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

Meyer RE, Chuong HH, Hild M, Hansen CL, Kinter M, Dawson DS - Mol. Biol. Cell (2015)

Bottom Line: In contrast, previous work with budding and fission yeast showed that some outer kinetochore proteins are lost in early meiosis.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.

View Article: PubMed Central - PubMed

Affiliation: Program in Cell Cycle and Cancer Biology, Oklahoma Medical Research Foundation, Oklahoma City, OK 73104.

No MeSH data available.


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 (PGAL1-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.
© Copyright Policy - creative-commons
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4551314&req=5

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 (PGAL1-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.

Mentions: We imagine two different ways in which Ipl1 activity in prophase might promote monopolin loading. First, Ipl1's releasing of kinetochore-microtubule attachments might allow monopolin to load onto kinetochores. This possibility is suggested by the observation that when monopolin is expressed ectopically in mitotic cells, higher levels of meiosis I–like segregation are observed if microtubules are briefly depolymerized before mitosis (Miller, Ünal, et al., 2012). Second, removing the outer kinetochore might directly improve accessibility of the Mtw1 complex for association with monopolin, independently of kinetochore–microtubule association. To study the relative effect on monopolin loading of removing either kinetochore–microtubule interactions or the Ndc80 complex (and therefore any Ndc80 complex–mediated microtubule interactions as well), we used two complementary strategies. The first one disrupted kinetochore–microtubule associations (Figure 7A). The second approach was to remove the Ndc80 outer kinetochore complex (ndc80-md; Figure 7B). For the first approach, we used a combined treatment with benomyl and nocodazole that efficiently destabilized microtubules (Supplemental Figure 6, A–C). The ipl1-md cells, which do not shed outer kinetochores and exhibit persistent kinetochore–microtubule attachments, were released from a prophase arrest for 2½ h (corresponding to the time with maximum loading of Mam1; Figure 6, B and D) in the presence or absence of microtubule-depolymerizing drugs. Treatment with microtubule- destabilizing agents resulted in a small but significant increase in monopolin loading (Figure 7A). To test the effect of removing the outer kinetochore, we used ndc80-md strains in which NDC80 is placed under the control of the CLB2 promoter and is therefore not expressed in meiotic cells. We showed previously, using the ndc80-md mutation, that the lack of centromere dispersion in ipl1-md mutants could be rescued by removing the outer kinetochore complex (Meyer et al., 2013). The ndc80-md mutation resulted in a strong rescue of monopolin loading in the ipl1-md strain (Figure 7B; see representative cell in Supplemental Figure 6D). The much greater rescue of monopolin loading that is seen in the ipl1-md mutants when Ndc80 is removed compared to when microtubules are destabilized suggests that even in the absence of kinetochore–microtubule associations, the Ndc80 complex can interfere with monopolin loading, possibly by obstructing access to the Mtw1 complex. This predicts that Ndc80 remains on the detached kinetochores after treatment with microtubule-destabilizing agents. To test this, we treated cells with benomyl/nocodazole and identified those with dispersed kinetochores (Supplemental Figure 7). In these cells, we scored the localization of Ndc80-GFP with individual dispersed Mtw1-3xmCherry foci (Figure 7C). Cells were categorized as having either intermediate dispersion (3–5 Mtw1 foci) or full dispersion (6–12 foci). In cells with intermediate dispersion, most Mtw1 foci that had been released from the SPB showed colocalization with Ndc80. With increased exposure time to the microtubule-destabilizing agents and in cells with greater dispersion, the level of colocalization was reduced (Figure 7C). Together the results suggest that loss of kinetochore–microtubule attachments leads to the accumulation of kinetochores that lack Ndc80.


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

Meyer RE, Chuong HH, Hild M, Hansen CL, Kinter M, Dawson DS - Mol. Biol. Cell (2015)

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 (PGAL1-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.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4551314&req=5

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 (PGAL1-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.
Mentions: We imagine two different ways in which Ipl1 activity in prophase might promote monopolin loading. First, Ipl1's releasing of kinetochore-microtubule attachments might allow monopolin to load onto kinetochores. This possibility is suggested by the observation that when monopolin is expressed ectopically in mitotic cells, higher levels of meiosis I–like segregation are observed if microtubules are briefly depolymerized before mitosis (Miller, Ünal, et al., 2012). Second, removing the outer kinetochore might directly improve accessibility of the Mtw1 complex for association with monopolin, independently of kinetochore–microtubule association. To study the relative effect on monopolin loading of removing either kinetochore–microtubule interactions or the Ndc80 complex (and therefore any Ndc80 complex–mediated microtubule interactions as well), we used two complementary strategies. The first one disrupted kinetochore–microtubule associations (Figure 7A). The second approach was to remove the Ndc80 outer kinetochore complex (ndc80-md; Figure 7B). For the first approach, we used a combined treatment with benomyl and nocodazole that efficiently destabilized microtubules (Supplemental Figure 6, A–C). The ipl1-md cells, which do not shed outer kinetochores and exhibit persistent kinetochore–microtubule attachments, were released from a prophase arrest for 2½ h (corresponding to the time with maximum loading of Mam1; Figure 6, B and D) in the presence or absence of microtubule-depolymerizing drugs. Treatment with microtubule- destabilizing agents resulted in a small but significant increase in monopolin loading (Figure 7A). To test the effect of removing the outer kinetochore, we used ndc80-md strains in which NDC80 is placed under the control of the CLB2 promoter and is therefore not expressed in meiotic cells. We showed previously, using the ndc80-md mutation, that the lack of centromere dispersion in ipl1-md mutants could be rescued by removing the outer kinetochore complex (Meyer et al., 2013). The ndc80-md mutation resulted in a strong rescue of monopolin loading in the ipl1-md strain (Figure 7B; see representative cell in Supplemental Figure 6D). The much greater rescue of monopolin loading that is seen in the ipl1-md mutants when Ndc80 is removed compared to when microtubules are destabilized suggests that even in the absence of kinetochore–microtubule associations, the Ndc80 complex can interfere with monopolin loading, possibly by obstructing access to the Mtw1 complex. This predicts that Ndc80 remains on the detached kinetochores after treatment with microtubule-destabilizing agents. To test this, we treated cells with benomyl/nocodazole and identified those with dispersed kinetochores (Supplemental Figure 7). In these cells, we scored the localization of Ndc80-GFP with individual dispersed Mtw1-3xmCherry foci (Figure 7C). Cells were categorized as having either intermediate dispersion (3–5 Mtw1 foci) or full dispersion (6–12 foci). In cells with intermediate dispersion, most Mtw1 foci that had been released from the SPB showed colocalization with Ndc80. With increased exposure time to the microtubule-destabilizing agents and in cells with greater dispersion, the level of colocalization was reduced (Figure 7C). Together the results suggest that loss of kinetochore–microtubule attachments leads to the accumulation of kinetochores that lack Ndc80.

Bottom Line: In contrast, previous work with budding and fission yeast showed that some outer kinetochore proteins are lost in early meiosis.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.

View Article: PubMed Central - PubMed

Affiliation: Program in Cell Cycle and Cancer Biology, Oklahoma Medical Research Foundation, Oklahoma City, OK 73104.

No MeSH data available.