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First published online 13 March 2007
doi: 10.1242/jcs.03425

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Aurora B controls the association of condensin I but not condensin II with mitotic chromosomes

Jesse J. Lipp^1,2,*, Toru Hirota^1,, Ina Poser² and Jan-Michael Peters^1,

¹ Research Institute of Molecular Pathology, Dr Bohrgasse 7, A-1030 Vienna, Austria
² Max Planck Institute of Molecular Cell Biology and Genetics Dresden, Pfotenhauerstr. 108, D-01307 Dresden, Germany

View larger version (90K): [in this window] [in a new window]   Fig. 1. Aurora B activity is required for loading condensin I in prometaphase. (A) HeLa cells stably expressing EGFP-tagged kleisin- (condensin II) were enriched in S-phase by a double-thymidine arrest and synchronously released into mitosis. The levels of EGFP-kleisin- associating with chromatin were monitored over time as cells entered mitosis. Chromosomes were visualised by adding the DNA-intercalating dye Hoechst 33342 (0.2 µg/ml). Aurora B activity was compromised either by adding Hesperadin (100 nM) 1 hour before the bulk of the cells entered mitosis or by depleting the protein with siRNAs specific to Aurora B for 30 hours. Movies were taken on an Olympus DeltaVision microscope (63x objective, three slices every 1 minute, 1x1 binning). Projected images are shown. (B) Quantification of chromatin-bound EGFP-kleisin- in control, Hesperadin-treated and Aurora B siRNA-transfected cells over time. Intensities were quantified using the Hoechst signal as a reference. The average cytoplasmic intensity was subtracted as background intensity. Averages of seven cells (n=7) for each condition are shown. Time point 0 (t=0) corresponds to nuclear envelope breakdown (NEBD). (C,D) Same experiment as in A and B using a HeLa cell line stably expressing kleisin- tagged with EGFP (condensin I). Curves represented in D are averages from seven cells (n=7) for each condition.

View larger version (65K): [in this window] [in a new window]   Fig. 2. Condensin I binding to chromatin is dependent on Aurora B activity. (A) Nocodazole-arrested human HeLa cells were either treated with 100 nM Hesperadin or DMSO for 1 hour, fixed and stained for the condensin subunits Smc2 (shared between condensin I and II), kleisin-, Cap-D2 (both condensin I specific) and Cap-D3 (condensin II specific). Chromosomes were counterstained with DAPI (1 µg/ml). Bar, 10 µm. (B) Average fluorescence intensities of condensin on chromatin were quantified using the DAPI channel as a reference and to determine the area occupied by chromatin. The background intensity was calculated by measuring the fluorescence intensity outside this area and subtracted from the condensin signal. The presented graphs are averaged from at least 30 different cells for each condition (n>30) and normalised to the control value ± s.d. (C) Biochemical fractionation of Nocodazole-arrested HeLa cells which had either been treated with 100 nM Hesperadin for 1 hour or left untreated before harvesting. The levels of condensin-I-(Cap-G and kleisin-) and condensin-II-specific subunits (Cap-D3 and Cap-G2) were detected by western blotting. Total mitotic extracts (ME), cytoplasm (Cyt) and chromatin (Chr) fractions are shown.

View larger version (56K): [in this window] [in a new window]   Fig. 3. Aurora B regulates centromeric enrichment of condensin I. (A) Mitotic HeLa cells were spread on glass slides under hypotonic conditions, fixed and stained with antibodies against condensin I (kleisin-). CREST autoimmune serum was used to detect centromeric regions. Inactivation of Aurora B kinase activity by Hesperadin treatment (100 nM) was monitored by staining for phosphorylated Histone H3 Serine 10 (H3S10ph). Chromosomes were counterstained with DAPI (1 µg/ml). Bar, 10 µm. (B) A magnified chromosome of the same experiment is shown stained with DAPI, antibodies against kleisin- and CREST autoimmune serum. Bar, 2 µm. (C) Quantification of condensin I (kleisin- and Cap-D2) and condensin II (Cap-D3) at centromeric regions was done using the CREST signal as a reference and to determine the location of centromeres during image segmentation. Mean ± s.d. from more than 100 different centromeres of at least ten different cells for each condition are shown. The presented graphs are normalised to the control value.

View larger version (31K): [in this window] [in a new window]   Fig. 4. Maintenance of condensin I is dependent on Aurora B activity. (A) Nocodazole-arrested HeLa cells either expressing EGFP-kleisin- (condensin I) or EGFP-kleisin- (condensin II). At time point 0 (t=0), 100 nM Hesperadin was added to the cells and the levels of condensin followed over time. Hoechst 33342 (0.2 µg/ml) was used to stain chromatin. (B) EGFP-kleisin- (condensin I) (red, n=25) and EGFP-kleisin- (condensin II) (blue, n=19) signals were quantified as in Fig. 1B and Fig. 1D over time. Note that the increase in EGFP-kleisin- (condensin II) visible in A upon Hesperadin treatment is masked by the normalisation with Hoechst, which likewise increases. Movies were taken on an Olympus DeltaVision microscope [40x objective, five slices every 2 minutes, 1x1 binning (EGFP-kleisin-) and 2x2 binning (EGFP-kleisin-)]. Projected images are shown.

View larger version (46K): [in this window] [in a new window]   Fig. 5. Aurora B phosphorylates condensin I regulatory subunits in vitro and in vivo. (A-C) Condensin was purified by immunoprecipitation from EGFP-FLAG-kleisin- (A), EGFP-FLAG-Cap-D2 (B) (both condensin I) and EGFP-FLAG-kleisin- (C) (condensin II) cell lines. Immunoprecipitates were incubated either with or without recombinant Aurora B and [-32P]ATP in vitro, resolved on a gel, silver stained (Silver) and exposed (32P). The identity of the bands was determined by mass spectrometry. (D) Condensin I (EGFP-FLAG-kleisin-, first panel) and condensin II (EGFP-FLAG-kleisin-, second panel) were immunoprecipitated either from interphase (hydroxyurea arrested; HU) or mitotic cells (Nocodazole arrested; Noc) and analysed for electrophoretic mobility shifts that are sensitive to pre-treatment of the HeLa cells with Hesperadin (100 nM for 1 hour) or to treatment of the immunoprecipitates with -phosphatase. Total mitotic extracts (ME) were probed with antibodies against H3S10ph to monitor Aurora B activity and against cyclin B to verify that cells were in mitosis.

View larger version (73K): [in this window] [in a new window]   Fig. 6. The persistence of cohesin on chromosome arms in Aurora B depleted cells does not depend on Sgo1. (A) In order to visualise cohesin in mitosis, expression of Scc1-9xMyc was induced by applying Doxycyclin (1 µg/ml for 48 hours). Cells were spread onto glass slides using hypotonic conditions, fixed and stained with antibodies against Myc, Sgo1 and the autoimmune serum CREST to detect centromeres. Sgo1 and Aurora B were depleted by siRNA transfection. 100 µm Hesperadin was used to inhibit Aurora B activity. Bar, 10 µm. (B) Number of cells (%) showing only centromeric cohesin versus cells which display cohesin also on chromosome arms. (C) Number of cells (%) showing paired versus single sister centromeres. (D) Images from a movie of HeLa cells stably expressing Sgo1-GFP. Centromeric enrichment of Sgo1-GFP is lost when Hesperadin (100 nM) is added to the medium (t=0). Chromosomes were visualised with Hoechst 33342 (0.2 µg/ml). Movies were taken on an Olympus DeltaVision microscope (63x objective, five slices every 2 minutes, 2x2 binning). Projected images at times indicated (in minutes) are shown.