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Figure 1. Effect of Aurora-B depletion on condensin I binding to chromosomes. (A) Mitotic extracts were depleted with control IgG (lane 7), anti-Aurora B (lane 8), anti-Aurora A (lane 9) and anti-Cdc2 (lane 10). Two microliter aliquots of each extract were analyzed by immunoblotting with the indicated antibodies. To estimate the efficiency of depletion, 2âμl (lane 1) of interphase extract, 2âμl (lane 2), 1âμl (lane 3), 0.5âμl (lane 4), 0.25âμl (lane 5) and 0.12âμl (lane 6) of mitotic extract were loaded in parallel (lanes 7â10). (B) Sperm nuclei were incubated with interphase extract (lane 1), mitotic extract (lanes 2â4), mock-depleted mitotic extract (lanes 5â7), Aurora B-depleted mitotic extract (lanes 8â10), Aurora A-depleted mitotic extract (lanes 11â13), or Cdc2-depleted mitotic extract (lanes 14â16). Assembled chromatin and chromosomes were isolated through a sucrose cushion, and 12.5% (lanes 2, 5, 8, 11 and 14), 25% (lanes 3, 6, 9, 12 and 15) and 50% (lanes 1, 4, 7, 10, 13 and 16) were separated by SDS-PAGE, and then detected by immunoblotting using anti-phospho H3 antibody (chromatin-bound; lower). To test the phosphorylation level in the extracts, 1âμl (lanes 2, 5, 8, 11 and 14), 2âμl (lanes 3, 6, 9, 12 and 15) and 4âμl (lanes 1, 4, 7, 10, 13 and 16) of the extracts were immunoblotted with anti-phospho H3 antibody (in extract; upper). (C) Chromatin-bound proteins were prepared the same as (B), and were separated by SDS-PAGE and immunoblotted by anti-XCAP-E (upper) or anti-XCAP-G antibodies (lower). (D) Sperm chromatin was incubated in interphase (a), mitotic (b), mock-depleted mitotic (c), Aurora B-depleted mitotic (d), Aurora A-depleted mitotic (e) or Cdc2-depleted mitotic (f) extracts at 22°C for 2âh, fixed, and then stained with Hoechst.
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Figure 2. Stimulation of chromosomal binding of condensin I by OA in interphase extracts. (A) Sperm nuclei were incubated with mitotic extract (lanes 1â3), interphase extract (lanes 4â6), interphase extract supplemented with 1.2 (lanes 7â9), 3.6 (lanes 10â12) or 12âμM (lanes 13â15) of OA at 22°C for 2âh. Chromatin-bound proteins were dissolved with SDS-PAGE sample buffer, and 12.5 (lanes 1, 4, 7, 10 and 13), 25 (lanes 2, 5, 8, 11 and 14) and 50% (lanes 3, 6, 9, 12 and 15) of each sample were separated by SDSâPAGE, and immunoblotted with anti-phospho H3. (B) Samples were prepared as described in (A), and 6.25% (lane 1), 12.5% (lanes 2, 4, 7, 10 and 13), 25% (lanes 3, 5, 8, 11 and 14) and 50% (lanes 6, 9, 12 and 15) of samples were blotted using anti-XCAP-E (upper), and anti-XCAP-G (lower) antibodies. (C) Sperm chromatin was assembled in the mitotic extract (a, d), interphase extract (b, e, g), and interphase extract supplemented with 3.6âμM OA (c, f, h). Samples were fixed and stained with Hoechst (a, b, c; first low), and anti-XCAP-H antibody (d, e, f; second low, short exposure: g, h; third low, long exposure). Bar, 10âμm. (D) Sperm chromatin was assembled in the mitotic extract (a, c, e) or interphase extract supplemented with 3.6âμM OA (b, d, f). After assembly, the reaction mixtures were supplemented with the indicated extra concentration of KCl at 22°C for 20âmin, fixed, and stained with Hoechst. Bar, 10âμm.
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Figure 3. Requirement of condensin in OA-dependent partial chromosome condensation in the interphase extract. (A) Mitotic (lanes 1â2) and interphase (lanes 3â4) extracts were mock-depleted (lanes 1 and 3) or depleted with a mixture of affinity-purified condensin antibodies (lanes 2 and 4). Equal volumes of these extracts were subjected to SDS-PAGE, blotted, and detected using the indicated antibodies. (B) Sperm chromatin was incubated with mock-depleted mitotic extract (lanes 1â4), condensin-depleted mitotic extract (lane 5), mock-depleted interphase extract (lane 6), mock-depleted interphase extract supplemented with OA (lane 7), condensin-depleted interphase extract (lane 8) or condensin-depleted interphase extract supplemented with OA (lane 9) at 22°C for 2âh. The assembled structures were isolated, and 25 (lane 1), 12.5 (lane 2), 6.3 (lane 3), 3.1 (lane 4) and 100% (lanes 5â9) were analyzed by immunoblotting with the indicated antibodies. (C) Sperm chromatin was incubated with mitotic extract (a), condensin-depleted mitotic extract (b), mock-depleted interphase extract (c), mock-depleted interphase extract with OA (d), condensin-depleted interphase extract (e), or condensin-depleted interphase extract with OA (f) at 22°C for 2âh. After incubation, assembled chromatin structures were fixed and stained with Hoechst. Bar, 10âμm.
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Figure 4. Requirement of Aurora B in the Cdc2-independent chromosomal targeting of condensin I. (A) Interphase extract was depleted with anti-Aurora B (lane 7), anti-Aurora A (lane 8) or anti-Cdc2 antibodies (lane 9). As a standard, 100% (lane 1) of mitotic extract, 100% (lane 2), 50% (lane 3), 25% (lane 4), 12.5% (lane 5) and 6.25% (lane 6) of interphase extract were loaded in parallel. Efficiency of immunodepletion was measured by quantitative immunoblotting using the antibodies indicated. (B) Sperm nuclei were incubated with mitotic extract (lanes 1â4), or interphase extract (lane 5), OA-treated interphase extract (lane 6), OA-treated interphase extract depleted of Aurora B (lane 7), OA-treated interphase extract depleted of Aurora A (lane 8), and OA-treated interphase extract depleted of Cdc2 (lane 9). Chromatin or chromosomes were isolated, and 100% (lanes 1, 5â9), 50% (lane 2), 25% (lane 3) and 12.5% (lane 4) of the samples were blotted by anti-phospho-H3 antibody (upper), anti-XCAP-C (middle) or anti-XCAP-E (lower) antibodies. In the case of blotting with anti-condensin subunit antibodies, a lower amount of samples was used as a standard, namely, 25% (lane 1), 12.5% (lane 2), 6.3% (lane 3) and 3.1% (lane 4). (C) Sperm chromatin was incubated with mitotic extract (a), interphase extract (b), OA-treated interphase extract (c), OA-treated interphase extract depleted of Aurora B (d), OA-treated interphase extract depleted of Aurora A (e) and OA-treated interphase extract depleted of Cdc2 (f). Samples were fixed and stained with Hoechst.
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Figure 5. Effect of OA on the supercoiling activity of condensin I. Condensin I was affinity-purified from mitotic (lanes 2â5), interphase (lanes 6â9), or OA-treated interphase extract (lanes 10â13) and standard supercoiling (upper) and DNA binding assays (lower) were performed; r, relaxed circular DNA; s, positively supercoiled DNA; arrow, free DNA; asterisk, DNA bound to condensin I. The approximate molar ratios of condensin I to DNA were 15:1 (lanes 2, 6 and 10), 30:1 (lanes 3, 7 and 11), 60:1 (lanes 4, 8 and 12) and 120:1 (lanes 5, 9 and 13). Condensin I was omitted from lane 1.
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