Construction of a meiotic SynSAC activation system

(A) Diagram of synthetic SAC dimerization system. (B) Spore viability of wild type (AM11189) and SynSAC dimer (AM30783) yeast strains (C) Meiosis spindle immunofluorescence timecourse of SynSAC dimer yeast strain (AM30783) under control (ethanol, left), meiosis I dimerizing (250μM ABA at release), or meiosis II dimerizing (250μM ABA at 100 minutes) conditions. (D) Meiosis spindle immunofluorescence timecourse in wild-type SynSAC (AM30783), SynSAC mad1Δ (AM33558), SynSAC mad2Δ (AM33559), and SynSAC mad3Δ (AM30784) strains. Vertical dotted line indicates time of ABA addition. 250μM abscisic acid (ABA) was added to each culture at 0 minutes upon addition of β-estradiol (prophase release). MI = metaphase I; AI = anaphase I; MII = metaphase II; AII = anaphase II.

Meiotic SynSAC delays degradation of Pds1securin.

(A-C) Meiosis timecourse with spindle immunofluorescence (left) and western blotting to visualize Pds1-Myc and Pgk1 loading control (right). (A) Control timecourse with ethanol added at prophase release (vertical dotted line). (B) Meiosis I SynSAC delay timecourse with 250μM ABA added at prophase release (vertical dotted line). (C) Meiosis II SynSAC delay meiosis timecourse with 250μm ABA added at 100 minutes after prophase release (vertical dotted line). Strain used in A-C was AM34398. MI = metaphase I; AI = anaphase I; MII = metaphase II; AII = anaphase II.

PP1 binding restrains SynSAC delay duration in meiotic metaphase

(A-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).

Kinetochore protein dynamics in meiotic prophase, metaphase I, metaphase II, and mitotic metaphase

(A and B) Knetochores purified by Dsn1-6His-3Flag immunoprecipation were analysed from cells arrested at the indicated stages using the SynSAC system by mass spectrometry. Strain used was AM33675. (A) Heatmap of individual protein levels of core kinetochore proteins (left). Heatmap of individual protein levels of transiently associated kinetochore 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 Wilcoxon two-sided test are shown.

Reduced kinetochore protein phosphorylation in metaphase II

(A-D) Phosphorylation analysis of kinetochores purified as in Figure 4 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 test are shown for all kinetochore phospho-sites (upper left). No other comparisons were significantly different by 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 4 stages together, at the top. Numbers within parentheses next to protein name indicate the sum of phospho-site abundances for all 4 stages. (C) Boxplots of maximum phospho-site range across the 4 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) Barplots of individual phospho-site abundances for the indicated sites at each of the 4 stages. Dots indicate the abundance in individual replicates.

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) Mo.f logos of amino acids surrounding kinetochore protein phospho-sites at each stage. (B) Barplots of the percent of phospho-sites which match indicated kinase consensus motifs at each stage. (C) Barplots 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.