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Wee1B, Myt1, and Cdc25 function in distinct compartments of the mouse oocyte to control meiotic resumption.

Oh JS, Han SJ, Conti M - J. Cell Biol. (2010)

Bottom Line: These movements are regulated by PKA inactivation and MPF activation, respectively.Mislocalized Wee1B or Myt1 is not able to maintain meiotic arrest.Thus, cooperation of Wee1B, Myt1, and Cdc25 is required to maintain meiotic arrest and relocation of these components before GVBD is necessary for meiotic reentry.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Obstetrics, Gynecology, and Reproductive Sciences, Center for Reproductive Sciences, University of California, San Francisco, San Francisco, CA 94143, USA.

ABSTRACT
After a long period of quiescence at dictyate prophase I, termed the germinal vesicle (GV) stage, mammalian oocytes reenter meiosis by activating the Cdc2-cyclin B complex (maturation-promoting factor [MPF]). The activity of MPF is regulated by Wee1/Myt1 kinases and Cdc25 phosphatases. In this study, we demonstrate that the sequestration of components that regulate MPF activity in distinct subcellular compartments is essential for their function during meiosis. Down-regulation of either Wee1B or Myt1 causes partial meiotic resumption, and oocytes reenter the cell cycle only when both proteins are down-regulated. Shortly before GV breakdown (GVBD), Cdc25B is translocated from the cytoplasm to the nucleus, whereas Wee1B is exported from the nucleus to the cytoplasm. These movements are regulated by PKA inactivation and MPF activation, respectively. Mislocalized Wee1B or Myt1 is not able to maintain meiotic arrest. Thus, cooperation of Wee1B, Myt1, and Cdc25 is required to maintain meiotic arrest and relocation of these components before GVBD is necessary for meiotic reentry.

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Localization and function of Wee1 kinases in meiotic arrest. (A and D) Oocytes were injected with the indicated Wee1B (A) or Myt1 (D) mRNAs, washed out of the inhibitor, and monitored for the absence of a GV (GVBD). These experiments were repeated three times, and the error bars indicate SEM. The expression levels of injected mRNAs were compared by immunoblotting with the GFP antibody. (B) The activity of Wee1B-targeting mutants was measured by H1 kinase assay. The data are representative of three independent experiments. The ratio of 32P incorporation into histone H1 and the amount of immunocomplexes were densitometrically measured and represented as a mean percentage ± SEM of control (Mock). (C) Localization of WT and NLS mutants of Myt1 was shown with brightfield and fluorescence images. Bar, 20 µm.
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fig5: Localization and function of Wee1 kinases in meiotic arrest. (A and D) Oocytes were injected with the indicated Wee1B (A) or Myt1 (D) mRNAs, washed out of the inhibitor, and monitored for the absence of a GV (GVBD). These experiments were repeated three times, and the error bars indicate SEM. The expression levels of injected mRNAs were compared by immunoblotting with the GFP antibody. (B) The activity of Wee1B-targeting mutants was measured by H1 kinase assay. The data are representative of three independent experiments. The ratio of 32P incorporation into histone H1 and the amount of immunocomplexes were densitometrically measured and represented as a mean percentage ± SEM of control (Mock). (C) Localization of WT and NLS mutants of Myt1 was shown with brightfield and fluorescence images. Bar, 20 µm.

Mentions: To investigate the functional significance of Wee1B export from the nucleus, we determined the biological activity of a cytoplasmic Wee1B. Active Wee1B-targeting mutants were microinjected into GV-arrested oocytes, and meiotic resumption was determined by scoring GVBD. Microinjection of nuclear Wee1B proteins (Wee1B-WT and Wee1B-NESmut) led to an efficient and complete block of meiotic resumption, whereas cytoplasmic Wee1B mutants (Wee1B-ΔNLS and Wee1B-ΔNLS/NESmut) failed to block maturation (Fig. 5 A). It is noteworthy that the level of protein expression was comparable for the different Wee1B mutants and that the activity of different Wee1B constructs measured by their effects on Cdc2-dependent histone H1 phosphorylation was not significantly different (Fig. 5 B). These results confirm that the inability of cytoplasmic Wee1B to block MPF activity is not simply caused by decreased activity associated with the disruption of the NLS or NES sequences but rather results from mislocalization of the mutant protein. Proper localization of Myt1 is also important for its function. Indeed, Myt1 chimeric proteins containing a C-terminal NLS sequence from SV40 large T antigen (Myt1-NLS) were designed, and appropriate nuclear expression of this Myt1 mutant by microinjection in GV oocytes was verified (Fig. 5 C). Microinjection of Myt1-NLS blocked GVBD less efficiently than WT (Myt1-WT; Fig. 5 D).


Wee1B, Myt1, and Cdc25 function in distinct compartments of the mouse oocyte to control meiotic resumption.

Oh JS, Han SJ, Conti M - J. Cell Biol. (2010)

Localization and function of Wee1 kinases in meiotic arrest. (A and D) Oocytes were injected with the indicated Wee1B (A) or Myt1 (D) mRNAs, washed out of the inhibitor, and monitored for the absence of a GV (GVBD). These experiments were repeated three times, and the error bars indicate SEM. The expression levels of injected mRNAs were compared by immunoblotting with the GFP antibody. (B) The activity of Wee1B-targeting mutants was measured by H1 kinase assay. The data are representative of three independent experiments. The ratio of 32P incorporation into histone H1 and the amount of immunocomplexes were densitometrically measured and represented as a mean percentage ± SEM of control (Mock). (C) Localization of WT and NLS mutants of Myt1 was shown with brightfield and fluorescence images. Bar, 20 µm.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2812522&req=5

fig5: Localization and function of Wee1 kinases in meiotic arrest. (A and D) Oocytes were injected with the indicated Wee1B (A) or Myt1 (D) mRNAs, washed out of the inhibitor, and monitored for the absence of a GV (GVBD). These experiments were repeated three times, and the error bars indicate SEM. The expression levels of injected mRNAs were compared by immunoblotting with the GFP antibody. (B) The activity of Wee1B-targeting mutants was measured by H1 kinase assay. The data are representative of three independent experiments. The ratio of 32P incorporation into histone H1 and the amount of immunocomplexes were densitometrically measured and represented as a mean percentage ± SEM of control (Mock). (C) Localization of WT and NLS mutants of Myt1 was shown with brightfield and fluorescence images. Bar, 20 µm.
Mentions: To investigate the functional significance of Wee1B export from the nucleus, we determined the biological activity of a cytoplasmic Wee1B. Active Wee1B-targeting mutants were microinjected into GV-arrested oocytes, and meiotic resumption was determined by scoring GVBD. Microinjection of nuclear Wee1B proteins (Wee1B-WT and Wee1B-NESmut) led to an efficient and complete block of meiotic resumption, whereas cytoplasmic Wee1B mutants (Wee1B-ΔNLS and Wee1B-ΔNLS/NESmut) failed to block maturation (Fig. 5 A). It is noteworthy that the level of protein expression was comparable for the different Wee1B mutants and that the activity of different Wee1B constructs measured by their effects on Cdc2-dependent histone H1 phosphorylation was not significantly different (Fig. 5 B). These results confirm that the inability of cytoplasmic Wee1B to block MPF activity is not simply caused by decreased activity associated with the disruption of the NLS or NES sequences but rather results from mislocalization of the mutant protein. Proper localization of Myt1 is also important for its function. Indeed, Myt1 chimeric proteins containing a C-terminal NLS sequence from SV40 large T antigen (Myt1-NLS) were designed, and appropriate nuclear expression of this Myt1 mutant by microinjection in GV oocytes was verified (Fig. 5 C). Microinjection of Myt1-NLS blocked GVBD less efficiently than WT (Myt1-WT; Fig. 5 D).

Bottom Line: These movements are regulated by PKA inactivation and MPF activation, respectively.Mislocalized Wee1B or Myt1 is not able to maintain meiotic arrest.Thus, cooperation of Wee1B, Myt1, and Cdc25 is required to maintain meiotic arrest and relocation of these components before GVBD is necessary for meiotic reentry.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Obstetrics, Gynecology, and Reproductive Sciences, Center for Reproductive Sciences, University of California, San Francisco, San Francisco, CA 94143, USA.

ABSTRACT
After a long period of quiescence at dictyate prophase I, termed the germinal vesicle (GV) stage, mammalian oocytes reenter meiosis by activating the Cdc2-cyclin B complex (maturation-promoting factor [MPF]). The activity of MPF is regulated by Wee1/Myt1 kinases and Cdc25 phosphatases. In this study, we demonstrate that the sequestration of components that regulate MPF activity in distinct subcellular compartments is essential for their function during meiosis. Down-regulation of either Wee1B or Myt1 causes partial meiotic resumption, and oocytes reenter the cell cycle only when both proteins are down-regulated. Shortly before GV breakdown (GVBD), Cdc25B is translocated from the cytoplasm to the nucleus, whereas Wee1B is exported from the nucleus to the cytoplasm. These movements are regulated by PKA inactivation and MPF activation, respectively. Mislocalized Wee1B or Myt1 is not able to maintain meiotic arrest. Thus, cooperation of Wee1B, Myt1, and Cdc25 is required to maintain meiotic arrest and relocation of these components before GVBD is necessary for meiotic reentry.

Show MeSH
Related in: MedlinePlus