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Inhibitory phosphorylation of separase is essential for genome stability and viability of murine embryonic germ cells.

Huang X, Andreu-Vieyra CV, York JP, Hatcher R, Lu T, Matzuk MM, Zhang P - PLoS Biol. (2008)

Bottom Line: We show that germ cells in the mutants are depleted during development.We further demonstrate that S1121A causes chromosome misalignment during proliferation of the postmigratory primordial germ cells, resulting in mitotic arrest, aneuploidy, and eventual cell death.Our results indicate that inhibitory phosphorylation of separase plays a critical role in the maintenance of sister chromatid cohesion and genome stability in proliferating postmigratory primordial germ cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Physiology and Biophysics, Baylor College of Medicine, Houston, Texas, United States of America.

ABSTRACT
Activity of separase, a cysteine protease that cleaves sister chromatid cohesin at the onset of anaphase, is tightly regulated to ensure faithful chromosome segregation and genome stability. Two mechanisms negatively regulate separase: inhibition by securin and phosphorylation on serine 1121. To gauge the physiological significance of the inhibitory phosphorylation, we created a mouse strain in which Ser1121 was mutated to Ala (S1121A). Here we report that this S1121A point mutation causes infertility in mice. We show that germ cells in the mutants are depleted during development. We further demonstrate that S1121A causes chromosome misalignment during proliferation of the postmigratory primordial germ cells, resulting in mitotic arrest, aneuploidy, and eventual cell death. Our results indicate that inhibitory phosphorylation of separase plays a critical role in the maintenance of sister chromatid cohesion and genome stability in proliferating postmigratory primordial germ cells.

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Persistent Expression of Aurora B Kinase in Mutant Germ Cells(A) Immunostaining using antibodies to Aurora B (red) and Mvh (green). Sections of 13.5 dpc gonads were used for the staining.(B) Western blot analysis of Aurora B, active caspase-3, p53, and Bax in extracts of 13.5 dpc genital ridges.(C) Spindle checkpoint–adapted HeLa cells maintain Aurora B expression. HeLa cells were stained for Aurora B (red) and γ-tubulin (green) after 36 h of nocodazole treatment.(D) Immunofluorescence detection of active caspase-3 in mutant genital ridge at 13.5 dpc.
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pbio-0060015-g005: Persistent Expression of Aurora B Kinase in Mutant Germ Cells(A) Immunostaining using antibodies to Aurora B (red) and Mvh (green). Sections of 13.5 dpc gonads were used for the staining.(B) Western blot analysis of Aurora B, active caspase-3, p53, and Bax in extracts of 13.5 dpc genital ridges.(C) Spindle checkpoint–adapted HeLa cells maintain Aurora B expression. HeLa cells were stained for Aurora B (red) and γ-tubulin (green) after 36 h of nocodazole treatment.(D) Immunofluorescence detection of active caspase-3 in mutant genital ridge at 13.5 dpc.

Mentions: To further demonstrate the mitotic arrest phenotype, we analyzed the expression of Aurora B kinase in PGCs. Aurora B is a chromosome passenger protein required for spindle assembly checkpoint [46,47]. In normal mitosis, Aurora B first associates with the metaphase chromosomes, departs the chromosomes and translocates to the cleavage furrow at anaphase, becomes concentrated in the mid-body during cytokinesis, and finally disappears in G1. When we immunostained with antibodies against Aurora B, we found that this mitotic kinase formed foci in all mutant PGCs with abnormal nuclear morphologies, although the cells were no longer in mitosis as their chromosomes had already decondensed (Figure 5A). Western blot analysis confirmed the immunostaining result (Figure 5B). These data suggest that mutant PGCs adapt to the spindle assembly checkpoint but retain Aurora B expression. To determine if the retention of Aurora B expression is always associated with cells having undergone adaptation to spindle checkpoint, we treated a panel of cell lines with nocodazole, let the cells adapt, and analyzed the pattern of Aurora B expression. All cell lines tested, including HeLa, U2OS, and mouse embryonic fibroblasts, showed the same nuclear Aurora B staining pattern and abnormal nuclear morphologies as did mutant PGCs (Figure 5C, results from HeLa cells are shown). We conclude that the formation of nuclear Aurora B foci is a common feature of mammalian cells adapted to the spindle assembly checkpoint. However, at present, it is unclear if the retention of Aurora B expression is necessary for adaptation.


Inhibitory phosphorylation of separase is essential for genome stability and viability of murine embryonic germ cells.

Huang X, Andreu-Vieyra CV, York JP, Hatcher R, Lu T, Matzuk MM, Zhang P - PLoS Biol. (2008)

Persistent Expression of Aurora B Kinase in Mutant Germ Cells(A) Immunostaining using antibodies to Aurora B (red) and Mvh (green). Sections of 13.5 dpc gonads were used for the staining.(B) Western blot analysis of Aurora B, active caspase-3, p53, and Bax in extracts of 13.5 dpc genital ridges.(C) Spindle checkpoint–adapted HeLa cells maintain Aurora B expression. HeLa cells were stained for Aurora B (red) and γ-tubulin (green) after 36 h of nocodazole treatment.(D) Immunofluorescence detection of active caspase-3 in mutant genital ridge at 13.5 dpc.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2214812&req=5

pbio-0060015-g005: Persistent Expression of Aurora B Kinase in Mutant Germ Cells(A) Immunostaining using antibodies to Aurora B (red) and Mvh (green). Sections of 13.5 dpc gonads were used for the staining.(B) Western blot analysis of Aurora B, active caspase-3, p53, and Bax in extracts of 13.5 dpc genital ridges.(C) Spindle checkpoint–adapted HeLa cells maintain Aurora B expression. HeLa cells were stained for Aurora B (red) and γ-tubulin (green) after 36 h of nocodazole treatment.(D) Immunofluorescence detection of active caspase-3 in mutant genital ridge at 13.5 dpc.
Mentions: To further demonstrate the mitotic arrest phenotype, we analyzed the expression of Aurora B kinase in PGCs. Aurora B is a chromosome passenger protein required for spindle assembly checkpoint [46,47]. In normal mitosis, Aurora B first associates with the metaphase chromosomes, departs the chromosomes and translocates to the cleavage furrow at anaphase, becomes concentrated in the mid-body during cytokinesis, and finally disappears in G1. When we immunostained with antibodies against Aurora B, we found that this mitotic kinase formed foci in all mutant PGCs with abnormal nuclear morphologies, although the cells were no longer in mitosis as their chromosomes had already decondensed (Figure 5A). Western blot analysis confirmed the immunostaining result (Figure 5B). These data suggest that mutant PGCs adapt to the spindle assembly checkpoint but retain Aurora B expression. To determine if the retention of Aurora B expression is always associated with cells having undergone adaptation to spindle checkpoint, we treated a panel of cell lines with nocodazole, let the cells adapt, and analyzed the pattern of Aurora B expression. All cell lines tested, including HeLa, U2OS, and mouse embryonic fibroblasts, showed the same nuclear Aurora B staining pattern and abnormal nuclear morphologies as did mutant PGCs (Figure 5C, results from HeLa cells are shown). We conclude that the formation of nuclear Aurora B foci is a common feature of mammalian cells adapted to the spindle assembly checkpoint. However, at present, it is unclear if the retention of Aurora B expression is necessary for adaptation.

Bottom Line: We show that germ cells in the mutants are depleted during development.We further demonstrate that S1121A causes chromosome misalignment during proliferation of the postmigratory primordial germ cells, resulting in mitotic arrest, aneuploidy, and eventual cell death.Our results indicate that inhibitory phosphorylation of separase plays a critical role in the maintenance of sister chromatid cohesion and genome stability in proliferating postmigratory primordial germ cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Physiology and Biophysics, Baylor College of Medicine, Houston, Texas, United States of America.

ABSTRACT
Activity of separase, a cysteine protease that cleaves sister chromatid cohesin at the onset of anaphase, is tightly regulated to ensure faithful chromosome segregation and genome stability. Two mechanisms negatively regulate separase: inhibition by securin and phosphorylation on serine 1121. To gauge the physiological significance of the inhibitory phosphorylation, we created a mouse strain in which Ser1121 was mutated to Ala (S1121A). Here we report that this S1121A point mutation causes infertility in mice. We show that germ cells in the mutants are depleted during development. We further demonstrate that S1121A causes chromosome misalignment during proliferation of the postmigratory primordial germ cells, resulting in mitotic arrest, aneuploidy, and eventual cell death. Our results indicate that inhibitory phosphorylation of separase plays a critical role in the maintenance of sister chromatid cohesion and genome stability in proliferating postmigratory primordial germ cells.

Show MeSH
Related in: MedlinePlus