Ipl1/Aurora-B is necessary for kinetochore restructuring in meiosis I in Saccharomyces cerevisiae

Abstract

In mitosis, the centromeres of sister chromosomes are pulled toward opposite poles of the spindle. In meiosis I, the opposite is true: the sister centromeres move together to the same pole, and the homologous chromosomes are pulled apart. This change in segregation patterns demands that between the final mitosis preceding meiosis and the first meiotic division, the kinetochores must be restructured. In budding yeast, unlike mammals, kinetochores are largely stable throughout the mitotic cycle. In contrast, previous work with budding and fission yeast showed that some outer kinetochore proteins are lost in early meiosis. We use quantitative mass spectrometry methods and imaging approaches to explore the kinetochore restructuring process that occurs in meiosis I in budding yeast. The Ndc80 outer kinetochore complex, but not other subcomplexes, is shed upon meiotic entry. This shedding is regulated by the conserved protein kinase Ipl1/Aurora-B and promotes the subsequent assembly of a kinetochore that will confer meiosis-specific segregation patterns on the chromosome.

FIGURE 1:

Centromere repositioning and outer kinetochore disassembly. (A) Representative pictures of diploid cells carrying markers for the kinetochore (MTW1-GFP) and the SPBs (SPC42-DsRed) progressing into meiosis. The cells enter meiosis with clustered centromeres, and centromeres disperse in prophase and then reattach, with sister chromatids attaching to microtubules from the same pole and homologues drawn to opposite poles. (B) A diploid strain with marked central and outer kinetochore proteins (MTW1-3xmCherry and NDC80-GFP) was released into meiosis, and cells with clustered centromeres and Ndc80 present, intermediate dispersion (<4 Mtw1 foci), or complete dispersion (>4 Mtw1 foci) and no Ndc80 were scored (n ≥ 100). Representative cells. Scale bar, 1 μm. (C) From B, the cells with clustered centromeres with (dark gray) or without Ndc80 (light gray) were scored. (D) From B, cells with intermediate dispersion and strong (dark gray) or weak (light gray) signals for Ndc80 were scored. (E) From B, cells with full dispersion and with (dark gray) or without Ndc80 (light gray) were scored. (F) In a diploid strain carrying the kinetochore markers MTW1-3xmCherry and NDC80-GFP, cells at different stages of synaptonemal complex (Zip1) assembly (none, dotty, or linear/full) were scored for the presence of Ndc80. Scale bar, 1 μm.

FIGURE 2:

Behavior of the constitutive-centromere associated network during early stages of meiosis I. (A) Cartoon of the kinetochore microtubule interface. The major complexes of the CCAN are represented. (B) Representative pictures of prophase diploid cells carrying markers for the central kinetochore component Mtw1 (MTW1-3xmCherry) and Mif2, Ndc10 (for the Cbf3 complex), or Cnn1 tagged with GFP. The yellow arrow indicates SPBs marked by SPC42-CFP in the first two strains, Scale bar, 1 μm. (C) Cartoon of the Ctf19 complex. (D, E) Diploid cells expressing Dsn1-FLAG were switched to sporulation medium (T = 0 h). The strains were ndt80 mutants, so they fail to exit from pachytene. The relative amounts of Ctf19 complex components in purified kinetochores at pachytene arrest vs. the time of meiotic entry were determined by MS-SRM. Results are averages of three independent experiments. The levels of individual components on dispersed prophase centromeres (detected with Mtw1-3xmCherry) were examined by fluorescence microscopy. (D) COMA complex MS-SRM results and levels Ctf19-GFP. (E) Additional Ctf19 complex MS-SRM results and levels of Ctf3-GFP. The yellow arrow indicates SPBs (Spc42-CFP). Scale bars, 1 μm.

FIGURE 3:

Behavior of the KMN network and Dam1 complex during early stages of meiosis I. (A–C) MS-SRM was used to determine the relative levels of proteins at pachytene relative to meiotic entry (three independent experiments), and fluorescence microscopy was used to visualize individual components tagged with GFP. Kinetochores were marked by Mtw1-3xmCherry, and SPBs by were marked by Spc42-CFP. (A) Mtw1 complex, Dsn1-GFP, and Nsl1-GFP. (B) Spc105 complex, Spc105-GFP. (C) Ndc80 complex, Spc24-GFP, Nuf2-GFP, and Ndc80-GFP. (D) Western blot analysis of the levels of GFP-tagged Ndc80 complex components. Samples were harvested from synchronous cultures, and extracts were prepared from samples harvested at the times indicated after meiotic induction. Blots were probed with antibodies against Pgk1 (loading control) and GFP. (E) Fluorescence microscopy examination of Dam1-GFP disposition in pachytene. (F) Quantification of fluorescence microscopy data (n ≥ 100). The yellow arrows indicate SPB marker when present. Scale bars, 1 μm.

FIGURE 4:

Blocking DNA replication does not block kinetochore disassembly. (A–D) Wild-type, cdc6-md, and cdc28-as1 diploid cells expressing a SPB marker (Spc42-DsRed) and either a central kinetochore marker, Mtw1-GFP (A, C), or an outer kinetochore marker, Ndc80-GFP (B, D), were sporulated. (B, C) The strains used were ndt80 mutants that arrest in pachytene. T = 0 h represents the time at which cells were switched to sporulation medium. (A, B) Cdc28-as1 was inhibited (red) or not (blue) by adding 5 μM 1NM-PP1 or DMSO, respectively, to the culture medium upon meiotic induction. The proportion of cells with dispersed kinetochores (A) or without outer kinetochores (B) was scored (n ≥ 100). Representative pictures of cell with dispersed kinetochores (A) or without the outer kinetochore (B). (C) The proportion of cells with dispersed kinetochores for wild-type (blue) or cdc6-md cells (red) was scored. A representative picture of a cell with dispersed kinetochores is shown (n ≥ 100/time point). (D) The proportion of cells at 4 h with (light gray) or without (dark gray) detectable outer kinetochores was scored in wild-type or cdc6-md cells (n ≥ 100). Representative pictures of cells with or without detectable Ndc80-GFP are shown. Inactivation of Cdc6 did not change the kinetics or level of dispersion and did not affect shedding of the outer kinetochore in detectable ways. Scale bars, 1 μm.

FIGURE 5:

Ipl1/Aurora-B is necessary for outer kinetochore disassembly. (A, C, D) Wild-type or ipl1-md diploid cells expressing Spc42-DsRed and outer kinetochore marker Ndc80-GFP (A), Spc24-GFP (C), or Dam1-GFP (D) were switched to sporulation medium (T = 0 h). (A–D) The strains used were ndt80 mutants that arrest in pachytene. (A) The proportion of cells with dispersed kinetochores was scored in wild-type (blue) or ipl1-md (red) diploid cells (n ≥ 100). A representative picture of a cell with dispersed kinetochores is shown. (B) The patterns of Ndc80-GFP staining were monitored in an ipl1-md diploid expressing Mtw1-3xmCherry and Ndc80-GFP. Cells were first categorized according to their stage in prophase by evaluating their Zip1 staining pattern (No/dotty or Linear/full). (C, D) The proportion of cells with (light gray) or without (dark gray) either Spc24 or Dam1 was scored (n ≥ 100). Representative pictures of cells with or without the designated kinetochore component are shown. (E) Wild-type or ipl1-md diploid cells carrying Dsn1-FLAG were sporulated. The quantification by mass spectrometry was done after purifying the kinetochore in five (ipl1-md) or seven (wild-type) independent experiments. The ratios represent the amount of each component at T = 3 h relative to the amount at T = 0 h. Scale bars, 1 μm.

FIGURE 6:

Loading of the monopolin complex. (A–E) Wild-type, ipl1-md, and ipl1-as5/ipl1-md diploid cells were sporulated and released from a pachytene arrest (P_GAL1-NDT80 GAL4-ER) at 6 h by the addition of 5 μM β-estradiol. Ipl1-as5 was inhibited by the addition of 50 μM 1NA-PP1 at the time of the release. All strains expressed Mam1-GFP and Mtw1-3xmCherry and Spc42-DsRed (SPB) when indicated. The protocol is summarized in A. T = 0 h represents the time when cells were released from the arrest. (B) Wild-type cells, harvested at timed intervals after pachytene release, were categorized according to Mam1-GFP distribution. (C) Images of Mam1-GFP patterns observed in wild-type cells. (D) Distribution of Mam1-GFP in ipl1-md (2) and ipl1-as5/ipl1-md mutants (3) after release from pachytene arrest. n ≥ 100 for all time points. (E) Representative images of Mam1-GFP distribution in ipl1 mutants. Note the bright Mam1-GFP focus in the leftmost image. These foci were common in ipl1-md mutants (see Supplemental Figure S5). Scale bars, 2 μm.

FIGURE 7:

Release of kinetochore–microtubule associations and shedding of outer kinetochores allow monopolin loading. (A) Monopolin loading on kinetochores with or without release of kinetochore–microtubule attachments. ipl1-md diploid cells were sporulated and released from a pachytene arrest (P_GAL1-NDT80 GAL4-ER) at 6 h after the induction of meiosis by the addition of 5 μM β-estradiol (T = 0 h). Microtubules were destabilized by the addition of benomyl (30 μg/ml) and nocodazole (15 μg/ml), which were added at the time of release from the pachytene arrest (protocol is summarized in Figure 6A). At 120 and 165 min after release from pachytene arrest, cells were categorized according to their Mam1-GFP localization (nuclear, low kinetochore localization, strong kinetochore colocalization; see Supplemental Figure 5 for representative images). n ≥ 83 cells for each time point. (B) Monopolin loading with and without the Ndc80 complex on kinetochores. Wild-type, ipl1-md, ndc80-md, or ipl1-md ndc80-md diploid strains were induced to enter meiosis and then released from pachytene by the addition of 5 μM β-estradiol at 4.5 h after the induction of meiosis. Cells were scored as in A. n ≥ 59 cells for each time point. (C) ipl1-md diploid cells expressing Ndc80-GFP and Mtw1-3xmCherry were switched to sporulation medium. The strains used were ndt80 mutants that arrest in pachytene. Benomyl (30 μg/ml) and nocodazole (15 μg/ml) were added to the cells 6 h after induction of meiosis (T = 0 h). The colocalization of Ndc80-GFP with individual Mtw1-mCherry foci was scored in cells with 3–5 dispersed kinetochores (intermediate dispersion) or those with 6–12 kinetochores (full dispersion). Colocalization is indicated by dark gray, and Mtw1-mCherry foci with no colocalizing Ndc80-GFP signal are indicated in white. Representative cells are shown. Scale bars, 1 μm. n ≥ 41 Mtw1 foci for each time point. Fisher's exact tests were used to evaluate the significance of observed differences in A–C. *p < 0.05, **p < 0.01, ***p < 0.001. (D) A model representing the steps of kinetochore remodeling in meiosis I.