Specialisation of meiotic kinetochores revealed through a synthetic spindle assembly checkpoint strategy
- Centre for Cell Biology, School of Biological Sciences, University of Edinburgh, United Kingdom
- Discovery Research Platform for Hidden Cell Biology, School of Biological Sciences, University of Edinburgh, United Kingdom
Figures
Figure 1
The meiotic synthetic SAC (SynSAC) activation system.
(A) Schematic of synthetic spindle assembly checkpoint (SAC) dimerisation system. (B) Spore viability of wild-type (AM11189) and SynSAC dimer (AM30783) yeast strains (C) Schematic showing spindle morphology at different stages in meiosis. (D and E) SynSAC strain AM30783 was released from prophase by β-estradiol addition under control (ethanol, left), meiosis I dimerising (250 μM ABA at release), or meiosis II dimerising (250 μM ABA at 100 min) conditions. (D) Representative images of fields of SynSAC cells at the indicated times after prophase release. Upper panels (ethanol) show control cells where SynSAC is not activated, lower panels (abscisic acid, ABA) show cells where SynSAC is activated from prophase release. Scale bar = 5 μm. (E) Scoring of spindle morphology after anti-tubulin immunofluorescence. 100 cells were scored at each timepoint. Arrow indicates time of ethanol or ABA addition.
Figure 2 with 1 supplement
Synthetic SAC (SynSAC) activation depends on checkpoint proteins.
(A–C) Meiosis spindle immunofluorescence timecourse in wild-type SynSAC (AM30783), SynSAC mad2∆ (AM33559), and SynSAC mad3∆ (AM30784) strains. All strains were released from prophase by β-estradiol addition. (A) Control cells where ethanol was added at release (B) SynSAC dimerisation was induced by addition of 250 μM abscisic acid at prophase release (C) SynSAC dimerisation was induced by addition of 250 μM of abscisic acid 100 min after release. 100 cells were scored at each timepoint.
Figure 2—figure supplement 1
Synthetic SAC (SynSAC) induces a robust abscisic acid (ABA)-induced mitotic arrest.
(A) Serial dilution mitotic growth assay of wild-type (AM11189), SynSAC (AM30783), SynSAC mad1∆ (AM33558), SynSAC mad2∆ (AM33559), and SynSAC mad3∆ (AM30784) strains on YPDA rich media (top) and YPDA with 250 μM ABA (bottom). (B) Mitotic SynSAC arrest timecourse. Yeast cultures of wild-type haploid SynSAC strain AM30663 were arrested in G1 with alpha factor and released into fresh media. Cultures were treated with either control solvent (ethanol) or ABA to induce SynSAC dimerisation. The fraction of cells with large buds and one nucleus after DAPI staining was determined after scoring 100 cells for each timepoint.
Figure 3 with 2 supplements
Meiotic synthetic SAC (SynSAC) delays degradation of Pds1securin.
(A–C) Meiosis timecourse with spindle immunofluorescence (left) and western blotting to visualize Pds1-18Myc and Pgk1 loading control (right). (A) Control timecourse with ethanol added at prophase release. (B) Meiosis I SynSAC delay timecourse with 250 μM abscisic acid (ABA) added at prophase release. (C) Meiosis II SynSAC delay meiosis timecourse with 250 μM ABA added at 100 min after prophase release (vertical dotted line). Strain used in A-C was AM34398. For the spindle counts, 100 cells were scored at each timepoint. Arrows indicate the time of ethanol or ABA addition.
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Figure 3—source data 1
Original files for western blot data shown in Figure 3A–C.
- https://cdn.elifesciences.org/articles/110117/elife-110117-fig3-data1-v1.zip
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Figure 3—source data 2
Original files for western blot data shown in Figure 3A–C with bands and treatments annotated.
- https://cdn.elifesciences.org/articles/110117/elife-110117-fig3-data2-v1.zip
Figure 3—figure supplement 1
Protein levels of the synthetic SAC (SynSAC) dimer constructs do not significantly change throughout meiosis.
(A and B) Meiotic timecourse after release from prophase of a strain expressing V5-tagged full-length endogenous Mps1 in addition to the Mps1(440-765)-ABI-3V5 SynSAC dimer construct (AM34816). (A) Spindle morphology after scoring 100 cells per timepoint. Arrows indicate time of ethanol addition. (B) Western blot analysis of samples at the indicated timepoints. Extracts from cycling wild-type (AM1828), MPS1-3V5 (34733) and his3::MPS1(440-765)–3V5-ABI (33850) strains were run as controls. (C and D) Meiotic timecourse of a strain expressing FLAG-tagged full-length endogenous Spc105 in addition to the Spc105(1-455)-PYL-3FLAG SynSAC dimer construct (AM34817). (C) Spindle morphology at the indicated timepoints were scored after tubulin immunofluorescence. (D) Western blot analysis of samples harvested at the indicated timepoints. Extracts from cycling wild-type (AM1828), SPC105-6His-3FLAG(AM34876) and leu2::Spc105(1-455)-PYL-3FLAG (AM30735) strains run as controls. * indicates a background band present in the untagged control strain.
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Figure 3—figure supplement 1—source data 1
Original files for western blot and ponceau staining data shown in Figure 3B and D.
- https://cdn.elifesciences.org/articles/110117/elife-110117-fig3-figsupp1-data1-v1.zip
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Figure 3—figure supplement 1—source data 2
Original files for western blot and ponceau staining data shown in Figure 3B and D with bands labelled.
- https://cdn.elifesciences.org/articles/110117/elife-110117-fig3-figsupp1-data2-v1.zip
Figure 3—figure supplement 2
Synthetic SAC (SynSAC) does not affect spore viability after meiosis.
Spore viability assay of SynSAC strains following meiosis I or meiosis II SynSAC activation. Following meiotic timecourses with the indicated treatments, tetrads were collected and dissected on rich media plates. The number of spores which grew colonies was counted. Strains analysed were wild-type (AM11189), SynSAC (AM30783), and SynSAC mad3∆ (30784).
Figure 4 with 1 supplement
PP1 binding restrains synthetic SAC (SynSAC) delay duration in meiotic metaphase.
(A and B) Meiosis I (A) and meiosis II (B) SynSAC spindle immunofluorescence timecourses in wild-type vs PP1 binding site mutant SynSAC strains. Top: Schematic indicating drug addition timing. Middle row: Control wild-type (AM30783), SynSAC wild-type (AM30783), SynSAC spc105-4A (AM34201). Bottom row: SynSAC spc105-RASA (AM34203), SynSAC spc105-4A-RASA (AM34202), SynSAC spc105-RVAF (AM34487). Arrows indicate the time of ethanol/abscisic acid (ABA) addition. 100 cells were scored at each timepoint.
Figure 4—figure supplement 1
Mutation of the PP1 binding sites in the Spc105 synthetic SAC (SynSAC) construct does not affect timing of meiosis, spore viability, or mitotic growth.
(A) Meiotic spindle immunofluorescence timecourses of control treated wild-type SPC105(1-455)-PYL (AM30783), spc105(1-455)–4A-PYL (AM34201), spc105(1-455)-RASA-PYL (AM34203), spc105(1-455)–4A-RASA-PYL (AM34202), and spc105(1-455)-RVAF-PYL (AM34487) strains. Arrows indicate time of ethanol addition. 100 cells were scored for each timepoint. (B) Spore viability of wild-type and PP1 binding site mutant SynSAC strains. Tetrads from control-treated meiotic timecourses as in (A) (ethanol added at prophase release), were collected and dissected on rich media. The number of colonies which grew from dissected spores was counted. (C) Serial dilution mitotic growth assay of wild-type and PP1 binding site mutant SynSAC strains as in (A) on YPDA and YPDA with 250 μM abscisic acid (ABA) media.
Figure 5 with 2 supplements
Kinetochore composition in mitotic metaphase, meiotic prophase, meiotic metaphase I, and meiotic metaphase II.
(A) Heatmap showing the fold change enrichment level of each kinetochore protein at each stage, for the tag vs no tag comparison. (B–D) Volcano plots comparing unscaled protein levels of Dsn1-6His-3FLAG (tag) to no tag after anti-FLAG immunoprecipitation from matched extracts of mitotic metaphase (B), meiotic prophase (C), metaphase I (D), and metaphase II (E). Strains used are no tag (AM30990) or DSN1-6His-3FLAG (AM33675).
Figure 5—figure supplement 1
Immunoprecipitation of kinetochores from meiotic prophase, metaphase I, metaphase II, and mitotic metaphase.
(A and B) Kinetochores were purified from the indicated cell cycle stages by anti-FLAG immunoprecipitation of Dsn1-6His-3FLAG (AM33675) and compared to no tag (strain AM30990). (A) Enrichment of cells at the indicated metaphase stages was confirmed by scoring spindle morphology after tubulin immunofluorescence. (B) Silver-stained gels of control no-tag and Dsn1-6His-3FLAG immunoprecipitations are shown.
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Figure 5—figure supplement 1—source data 1
Original files for silver-stained gels shown in B.
- https://cdn.elifesciences.org/articles/110117/elife-110117-fig5-figsupp1-data1-v1.zip
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Figure 5—figure supplement 1—source data 2
Original files for silver-stained gels shown in B with position of BSA and conditions labelled.
- https://cdn.elifesciences.org/articles/110117/elife-110117-fig5-figsupp1-data2-v1.zip
Figure 5—figure supplement 2
Kinetochore composition in meiosis.
(A and B) Analysis of kinetochore composition after Dsn1 immunoprecipitation at different cell cycle stages. (A) Bar plot of number of proteins detected in each Dsn1-6His-3FLAG IP sample from the indicated stage and replicate. (B) Venn diagram showing the number of proteins detected in each Dsn1-6His-3FLAG IP sample and the overlap of which proteins were unique to each sample or shared between samples. Proteins were counted as included in a group if they were detected in all replicates of that sample type. (C) Kinetochore protein enrichment in kinetochore and control immunoprecipitations. Heatmap showing the abundance of each kinetochore protein in all immunoprecipitation samples analysed. Grey indicates missing values.
Figure 6 with 2 supplements
Volcano plots of differential protein enrichment for each pair-wise comparison between different cell cycle stages.
Comparisons of protein abundance detected by mass spectrometry in kinetochore purifications at the different stages. (A) Volcano plot comparing Dsn1-scaled protein levels in meiotic prophase (left) vs metaphase I (right). (B) Volcano plot comparing Dsn1-scaled protein levels in mitotic metaphase (left) vs meiotic prophase (right). (C) Volcano plot comparing Dsn1-scaled protein levels in meiotic prophase (left) vs metaphase II (right). (D) Volcano plot comparing Dsn1-scaled protein levels in metaphase I (left) vs metaphase II (right). (E) Volcano plot comparing Dsn1-scaled protein levels in mitotic metaphase (left) vs metaphase II (right). (F) Volcano plot comparing Dsn1-scaled protein levels in mitotic metaphase (left) vs metaphase I (right).
Figure 6—figure supplement 1
Total protein and kinetochore protein abundances before and after scaling to Dsn1.
(A–F) Mass spectrometry analysis of FLAG immunoprecipitations from no tag (AM30990) or DSN1-6His-3FLAG (AM33675) strains. (A) Protein abundances (intensities) of all IP samples analysed by mass spectrometry. (B) Kinetochore protein abundances of all IP samples analysed by mass spectrometry. (C) Protein abundances after scaling to the Dsn1 protein level in each sample. (D) Kinetochore protein abundances after scaling to the Dsn1 protein level in each sample. (E) Protein abundances of the 4 DSN1-6His-3FLAG sample types. Proteins were included if they were detected in all replicates of that stage. (F) Kinetochore protein abundances of the 4 DSN1-His-3FLAG sample types. Proteins were included if they were detected in all replicates of that stage.
Figure 6—figure supplement 2
Identity and function of proteins enriched on kinetochores at distinct stages.
(A) GO term enrichment of the selected terms among the top 50 non-kinetochore proteins co-purified at each stage. (B) Top 20 non-kinetochore proteins, ranked by increasing fold change enrichment in the first stage listed vs the second stage. (For example, prophase vs MI, proteins are ranked by enrichment in prophase).
Figure 7
Kinetochore protein dynamics in meiotic prophase, metaphase I, metaphase II, and mitotic metaphase.
(A and B) Kinetochores purified by Dsn1-6His-3FLAG immunoprecipitation were analysed from cells arrested at the indicated stages using the SynSAC system by mass spectrometry. Strain used was AM33675. Protein levels are Dsn1-scaled. (A) Heatmap of individual protein levels of core kinetochore proteins (left). Heatmap of individual protein levels of kinetochore-associated proteins at each stage (right). (B) Boxplots of groups of kinetochore proteins at each stage. Dots indicate individual proteins and the numbers above each plot indicate the number of proteins included in each group at that stage. P-values from the Wilcoxon two-sided test are shown.
Figure 8 with 1 supplement
Reduced kinetochore protein phosphorylation in metaphase II.
(A–D) Phosphorylation analysis of kinetochores purified as in Figure 5 and subjected to phospho-enrichment prior to mass spectrometry. (A) Boxplots of groups of kinetochore protein phosphorylation sites at each stage. Numbers above each plot indicate the number of phospho-sites included in the group at each stage. P-values from two-sided Wilcoxon tests are shown for all kinetochore phospho-sites (upper left). No other comparisons were significantly different by the Wilcoxon test in any other group/stage. (B) Heatmap of total sum of phospho-site abundance for each kinetochore protein at each stage. Phospho-proteins are ranked, with proteins with the highest sum of phospho-site abundances, for all four stages together, at the top. Numbers within parentheses next to protein name indicate the sum of phospho-site abundances for all four stages. (C) Boxplots of maximum phospho-site range across the four stages for each kinetochore protein. Phospho-proteins are ranked so that proteins with the highest median phospho-site dynamic range across stages are at the top. Numbers in parentheses indicate the number of phospho-sites considered to calculate maximum phospho-site range. (D) Bar plots of individual phospho-site abundances for the indicated sites at each of the four stages. Dots indicate the abundance in individual replicates. P-values from two-sided t-tests are indicated.
Figure 8—figure supplement 1
Identification of phospho-sites co-isolated with purified kinetochores in meiotic prophase, metaphase I, metaphase II, and mitotic metaphase.
(A) Bar plot of the number of phospho-sites quantified in each Dsn1-6His-3FLAG immunoprecipitation. (B) Venn diagram of the number of phospho-sites identified in each Dsn1-6His-3FLAG immunoprecipitation and whether they were unique to sample type or shared between groups. A phospho-site was included in a group if it was detected in all replicates of that sample type. (C) GO term enrichment of the selected terms among the top 50 non-kinetochore phospho-proteins co-purified at each stage. (D) Top 20 non-kinetochore phospho-sites, ranked by increasing fold change enrichment in the first stage listed vs the second stage. (For example, prophase vs MI, phospho-sites are ranked by enrichment in prophase).
Figure 9
Kinetochore phospho-site motifs do not vary significantly by stage.
(A–D) Features of kinetochore phosphorylation after phospho-analysis of kinetochore purifications in Figure 5. (A) Motif logos of amino acids surrounding kinetochore protein phospho-sites at each stage. (B) Bar plots of the percent of phospho-sites which match indicated kinase consensus motifs at each stage. (C) Bar plots of the percent of phospho-sites which match the Polo kinase consensus motif (top) or minimal Cdk consensus motif (bottom), sorted by kinetochore sub-complex and cell cycle stage. (D) Heatmap showing the abundance of individual kinetochore phospho-sites matching the minimal Polo kinase consensus. (E) Heatmap showing the abundance of individual kinetochore phospho-sites matching the minimal Cdk kinase consensus.
Additional files
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Supplementary file 1
List of plasmids used in this study.
- https://cdn.elifesciences.org/articles/110117/elife-110117-supp1-v1.xlsx
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Supplementary file 2
List of strains used in this study.
- https://cdn.elifesciences.org/articles/110117/elife-110117-supp2-v1.xlsx
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MDAR checklist
- https://cdn.elifesciences.org/articles/110117/elife-110117-mdarchecklist1-v1.pdf
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Source code 1
R script used for mass spectrometry analysis and figure generation in this study.
- https://cdn.elifesciences.org/articles/110117/elife-110117-code1-v1.zip
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Specialisation of meiotic kinetochores revealed through a synthetic spindle assembly checkpoint strategy
eLife 15:RP110117.
https://doi.org/10.7554/eLife.110117.3
