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First published online December 11, 2006
doi: 10.1242/10.1242/jcs.03277

Journal of Cell Science 119, 5057-5066 (2006)
Published by The Company of Biologists 2006
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Essential roles for cohesin in kinetochore and spindle function in Xenopus egg extracts

Renée Deehan Kenney and Rebecca Heald*

Department of Molecular and Cell Biology, 311 Life Sciences Addition, University of California, Berkeley, CA 94720-3200, USA


Figure 1
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  		Fig. 1. Sister chromatid cohesion defects in Xenopus egg extracts depleted of cohesin. (A) Upper panel. Western blot of untreated total (total extract) and IgG-depleted ({Delta}Mock) Xenopus egg extract, and Xenopus egg extract depleted by antibodies against XSmc1 and XRad21 ({Delta}Cohesin). The blot was probed with an affinity-purified XSmc1 antibody that recognizes a single band of 155 kDa (present in total extract and {Delta}Mock), which is absent in cohesin-depleted extract ({Delta}Cohesin). Lower panel. The same blot probed with an affinity-purified antibody against the XCAP-G condensin subunit, recognizing a single band of 130 kDa (present in control, IgG- and cohesin-depleted extracts. (B) Immunofluorescence images of sperm chromosomes assembled in mock- and cohesin-depleted extracts that had been cycled through interphase to allow DNA replication, then back into mitosis. Chromosomes were spun down onto coverslips 60 minutes after induction of mitosis and processed for immunofluorescence using an antibody that recognizes a subunit of the condensin complex, XCAP-G (blue) and the kinetochore protein BubR1 (red). Bar, 10 µm.

 

Figure 2
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  		Fig. 2. Defects in metaphase chromosome alignment and anaphase segregation in the absence of cohesin.(A) Fluorescence images showing spindles assembled around Xenopus sperm nuclei in mock- and cohesin-depleted extracts, in the presence of Rhodamine-labeled tubulin (red). After 60 minutes in metaphase, reactions were spun down onto coverslips and processed for immunofluorescence with an antibody against the kinetochore protein Ndc80 (green). DNA was stained with Hoechst-33258 dye (blue), and microtubules are red. Bar, 10 µm. (B) Quantification of chromosome misalignment. The number of chromosomes that had strayed completely away from the metaphase plate in each spindle were counted. Bars represent averages for three independent experiments and a total of n=328 and n=317 spindles in mock- and cohesin-depleted extracts, respectively. Error bars are standard deviations (± s.d.). (C) Quantification of metaphase plate compaction relative to spindle size in mock- and cohesin-depleted spindles. Bars represent the area of the metaphase plate divided by the area of the spindle, to account for differences in spindle size. At least ten spindles were measured in each of three independent experiments, using Metamorph software (Molecular Devices). Error bars are standard deviations. (D) Fluorescence images showing microtubules (red), kinetochore component Ndc80 (green) and DNA (blue) in spindles undergoing anaphase in mock- and cohesin-depleted extracts. Reactions were spun onto coverslips 20 minutes post anaphase induction and processed for immunofluorescence. Bar, 10 µm.

 

Figure 3
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  		Fig. 3. Examples of anaphase chromosome segregation defects observed in the absence of cohesin; mock- and cohesin-depleted extracts containing spindles fixed 15 minutes after anaphase induction. Note that, chromosomes clearly segregated in three out of four control spindles, whereas cohesin-depletion causes segregation failure and/or unequal distribution of chromatids. Microtubules are red and chromosomes are blue-green.

 

Figure 4
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  		Fig. 4. Kinetochore-spindle-attachment defects in the absence of cohesin are not rescued by topologically linking sister chromatids. (A) Fluorescence images showing microtubules (red) and the kinetochore component Ndc80 (green) in spindles formed in mock- or cohesin-depleted extracts that had been treated with 0.3 µM nocodazole to preferentially depolymerize non-kinetochore microtubules so that kinetochore-microtubule attachments could be visualized. Reactions were spun onto coverslips and processed for immunofluorescence 20 minutes post drug addition. Bar, 5 µm. (B) Fluorescence images showing microtubules (red), Ndc80 (green) and DNA (blue) in control ({Delta}Mock) and cohesin ({Delta}Cohesin) depleted spindle assembly reactions that had been incubated with a solvent control (DMSO) or the topoismerase II inhibitor etoposide (50 µM) to prevent DNA decatenation. Drug was added after DNA replication, upon induction of mitosis. Bar, 10 µm.

 

Figure 5
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  		Fig. 5. Centromeric localization of INCENP and aurora B is fragmented in cohesin-depleted spindles. (A) Fluorescence images showing DNA (blue), tubulin (red) and Ndc80, INCENP or aurora B (green) in spindles formed in cycled extracts in the presence ({Delta}Mock) or absence ({Delta}Cohesin) of cohesin. Reactions were spun onto coverslips 60 minutes into metaphase. Bar, 10 µm. (B) Fluorescence images of replicated, isolated sperm chromosomes assembled in mock- and cohesin-depleted extracts, spun onto coverslips 60 minutes after induction of mitosis and processed for immunofluorescence with antibodies to INCENP (green) and the kinetochore protein BubR1 (red). DNA was counterstained with Hoechst-33258 dye (blue). Bar, 2.5 µm. (C) Bipolar spindles assembled in the presence of Rhodamine-labeled tubulin (red) in control ({Delta}Mock) or cohesin ({Delta}Cohesin) depleted extracts were induced to enter anaphase, fixed after 20 minutes and spun onto coverslips for immunofluorescence using antibodies against INCENP and aurora B (green). DNA was counterstained with Hoechst-33258 dye (blue) and microtubules are shown in red. Bar, 10 µm.

 

Figure 6
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  		Fig. 6. Inhibition of bipolar spindle attachments can rescue inner centromeric cohesion and protein passenger localization in the absence of cohesin. (A-D) Fluorescence images of spindle reactions treated with the Eg5 inhibitor monastrol (125 µM) either prior to spindle assembly for 30 minutes (A,B), or for 30 minutes following spindle assembly (C,D) in mock- and cohesin-depleted extracts. DNA is blue, microtubules are red and Ndc80, INCENP or aurora B are green. Bar, 10 µm.

 

Figure 7
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  		Fig. 7. Effects of co-depleting both condensin and cohesin from Xenopus egg extracts. (A) Western blot of mock- and double-depleted (DD) extracts showing that a cohesin subunit (SMC1), and a condensin subunit (XCAP-G) have been depleted. Background bands are marked with asterisks. (B) Spindle assembly defects are additive. Spindles assembled in extracts depleted using control ({Delta}Mock), cohesin ({Delta}Cohesin), condensin ({Delta}Condensin) or both ({Delta}Cohesin+{Delta}Condensin) antibodies were evaluated for spindle assembly defects. Bars represent the average of two experiments, with more than 400 microtubule structures evaluated under each condition. Error bars represent standard deviation (± s.d.). (C) Chromosome morphology in single- and double-depleted extracts. Right set of panels show the position of chromosomes stained with Hoechst-33258 dye (blue) within metaphase spindles (microtubules are red). Left set of panels show higher magnification images of the same chromosomes (blue) stained with antibodies to kinetochore checkpoint protein BubR1 (red). Note misaligned chromosomes in the absence of cohesin. Arrowhead shows chromosome distortion apparent in the absence of condensin, probably due to pulling at kinetochores by spindle microtubules. Both the alignment and distortion defects are rescued in the double depletion. Bars, 10 µm.

 

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© The Company of Biologists Ltd 2006