Shugoshin biases chromosomes for biorientation through condensin recruitment to the pericentromere
- University of Edinburgh, United Kingdom
- Institute of Bioanalytics, Technische Universität Berlin, Germany
Figures
Figure 1 with 1 supplement
Sgo1 alleles that affect biorientation.
(A) Sgo1-3A, but not Sgo1-100 or Sgo1-700 are maintained at the centromere in cells arrested in mitosis by treatment with nocodazole. Cells carrying SGO1-6HA (AM906), SGO1-100-6HA (AM6956), SGO1-700-…
Figure 1—figure supplement 1
The sgo1-100 and sgo1-700 mutations do not affect the timing of cell cycle entry.
Strains as in Figure 1A were released from G1 as described in Figure 1B and DNA content was measured at the indicated times by FACS.
Figure 2
PP2A-Rts1 recruitment to the centromere by Sgo1 is not required for biorientation.
(A) Sgo1-100 and Sgo1-700, but not Sgo1-3A, associate with Rts1. Cells carrying RTS1-9MYC and SGO1-SZZ(TAP) (AM9144), sgo1-100-SZZ(TAP) (AM9272), sgo1-700-SZZ(TAP) (AM9142), sgo1-3A-SZZ(TAP) …
Figure 3 with 2 supplements
Sgo1 is required for the maintenance of Ipl1 at centromeres, but is dispensable for its initial recruitment.
(A) Ipl1/aurora B co-immunoprecipitates with Sgo1. Cells producing SZZ(TAP)-tagged Sgo1 (AM8975), Sgo1-100 (AM8971), Sgo1-700 (AM8969), Sgo1-3A (AM8973) or no TAP (AM3513) and carrying IPL1-6HA were …
Figure 3—figure supplement 1
The Haspin homologs, Alk1 and Alk2, are not required for the initial recruitment of Ipl1 to centromeres.
Wild-type (AM3513), alk1Δ alk2Δ (AM10612) and alk1Δ alk2Δ SGO1-aid (AM10393) cells carrying IPl1-6HA as well as a no tag control (AM1176) were arrested in G1 by treatment with alpha factor. Samples …
Figure 3—figure supplement 2
Defective biorientation in ipl1-as mutants.
Biorientation assay showing CEN4-GFP separation in wild-type (AM4643) and ipl1-as (AM10374) cells carrying SPB (SPC42-tdTomato) markers. Cells were released from G1 into nocodazole and NAPP1, before …
Figure 4 with 3 supplements
Sgo1 interacts with condensin and recruits it to the pericentromere.
(A and B) Condensin and PP2A co-purify with Sgo1. Sgo1 was purified from wild-type or protease-deficient cells that were (A) cycling or (B) arrested in mitosis using the cold-sensitive tubulin …
Figure 4—figure supplement 1
The Sgo1-condensin interaction is not dependent on DNA or treatment with the cross-linking agent, DSP.
Cells carrying YCS4-6HA and SGO1-SZZ(TAP) (AM9138) or no TAP (AM5705) were arrested in mitosis by treatment with nocodazole for 2 hr. Cultures were either harvested and directly drop-frozen (−DSP) …
Figure 4—figure supplement 2
Removal of PCR duplicates does not alter the conclusion that Sgo1 is important for Brn1 enrichment in the pericentromere.
This is the same analysis as in Figure 3H, except that only unique reads are included (therefore eliminating duplicate samples generated during the PCR amplification step). Both methods of analysis …
Figure 4—figure supplement 3
Brn1 is reduced around all 16 individual centromeres in sgo1Δ cells.
Brn1 enrichment in a 20 kb region surrounding all 16 individual centromeres in wild type and sgo1Δ cells is shown for the experiment described in Figure 4F–H. All reads were included in this analysis.
Figure 5 with 4 supplements
Sgo1 is not required for cohesin association with chromosomes.
Wild-type (AM1145) and sgo1Δ (AM1474) cells carrying SCC1-6HA were arrested in mitosis by treatment with nocodazole for 3 hr. Samples were harvested, anti-HA ChIP was performed and both input and IP …
Figure 5—figure supplement 1
Scc1 association with all 16 centromeres is unaffected by SGO1 deletion.
Scc1 enrichment in a 20 kb region surrounding all 16 individual centromeres in wild type and sgo1Δ cells is shown from the experiment in Figure 5. All reads were included in this analysis.
Figure 5—figure supplement 2
ChIP-qPCR analysis showing Scc1-6HA levels at the centromere and pericentromere.
Wild type and sgo1Δ cells were treated as described in Figure 5 except that ChIP samples were analyzed by qPCR using primer sets at CEN4, CEN5 and a site in the pericentromere of chromosome IV. The …
Figure 5—figure supplement 3
Cohesin is required for normal Sgo1 association with the pericentromere.
Wild type (AM906) and pMET-SCC1 (AM6673) strains carrying SGO1-6HA together with no tag wild type (AM1176) and pMET-SCC1 controls (AM1599) were arrested in G1 using alpha factor in the presence of …
Figure 5—figure supplement 4
Cohesin loading factors, but not monopolin, are important for proper condensin association with the centromere.
Wild type (AM5708), SCC2-aid (AM8918), chl4Δ (AM8885), iml3Δ (AM5710) and lrs4Δ (AM9766) strains carrying BRN1-6HA, as well as a no tag control (AM1176) were treated with nocodazole and NAA (to …
Figure 6 with 1 supplement
Sgo1 is sufficient for condensin recruitment.
(A and B) Sgo1 overproduction leads to increased levels of Brn1 on chromosomes. Cells carrying BRN1-6HA and that were otherwise wild type (AM5708) or carrying pGAL-SGO1 (AM10859) integrated an …
Figure 6—figure supplement 1
SGO1 overexpression in metaphase-arrested cells increases Brn1 association with the centromere.
Strains AM5708 (BRN1-6HA), AM10859 (BRN1-6HA pGAL-SGO1) were pre-cultured in YEP + R + Ade medium before supplementing with nocodazole and galactose (2 hr) for 3 hr and then harvesting for anti-HA …
Figure 7 with 1 supplement
Condensin facilitates effective error correction.
(A) Sister kinetochore biorientation is defective after nocodazole washout in cells lacking condensin. Strains carrying SPB (Spc42-tdTomato) and CEN4 (CEN4-GFP) markers were released from a G1 …
Figure 7—figure supplement 1
Deletion of SGO1 impairs biorientation rather than centromere cohesion.
The distance from CEN4-GFP to the nearest SPC42-tdTomato focus was measured for cells with just one visible CEN4-GFP foci from the experiment shown in Figure 7C–F. The fraction of cells with a CEN4-G…
Figure 8
Condensin biases chromosomes to biorient.
(A–C) Condensin and Sgo1, but not Ipl1, are required to bias sister kinetochores towards biorientation. (A) Scheme of the microfluidics assay to test sister kinetochore bias. Wild-type, sgo1Δ, YCS5-a…
Figure 9 with 2 supplements
Shugoshin enables the bias towards sister kinetochore biorientation by condensin recruitment.
(A) Schematic diagram showing Sgo1-associated complexes and their functions at the pericentromere. (B) Sgo1-100 and Sgo1-3A, but not Sgo1-700, retain association with Brn1. Cells carrying BRN1-6HA …
Figure 9—figure supplement 1
The Sgo1-100 protein can recruit condensin to kinetochores.
(A and B) Condensin is at least partially recruited to the centromere after release from G1 in sgo1-100 cells. Wild-type (AM5708), sgo1-100 (AM9442) and sgo1-700 (AM9291) cells carrying BRN1-6HA …
Figure 9—figure supplement 2
Sgo1-tetR-GFP, but not Sgo1-100-tetR-GFP, Sgo1-700-tetR-GFP or Sgo1-3A-tetR-GFP tethered to CEN4 can partially rescue the mis-segregation of chromosome IV after nocodazole washout in otherwise sgo1Δ cells.
Diploid cells carrying tetR-tdTomato and SGO1-tetR-GFP (AM14005), sgo1-100-tetR-GFP (AM14006), sgo1-700-tetR-GFP (AM14007), sgo1-3A-tetR-GFP (AM14008) or no Sgo1-TetR fusion (AM14009), with tetOs …
Figure 10
Model for dual role of Sgo1 in biorientation is shown.
Shugoshin ensures sister kinetochore biorientation through two mechanisms. First, early in the cell cycle, shugoshin mediates the enrichment of condensin within the pericentromere. We propose that …
Videos
Video 1
Example video of a wild-type cell in the error correction assay.
The video corresponds to the image gallery in Figure 5C (upper panel).
Video 2
Example video of an ipl1-as cell in the error correction assay.
The video corresponds to the image gallery in Figure 5C (lower panel).
Video 3
Example video of a wild type cell in the assay to test for a bias towards sister kinetochore biorientation.
The video corresponds to the image gallery in Figure 6C (upper panel).
Video 4
Example video of a YCS5-aid cell in the assay to test for a bias towards sister kinetochore biorientation.
The video corresponds to the image gallery in Figure 6C (lower panel).
Additional files
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Supplementary file 1
Complete list of peptides identified in the experiments shown in Figure 4A,B.
- https://doi.org/10.7554/eLife.01374.031
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Supplementary file 2
(A) Yeast strains used in this study. (B) qPCR primers used in this study. (C) Genome summary table for Brn1-6HA ChIP-seq.
- https://doi.org/10.7554/eLife.01374.032
