 hours after treatment and stained for CAP-D2 (green), CID (red), and DNA (blue). (C) CAP-D2-depleted cells have no CAP-D2 on condensed chromosomes and exhibit abnormal chromosome morphology. Sister chromatids fail to resolve and segregate properly, and chromosomes are not properly aligned on the metaphase plate. Centromeres appear stretched. (D,E) Hypotonic treatment of control (D) and CAP-D2-depleted (E) cells 65 hours after dsRNA treatment shows that chromatid resolution is defective in the CAP-D2-depleted cells. Cells were hypotonically treated, centrifuged onto poly-L-lysine slides, fixed and stained for CAP-D2 (red), P-H3 (green) and DNA (blue). Note that individual chromatids are not readily apparent after depletion of CAP-D2. Scale bar: 10 µm.

View larger version (58K): [in a new window]   Fig. 4. Chromosome arm and centromere resolution are compromised after CAP-D2 depletion. (A) Immunoblotting showing that the levels of DSA1 were not affected 72 hours after CAP-D2 RNAi treatment. Lanes: C, control dsRNA; –, no dsRNA; D, CAP-D2 dsRNA. -tubulin was used as a loading control. (B,C) Centromere resolution is disrupted in the CAP-D2-depleted cells. Control (B) and CAP-D2-depleted (C) cells were cytospun onto poly-L-lysine slides 72 hours after dsRNAi treatment, fixed and stained for DSA1 (green), CID (red) and DNA (blue). White boxes indicate regions shown at higher magnification in the panels on the right. The majority of prometaphase/metaphase cells had normal CID staining between sister chromatids (C1 and higher magnification), while a small percentage showed a slight stretching (C2 and higher magnification). Cells in anaphase showed severe centromere stretching on chromosomes (C3-5 and higher magnification). Scale bar: 10 µm. (D) The distance (in µm) between paired CID spots of cells in prometaphase/metaphase after staining for DSA1/CID/DAPI. The distance in CAP-D2-depleted cells is more than twofold higher than in control cells.

View larger version (50K): [in a new window]   Fig. 5. Effect on Barren and SMC4 of CAP-D2 depletion. (A) Immunoblotting of control and CAP-D2-depleted extracts with Barren and SMC4 antibodies demonstrates that Barren levels are reduced while SMC4 levels appear largely unaffected after loss of CAP-D2. -tubulin was used as a loading control. C, control dsRNA; –, no RNA; D, CAP-D2 dsRNA. (B,C) Cells from both control (B) and CAP-D2 RNAi (C) experiments were cytospun onto poly-L-lysine slides 72 hours after treatment and stained for Barren (red), Cyclin B (green) and DNA (blue). Barren is absent from mitotic chromosomes and localised diffusely in the cytoplasm after CAP-D2 RNAi. (D,E) Control (D) and CAP-D2 RNAi (E) cells were cytospun onto poly-L-lysine slides 65 hours after treatment, extracted during fixation, and stained for SMC4 (red), CAP-D2 (green) and DNA (blue). While cells have varying levels of SMC4 on chromatin, any axial localisation of SMC4 is lost in the CAP-D2-depleted cells. Scale bar: 10 µm.

View larger version (54K): [in a new window]   Fig. 6. Effect on SMC2 and DNA topoisomerase II in CAP-D2-depleted cells. (A) Immunoblotting of control and CAP-D2-depleted extracts with an antibody to SMC2 shows that the protein level is largely unaffected by the CAP-D2 depletion. -tubulin was used as a loading control. C (control dsRNA), – (no RNA), D (CAP-D2 dsRNA). (B,C) Immunolocalisation of SMC2 and topo II in control (B) and CAP-D2-depleted (C) cells after 65 hours of dsRNA treatment. Cells were cytospun onto poly-L-lysine slides, extracted during fixation and stained for SMC2 (green), topo II (red), and DNA (blue). Despite the fact that SMC2 and topo II still localise to chromatin, albeit at varying levels, any axial definition is lost. The scale bar is 10 µm. (D,E) Instability of condensin subunits after mutation of CAP-H and SMC4 in Drosophila. (D) CAP-D2 is unstable when Barren is absent in a barrL305 mutant. Immunoblot of 12- to 13-hour wild-type (Canton S) and barrL305 homozygous embryo extracts shows that Barren is almost undetectable in mutant embryos. The remaining protein probably represents maternal contribution to the embryos. The membrane was probed for Barren, CAP-D2, SMC2, SMC4 and Scc1. The decrease in protein level was determined using phosphorimaging (5 embryos were loaded per lane). Barren levels decreased eightfold, CAP-D2 levels decreased sixfold and SMC2 and SMC4 levels decreased fourfold and 3.5-fold respectively. -tubulin was used as a loading control. (E) Stability of condensin subunits in an SMC4 (gluon17C) mutant. Immunoblot of 12- to 13-hour wild-type (Canton S) and gluon17C homozygous embryo extract with the SMC4, SMC2, CAP-D2 and Barren antibodies. The levels of the four proteins were calculated using phosphorimaging (5 embryos were loaded per lane). SMC4 shows a 5.9-fold decrease in protein levels, SMC2 shows a 5.6-fold decrease, while Barren and CAP-D2 show a 4.7-fold decrease. -tubulin was used as a loading control.

View larger version (49K): [in a new window]   Fig. 7. Depletion of CAP-D2 and DNA topoisomerase II by double RNAi in S2 cells. (A) The depletion of CAP-D2 and topo II after treatment with two dsRNAs was assessed at 72 and 96 hours by immunoblot analysis. Both proteins were similarly depleted and almost undetectable by 96 hours. -tubulin was used as a loading control. –, no dsRNA; C, control dsRNA; D, CAP-D2 dsRNA; T, topoII dsRNA; D/T, CAP-D2/topo II dsRNA. (B,C) Analysis of chromosome morphology in control (B) and CAP-D2/Topo II doubly-depleted (C) cells. Cells were cytospun onto poly-L-lysine slides, extracted during fixation and stained for topo II (red), CAP-D2 (green), CID (white) and DNA (blue), 96 hours after dsRNA treatment. Chromosome morphology was abnormal and remarkably similar to that observed after single CAP-D2 RNAi. Centromeres were stretched along the chromatin in anaphase as observed after CAP-D2 depletion alone. Scale bar: 10 µm.

View larger version (66K): [in a new window]   Fig. 8. Examination of CAP-D2 and DNA topoisomerase II in metaphase chromosomes after dsRNA treatment. (A,B) Metaphase chromosome spreads of control (1,2), topo II-depleted (3), CAP-D2-depleted (4) and CAP-D2/Topo II doubly depleted (5) cells, after cells were arrested with colchicine. Cells were cytospun onto poly-L-lysine slides, extracted during fixation and stained for topo II (red), CAP-D2 (green) and DNA (blue), 74 hours after dsRNA treatment. (A) Control cells displayed the typical X-shape and sister chromatids could be easily distinguished. Both CAP-D2 and topo II localised axially along the length of the chromosomes with partial colocalisation (A1,2 and B, Control). In topo II, CAP-D2 single depletions, and in the CAP-D2/Topo II double depletion, no sister chromatid resolution could be observed and chromosomes appeared fuzzy. Scale bar: 10 µm. (B) Individual chromosomes observed in chromosome spreads. The chromosome phenotype was similar in the two single and the double RNAi experiments.