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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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Securin Is Not Required for Germline Development(A) Early embryonic lethality in mice lacking both securin and the inhibitory phosphorylation of separase. Embryos shown were at 10.5 dpc. The double mutant was much smaller than the control and was retarded in development.(B) No depletion of PGCs is caused by the lack of securin. Genital ridges from 12.5 dpc embryos were whole-mount stained for AP activity.(C) Expression of securin in different stages of the male germ cell development. Sections of both fetal and postnatal testes were immunostained for securin. Testis from a 3-wk-old securin–/– mouse was included as a negative control.(D) Western blotting analysis of securin levels in fetal (13.5 dpc) and postnatal testes.(E) Western blotting analysis of separase and securin levels in 13.5 dpc embryos.
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pbio-0060015-g006: Securin Is Not Required for Germline Development(A) Early embryonic lethality in mice lacking both securin and the inhibitory phosphorylation of separase. Embryos shown were at 10.5 dpc. The double mutant was much smaller than the control and was retarded in development.(B) No depletion of PGCs is caused by the lack of securin. Genital ridges from 12.5 dpc embryos were whole-mount stained for AP activity.(C) Expression of securin in different stages of the male germ cell development. Sections of both fetal and postnatal testes were immunostained for securin. Testis from a 3-wk-old securin–/– mouse was included as a negative control.(D) Western blotting analysis of securin levels in fetal (13.5 dpc) and postnatal testes.(E) Western blotting analysis of separase and securin levels in 13.5 dpc embryos.

Mentions: The above analyses demonstrate that the inhibitory phosphorylation of separase plays a critical role in the maintenance of male embryonic germ cell genome stability by preventing premature separation of sister chromatids. Besides sterility, mice carrying one S1121A separase allele are essentially normal, suggesting redundancy between inhibitory phosphorylation and securin in the regulation of separase in somatic cells. Indeed, elimination of both inhibitory mechanisms causes early lethality (Figure 6A). However, inhibitory phosphorylation is uniquely required for germline development, since lack of securin causes no appreciable depletion of germ cells (Figure 6B) nor infertility [16,19]. The question that remains open is the origin of the germline vulnerability to the loss of the inhibitory phosphorylation of separase. One possibility is that post-migratory germ cells express relatively low levels of securin so that inhibitory phosphorylation is the primary mechanism that inhibits separase in these cells. To test this possibility, we examined the expression of securin in genital ridges. Immunostaining analysis showed that securin levels were undetected in both germ cells and somatic cells (Figure 6C). However, securin was readily detected in postnatal testes (Figure 6C). In agreement with these results, western blotting showed much lower levels of securin expression in the fetal testes than in the postnatal testes (Figure 6D). Furthermore, securin levels in genital ridges were lower than those observed in whole embryos or embryo heads, whereas the level of separase expression was similar in fetal genital ridges and other parts of the body (Figure 6E). Since somatic cells in separase point mutant genital ridges appear normal and do not display any mitotic defects, these data suggest that the stoichiometric ratio of securin to separase in postmigratory germ cells is lower compared with somatic cells, and these cells must rely completely on the inhibitory phosphorylation of separase to prevent premature activation of the protease.


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)

Securin Is Not Required for Germline Development(A) Early embryonic lethality in mice lacking both securin and the inhibitory phosphorylation of separase. Embryos shown were at 10.5 dpc. The double mutant was much smaller than the control and was retarded in development.(B) No depletion of PGCs is caused by the lack of securin. Genital ridges from 12.5 dpc embryos were whole-mount stained for AP activity.(C) Expression of securin in different stages of the male germ cell development. Sections of both fetal and postnatal testes were immunostained for securin. Testis from a 3-wk-old securin–/– mouse was included as a negative control.(D) Western blotting analysis of securin levels in fetal (13.5 dpc) and postnatal testes.(E) Western blotting analysis of separase and securin levels in 13.5 dpc embryos.
© Copyright Policy
Related In: Results  -  Collection

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

pbio-0060015-g006: Securin Is Not Required for Germline Development(A) Early embryonic lethality in mice lacking both securin and the inhibitory phosphorylation of separase. Embryos shown were at 10.5 dpc. The double mutant was much smaller than the control and was retarded in development.(B) No depletion of PGCs is caused by the lack of securin. Genital ridges from 12.5 dpc embryos were whole-mount stained for AP activity.(C) Expression of securin in different stages of the male germ cell development. Sections of both fetal and postnatal testes were immunostained for securin. Testis from a 3-wk-old securin–/– mouse was included as a negative control.(D) Western blotting analysis of securin levels in fetal (13.5 dpc) and postnatal testes.(E) Western blotting analysis of separase and securin levels in 13.5 dpc embryos.
Mentions: The above analyses demonstrate that the inhibitory phosphorylation of separase plays a critical role in the maintenance of male embryonic germ cell genome stability by preventing premature separation of sister chromatids. Besides sterility, mice carrying one S1121A separase allele are essentially normal, suggesting redundancy between inhibitory phosphorylation and securin in the regulation of separase in somatic cells. Indeed, elimination of both inhibitory mechanisms causes early lethality (Figure 6A). However, inhibitory phosphorylation is uniquely required for germline development, since lack of securin causes no appreciable depletion of germ cells (Figure 6B) nor infertility [16,19]. The question that remains open is the origin of the germline vulnerability to the loss of the inhibitory phosphorylation of separase. One possibility is that post-migratory germ cells express relatively low levels of securin so that inhibitory phosphorylation is the primary mechanism that inhibits separase in these cells. To test this possibility, we examined the expression of securin in genital ridges. Immunostaining analysis showed that securin levels were undetected in both germ cells and somatic cells (Figure 6C). However, securin was readily detected in postnatal testes (Figure 6C). In agreement with these results, western blotting showed much lower levels of securin expression in the fetal testes than in the postnatal testes (Figure 6D). Furthermore, securin levels in genital ridges were lower than those observed in whole embryos or embryo heads, whereas the level of separase expression was similar in fetal genital ridges and other parts of the body (Figure 6E). Since somatic cells in separase point mutant genital ridges appear normal and do not display any mitotic defects, these data suggest that the stoichiometric ratio of securin to separase in postmigratory germ cells is lower compared with somatic cells, and these cells must rely completely on the inhibitory phosphorylation of separase to prevent premature activation of the protease.

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