The molecular basis of monopolin recruitment to the kinetochore

Abstract

The monopolin complex is a multifunctional molecular crosslinker, which in S. pombe binds and organises mitotic kinetochores to prevent aberrant kinetochore-microtubule interactions. In the budding yeast S. cerevisiae, whose kinetochores bind a single microtubule, the monopolin complex crosslinks and mono-orients sister kinetochores in meiosis I, enabling the biorientation and segregation of homologs. Here, we show that both the monopolin complex subunit Csm1 and its binding site on the kinetochore protein Dsn1 are broadly distributed throughout eukaryotes, suggesting a conserved role in kinetochore organisation and function. We find that budding yeast Csm1 binds two conserved motifs in Dsn1, one (termed Box 1) representing the ancestral, widely conserved monopolin binding motif and a second (termed Box 2-3) with a likely role in enforcing specificity of sister kinetochore crosslinking. We find that Box 1 and Box 2-3 bind the same conserved hydrophobic cavity on Csm1, suggesting competition or handoff between these motifs. Using structure-based mutants, we also find that both Box 1 and Box 2-3 are critical for monopolin function in meiosis. We identify two conserved serine residues in Box 2-3 that are phosphorylated in meiosis and whose mutation to aspartate stabilises Csm1-Dsn1 binding, suggesting that regulated phosphorylation of these residues may play a role in sister kinetochore crosslinking specificity. Overall, our results reveal the monopolin complex as a broadly conserved kinetochore organiser in eukaryotes, which budding yeast have co-opted to mediate sister kinetochore crosslinking through the addition of a second, regulatable monopolin binding interface.

Keywords: Kinetochore; Meiosis; Monopolin; RWD domain.

Fig. 1

Identification of Csm1 and a conserved N-terminal Dsn1 motif in a wide range of eukaryotes. a Speculative model for intra-kinetochore crosslinking by monopolin in mitosis, based on prior observations that the budding yeast monopolin complex subunit Csm1 interacts with the kinetochore through a disordered region in the Mis12 complex subunit Dsn1. Using our previously established workflow ConFeaX (Tromer et al. 2016), we uncovered a short motif (Dsn1-N) that is conserved in a wide range of eukaryotic Dsn1 orthologs (Fig. S1). b Presence-absence profiles of the KMN network (including Knl1/Zwint-1, Mis12 complex, and the Ndc80 complex), CCAN/Ctf19 complex, plus Csm1 and Dsn1-N in 109 eukaryotic proteomes. White squares indicate absence and coloured squares presence of the proteins in a particular species (colours correspond to complexes in panel A). The tree to the right depicts the various eukaryotic supergroups. Encephalitozoon and Oomycetes are highlighted to indicate that these species’ Dsn1 proteins appear to possess two Dsn1-N motifs (Fig. S1b, c). ct-SNE projection and 2 dimensional representation of phylogenetic profile similarity (Pearson distance [D = 1- − r]) of kinetochore proteins depicted in panel b. The table in the lower left corner summarises the frequencies of Csm1, Dsn1 and Dsn1-N in 109 eukaryotic species (panel b). While the presence-absence profiles of Dsn1 and Csm1 are not similar (Pearson correlation coefficient, r = 0.339), the presence-absence profiles of Csm1 and Dsn1-N are highly similar (r = 0.799). In species with both Csm1 and Dsn1, only 6 do not have a Dsn1-N motif (6 of 55), while in species with Dsn1 that lack Csm1, none have the Dsn1-N motif (0 of 30)

Fig. 2

Structure of the Csm1-Dsn1 complex. a Sequence logos for eukaryotic Dsn1-N (Fig. S1) and budding yeast Dsn1 Box 1 (Fig. S2a, b), demonstrating high homology between the two motifs; b Domain schematic of Dsn1 from S. cerevisiae and C. glabrata, with conserved regions shown in orange. The Dsn1 C-terminal domain forms a folded complex with other MIND complex subunits, while the N-terminal conserved region interacts with Csm1. Bottom: Sequence alignment of the S. cerevisiae and C. glabrata Box 1-2-3 region (see Fig. S2 for larger sequence alignments); c Overall view of the Cg Csm1^69–181:Cg Dsn1^14–72 complex, showing the Dsn1 Box 2-3 region (orange) interacting with a Csm1 dimer (blue with white surface); d Overall view of the Cg Csm1^69–181:Sc Dsn1^71–110 complex, showing the Dsn1 Box 2-3 region. See Fig. S5 for more details on Csm1-Dsn1 Box 2-3 interactions, and Fig. S6 for crystal packing interactions; e Overall view of the Cg Csm1^69–181:Cg Dsn1^14–72 complex, showing the Dsn1 Box 1 region (orange) interacting with a Csm1 dimer (teal with white surface). See Fig. S5a for crystal packing interactions for this complex; f Ni²⁺-pulldown of in vitro translated S. cerevisiae Dsn1 N-terminal region constructs by Sc His₆-Csm1^69–190

Fig. 3

Dsn1 Box 2 contributes to successful meiosis. a Close-up view of the Cg Dsn1 Box 2 region (orange) interacting with the “side” of a Csm1 protomer (blue with white surface) in the Cg Csm1^69–181:Cg Dsn1^14–72 complex. Residue numbers shown are for Cg Dsn1, with Sc Dsn1 equivalents shown in orange text. See Fig. S5d–f for equivalent views of the Cg Csm1^69–181:Sc Dsn1^71–110 and Cg Csm1^69–181:Cg Dsn1^43–67DD complexes; b Point mutations in Dsn1 affect spore survival. Diploid cells carrying the indicated homozygous mutations in DSN1 were sporulated, dissected, and the number of spores that formed colonies from each tetrad was scored. Between 38 and 56 tetrads were dissected for each condition, from a minimum of two independent diploid strains. Diploid strains used were generated from matings between AMy1827 and AMy1828 or AMy1835 (wild type), AMy1932 and AMy1947 (mam1Δ), AMy21921 and AMy22719 (DSN1-L88A L92A L95A), AMy23151 and AMy23152 (DSN1-E90A N94A D97A), and AMy24629 and AMy24632 (DSN1-E90K N94K D97K); c, d Live cell imaging of heterozygous CEN5-GFP foci during meiosis reveals defective monoorientation in the presence of Dsn1 Box 2 mutations. Cells also carry Mtw1-tdTomato to label kinetochores and Pds1-tdTomato, the destruction of which marks anaphase I onset; c Representative images of strains producing either wild type Dsn1 or Dsn1-L88A L92A L95A. While wild type cells segregate a single CEN5-GFP focus to one pole, some DSN1-L88A L92A L95A cells split GFP foci and exhibit delayed meiosis II. Arrowheads indicate position of CEN5-GFP foci during anaphase I, revealing whether they segregate to the same pole (monooriented, as in the wild type example) or opposite poles (bioriented as in DSN1-L88A L92A L95A cells). Images are from frames taken at 15 min intervals; d Scoring of GFP foci position at anaphase I onset, defined as the first occasion on which Mtw1-tdTomato segregate. Strains used were AMy25832 (wild type; n = 78), AMy25881 (DSN1-L88A L92A L95A; n = 26), AMy25763 (DSN1-E90A N94A D97A; n = 39) and AMy25881 (DSN1-E90K N94K D97K; n = 93)

Fig. 4

Dsn1 Box 3 residues are critical for meiosis. a Close-up view of the Cg Dsn1 Box 3 region (orange) interacting with the Csm1 conserved hydrophobic cavity (blue with white surface) in the Cg Csm1^69–181:Cg Dsn1^14–72 complex. Residue numbers shown are for Cg Dsn1, with Sc Dsn1 equivalents shown in orange text. See Fig. S5 g–i for equivalent views of the Cg Csm1^69–181:Sc Dsn1^71–110 and Cg Csm1^69–181:Cg Dsn1^43-67DD complexes; b Diploid cells with heterozygous or homozygous mutations in DSN1 were sporulated, dissected and the number of spores which grew up from each tetrad scored. Between 38 and 78 tetrads were dissected for each condition, from a minimum of two independent diploids. Data for wild type and mam1Δ is reproduced from Fig. 3b. Heterozygous diploids were generated from crosses between AMy1827 and AMy24652 (DSN1-V104A F107A), AMy1827 and AMy25110 (DSN1-V104D F107D), AMy1827 and AMy26803 (DSN1-S109A S110A), AMy1827 and AMy24744 (DSN1-S109D S110D). Homozygous diploids were generated from crosses between AMy24624 and AMy24652 (DSN1-V104A F107A), AMy24858 and AMy25110 (DSN1-V104D F107D), AMy26426 and AMy26803 (DSN1-S109A S110A), AMy24744 and AMy24688 (DSN1-S109D S110D); c Live cell imaging was used to score sister chromatid co-segregation during anaphase I in cells carrying heterozygous CEN5-GFP foci and Dsn1 Box 3 mutations as described in Fig. 3 c, d. Data for wild type and mam1Δ is reproduced from Fig. 3 d, other strains analysed and number of cells counted were AMy25762 (DSN1-V104A F107A) n = 51, AMy26475 (DSN1-V104D F107D) n = 61 and AMy26828 (DSN1-S109A S110A) n = 50, AMy27009 (DSN1-S109D S110D) n = 64; d Analysis of Mam1-9Myc association with a representative centromere (CEN4) by anti-Myc chromatin immunoprecipitation followed by quantitative PCR (ChIP-qPCR). Wild type (AM25617), DSN1-V104A F107A (AM24669), DSN1-V104D F107D (AMy26778), DSN1-S109A S110A (AMy26800) and DSN1-S109D S110D (AMy26476) cells carrying MAM1-9MYC were arrested in metaphase I of meiosis by depletion of Cdc20. Strain AMy8067 was used as a no tag control. Shown is the average from 8 biological replicates for wild type and no tag. The average from 3 experiments is shown for all DSN1 mutants with the exception of DSN1-S109D S110D where the average from 5 biological replicates is shown. Error bars indicate standard error; e Close-up view of the Cg Dsn1 Box 3 region with conserved serine residues mutated to aspartate (from the structure of Cg Csm1^69–181:Cg Dsn1^43–67DD). Residue D66 is visible forming hydrogen-bond interactions with Csm1 K172. The side-chain for residue D67 is disordered, and is modelled as alanine. Csm1 is shown in white with surface coloured by charge. f Live cell imaging of Mam1-GFP. Cells carrying Mtw1-tdTomato were released from a prophase block by β-oestradiol-dependent inducible expression of Ndt80 (Carlile and Amon 2008). Representative images are shown for the indicated genotypes. Graph displays the fraction of cells with the localisation pattern depicted in the schematic. Strains used were wild type (AMy14942; n = 40), csm1∆ (AMy15096; n = 37), DSN1-S109A S110A (AMy26963, n = 50), and DSN1-S109D S110D (AMy26947, n = 39)

Fig. 5

The Csm1-Dsn1 Box 1 interface a Close-up view of the Cg Dsn1 Box 1 region (orange) interacting with the Csm1 conserved hydrophobic cavity (blue with white surface) in the Cg Csm1^69–181:Cg Dsn1^14–72 complex. Residue numbers shown are for Cg Dsn1, with Sc Dsn1 equivalents shown in orange text; b Dsn1 Box 1 is critical for meiosis. Spore viability of diploid strains with the indicated genotypes were analysed as described in Fig. 3b. Between 38 and 68 tetrads were dissected for each condition, from a minimum of two independent diploids. Data for wild type and mam1Δ is reproduced from Fig. 3b. Other diploids were generated from matings between AMy1827 and AMy11417 (heterozygous mam1Δ), AMy1827 and AMy17222 (heterozygous DSN1-L72A F74A), AMy1827 and AMy17123 or AMy17313 (heterozygous DSN1-L72D L74D), AMy17222 and AMy17223 (homozygous DSN1-L72A F74A) or AMy17313 and AMy17373 (homozygous DSN1-L72D F72D). c Live cell imaging was used to score sister chromatid co-segregation during anaphase I in cells carrying heterozygous CEN5-GFP foci and Dsn1 Box 1 mutations as described in Fig. 3 c, d. Data for wild type and mam1Δ is reproduced from Fig. 3 d, other strains analysed were AMy25821 (DSN1-L72A F74A; n = 78) and AMy26543 (DSN1-L72D F72D; n = 59); d Mam1 association with a representative centromere in a metaphase I arrest was analysed by ChIP-qPCR as described in Fig. 4d. Data for wild type and no tag is reproduced from Fig. 4d. Other strains used were AMy25618 (DSN1-L72A F74A) and AMy26543 (DSN1-L72D F72D) and the average of 3 or 5 biological replicates, respectively, is shown with standard error bars

Fig. 6

S. pombe Mis13 binds Csm1 through a Dsn1-N/Box 1 motif. a Sequence alignment of the Cg Dsn1 Box 1 region with the N-terminal region of S. pombe Mis13 (See Fig. S2a–e for sequence alignment of fungal Dsn1 N-termini); b Close-up view of the Cg Dsn1 Box 1 region (orange) interacting with the Csm1 conserved hydrophobic cavity (blue with white surface). Residue numbers shown are for Cg Dsn1 (orange) and Csm1 (blue), with S. pombe Mis13 equivalents shown in grey text; c Fluorescence polarisation peptide-binding assay showing interaction of the isolated S. pombe Mis13 Dsn1-N region (residues 5–17, sequence PEEQEGFVFVRKG) with purified S. pombe Csm1 C-terminal globular domain (residues 125–261). The Csm1 I241D mutant mimics the S. cerevisiae L161D mutant, known to disrupt binding of Dsn1 in vitro (Corbett et al. 2010) by disrupting the conserved hydrophobic cavity (L159 in Cg Csm1; see panel B); d Ni²⁺-pulldown of in vitro translated S. pombe Mis13 N-terminal region (wild type and mutants) by His₆-Csm1^125–261. Mis13 residues whose mutation to alanine disrupts Csm1 binding are marked by asterisks in panel a

Fig. 7

Dsn1 Box 1 and Box 3 perform independent roles in meiosis. a Ni²⁺-pulldown of in vitro translated S. cerevisiae Dsn1 N-terminal region constructs (fused to an N-terminal maltose binding protein tag) by Sc His₆-Csm1^69–190; b Combination of Box 1 and Box 3 mutations lead to an additive effect on meiosis. Spore viability of diploid strains with the given heterozygous mutations in DSN1 were analysed as described in Fig. 3b. Between 38 and 68 tetrads were dissected for each Dsn1 point mutant diploid, from a minimum of two independent diploids, while more than 300 tetrads were scored for the truncations. Data for wild type and mam1Δ is reproduced from Fig. 3b. Other diploids were generated from matings between AMy17232 and AMy1827 (heterozygous Δ78-DSN1), AMy17230 and AMy1827 (Δ110-DSN1), AMy17505 and AMy17507 (homozygous Δ110-DSN1), AMy1828 and AMy26727 (wild type and DSN1-L72A F74A V104A F107A), AMy1828 and AMy25883 (wild type and DSN1-L72A F74A S109A S110A), AMy1828 and AMy26728 (wild type and L72A F74A V104A F107A S109A S110A); AMy17505 and AMy26727 (Δ110-DSN1 and DSN1-L72A F74A V104A F107A), AMy17505 and AMy25883 (Δ110-DSN1 and DSN1-L72A F74A S109A S110A), AMy17505 and AMy26728 (Δ110-DSN1 and L72A F74A V104A F107A S109A S110A). Data for DSN1-L72A F74A, DSN1-V104A F107A, and DSN1-S109A S110A is reproduced from Fig. 3b and 4b; c Model for sister kinetochore monoorientation by the monopolin complex. Initial unstable kinetochore association of the monopolin complex (1) occurs via Dsn1 Box 1 (yellow). While initial binding is non-specific, association of a single complex with sister kinetochores triggers a switch to a more stable binding mode (2) involving Dsn1 Box 2-3 (orange). The association is further stabilised by phosphorylation of Dsn1 S109/S110 by Hrr25 or another kinase, resulting in stable sister kinetochore monoorientation (3). While we draw a single Dsn1 interacting with each globular head of the monopolin complex, each head possesses two conserved hydrophobic cavities and therefore could bind two copies of Dsn1