Wignall SM et al. (2003), The condensin complex is required for proper sp...

XB-ART-5103

J Cell Biol 2003 Jun 23;1616:1041-51. doi: 10.1083/jcb.200303185.

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The condensin complex is required for proper spindle assembly and chromosome segregation in Xenopus egg extracts.

Wignall SM, Deehan R, Maresca TJ, Heald R.

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Chromosome condensation is required for the physical resolution and segregation of sister chromatids during cell division, but the precise role of higher order chromatin structure in mitotic chromosome functions is unclear. Here, we address the role of the major condensation machinery, the condensin complex, in spindle assembly and function in Xenopus laevis egg extracts. Immunodepletion of condensin inhibited microtubule growth and organization around chromosomes, reducing the percentage of sperm nuclei capable of forming spindles, and causing dramatic defects in anaphase chromosome segregation. Although the motor CENP-E was recruited to kinetochores pulled poleward during anaphase, the disorganized chromosome mass was not resolved. Inhibition of condensin function during anaphase also inhibited chromosome segregation, indicating its continuous requirement. Spindle assembly around DNA-coated beads in the absence of kinetochores was also impaired upon condensin inhibition. These results support an important role for condensin in establishing chromosomal architecture necessary for proper spindle assembly and chromosome segregation.

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Species referenced: Xenopus laevis
Genes referenced: btg3 cenpe rad21 ran

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	Figure 1. Condensin depletion from crude Xenopus egg extracts causes defects in chromosome condensation. (A) Chromosomes assembled in clarified extract to which buffer (Control) or 0.26 mg/ml XCAP-E antibody (+ α-XCAP-E) was added. Xenopus sperm nuclei were incubated in CSF extract for 90 min, and fixed and stained with Hoechst 33258. Bar, 10 μm. (B) Western blot analysis of crude Xenopus egg extracts depleted using IgG (Control) or XCAP-E and XCAP-G antibodies (ΔXCAP), and probed with affinity-purified XCAP-E antibody, which recognized a single band of 140 kD in control extract. (C) Silver-stained 8% gel of Xenopus condensin complex purified from Xenopus egg extract using XCAP-G antibody–coupled beads and eluted using XCAP-G peptide. All five subunits of the complex are present, though XCAP-H appears lighter and more diffuse. (D) Chromosomes assembled in control and condensin-depleted crude extracts. Xenopus sperm nuclei were added to extracts, which were then cycled through interphase and back into mitosis. The reactions were fixed in metaphase, 60 min after initiation of mitosis. Bar, 10 μm.
	Figure 2. Condensin inhibition reduces the fidelity of sperm spindle assembly. (A) Mitotic structures formed in extracts depleted using IgG (Control) or condensin antibodies (ΔXCAP) that were fixed 60 min after the induction of mitosis. The three ΔXCAP images show structures representative of the three categories quantified in B and C: normal spindles, abnormal structures, and reduced microtubules. In all images, microtubules appear red and chromosomes blue. Bar, 10 μm. (B) Quantification of the effect of condensin depletion on spindle assembly 60 min after the start of mitosis. Two experiments (red and blue bars) showing the percent sperm nuclei in each category depicted in A. For each condition, >100 structures were counted. (C) Quantification of the effect of XCAP-E antibody addition on spindle assembly. Two separate experiments are shown. To each spindle reaction, either buffer (Control) or 0.26 mg/ml α-XCAP-E antibody (α-XCAP) was added at the start of mitosis. Samples were fixed at 60 min and the microtubule structures were assigned to the categories depicted in A. (D) Spindle assembly reactions in extracts depleted with IgG (Control) or XCAP antibodies (ΔXCAP), and in XCAP-depleted extract to which purified Xenopus condensin complex was added (Add-back). Samples were fixed at 60 min after the start of mitosis and the microtubule structures were examined. Percent normal spindles is shown.
	Figure 3. Depletion of the condensin complex from Xenopus egg extracts causes defects in anaphase chromosome segregation. (A) Chromosome segregation in extracts depleted with IgG (Control) or condensin antibodies (ΔXCAP). Once metaphase spindles were assembled (t = 0'), anaphase was induced and samples were fixed at 30, 35, and 40 min. Bar, 10 μm. (B) Quantification of the effect of condensin depletion on chromosome segregation. One representative experiment is shown. Samples were fixed 40 min after anaphase initiation, and the chromosomes in each spindle were classified as completely separated, partially separated, or unseparated. 188 and 162 structures were counted for the control and condensin depletion (ΔXCAP), respectively. Percent sperm nuclei in each category is depicted. (C) Deconvolution microscopy of chromosomes during anaphase in control and condensin-depleted (ΔXCAP) extracts. The samples were fixed 35 min after anaphase induction. The poles of each spindle are marked with asterisks (*). Two side-by-side spindles are shown in the condensin-depleted extract. Bar, 10 μm. (D) Chromosome segregation in extracts depleted with IgG (Control) or XCAP antibodies (ΔXCAP), and in the XCAP-depleted extract to which purified Xenopus condensin complex was added (Add-back). The structures were fixed 30 min after anaphase initiation. Bar, 10 μm.
	Figure 4. Inhibition of the condensin complex either before mitosis or after anaphase initiation causes chromosome segregation defects. (A) Segregation of sperm chromosomes in Xenopus egg extracts in the presence of buffer or 0.26 mg/ml XCAP-E antibody, added either as the extract was entering mitosis (α-XCAP) or immediately after anaphase induction (α-XCAP ana). Samples were fixed at metaphase (Metaphase), and early and late anaphase (Early Ana and Late Ana). Bar, 10 μm. (B) Quantification of the effect of condensin antibody addition on chromosome segregation. Spindles were fixed 40 min after anaphase induction, and the chromosomes were classified as completely separated, partially separated, or unseparated. For each reaction, >100 structures were counted. (C) Chromosome segregation in the presence of IgG (Control), XCAP-E antibody preincubated with buffer (α-XCAP), or XCAP-E antibody preincubated with antigen (α-XCAP + pep). Samples were fixed 35 min after anaphase induction. Bar, 10 μm. Videos 1–6 are available at http://www.jcb.org/cgi/content/full/jcb.200303185/DC1.
	Figure 5. Depletion of the condensin complex does not affect the Ran-GTP gradient, but disrupts kinetochore morphology. (A) RanGTP was visualized by addition of a probe that shows a loss of FRET when bound to RanGTP. Fluorescence images show sperm nuclei, the FRET ratio signal (IFRET/ICFP), and an overlay. Ratios range from low FRET (blue) to high FRET (red). Gradients appeared indistinguishable surrounding chromosomes in clarified extracts treated with buffer or α-XCAP-E. Bar, 10 μm. (B) Immunofluorescence images of CENP-E (green) in spindles formed in extracts depleted using IgG (Control) or condensin antibodies (ΔXCAP), revealing aberrant kinetochore morphology in the absence of condensin. Samples were fixed 60 min after the induction of mitosis. Bar, 10 μm. (C) Addition of 1 μM nocodazole to metaphase reactions increased the CENP-E signal and partially alleviated its distortion in ΔXCAP extracts. Bar, 10 μm.
	Figure 6. Condensin inhibition reduces the fidelity of DNA bead spindle assembly. (A) Microtubule structures formed around DNA beads in extracts depleted using IgG (Control) or condensin antibodies (ΔXCAP) that were fixed in metaphase 90 min after initiation of mitosis. The three ΔXCAP images show structures representative of the three categories quantified in B and C: normal spindles, abnormal structures, and reduced microtubules. Bar, 10 μm. (B) Quantification of the effect of condensin depletion on bead spindle assembly in two separate experiments (red and blue bars). Samples were fixed 90 min after the start of mitosis. Each cluster of 10 or more DNA beads was examined and the microtubule array surrounding it was assigned to the categories depicted in A. For each reaction, >100 structures were counted. Percent of bead clusters in each category are depicted. (C) Quantification of the effect of XCAP-E antibody addition on bead spindle assembly in two separate experiments. To each spindle assembly reaction, either buffer (Control) or 0.26 mg/ml α-XCAP-E antibody (α-XCAP) was added at the start of mitosis. Samples were fixed at 90 min, and the microtubule structures were grouped into the categories depicted in A.
	Figure 7. Condensin depletion does not affect CENP-E localization in anaphase or XRad21 degradation. (A) CENP-E staining (green) in spindles formed in extracts depleted using IgG (Control) or condensin antibodies (ΔXCAP). Samples were fixed 15 min after anaphase initiation. Bar, 10 μm. CENP-E is localized to regions of DNA that is stretched toward the spindle poles (arrow), suggesting that the kinetochores are still functional. (B) Analysis of XRad21 degradation in anaphase. Chromosomes were purified from extracts depleted with IgG (control) or condensin antibodies (ΔXCAP) in either metaphase or anaphase. Proteins were separated on an 8% gel and blotted using affinity-purified XRad21 antibodies, revealing a similar reduction of XRad21 in the presence and absence of condensin. The blot was also probed with antibodies against topoII as a loading control (not depicted).

References [+] :

Andersen, Balanced regulation of microtubule dynamics during the cell cycle: a contemporary view. 1999, Pubmed