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Chromatin Immunoprecipitation Assay for Tissue-specific Genes using Early-stage Mouse Embryos
Published on: April 29, 2011
Characterization of p70 S6 kinase 1 in early development of mouse embryos
Xiao-Yan Xu1, Zhe Zhang, Wen-Hui Su
1Department of Pathophysiology, College of Basic Medicine, China Medical University, Shenyang, Liaoning Province, PR China.
Abstract:
The mTOR kinase controls cell growth, proliferation, and survival through two distinct multiprotein complexes mTORC1 and mTORC2. p70 S6 Kinase 1 (S6K1) is characterized as downstream effector of mTOR. Until recently, the connection between S6K1 and mTORC1 /mTORC2 during the early development of mouse embryos has not been well elucidated. Here, the expression level of total S6K1 and its phosphorylation at Thr389 was determined in four phases of one-cell embryos. S6K1 was active throughout the cell cycle especially with higher activity in G2 and M phases. Rapamycin decreased the activity of M-phase promoting factor (MPF) and delayed the first mitotic cleavage. Down-regulating mTOR and raptor reduced S6K1 phosphorylation at Thr389 in one-cell embryos. Furthermore, rapamycin and microinjection of raptor shRNA decreased the immunofluorescent staining of Thr389 phospho-S6K1. It is proposed that mTORC1 may be involved in the control of MPF by regulating S6K1 during the early development of mouse embryos.
Insights
Mammalian target of rapamycin (mTOR) signaling, specifically mTORC1, regulates early mouse embryo development by influencing S6 Kinase 1 (S6K1) activity. This pathway impacts cell cycle progression and mitotic cleavage.
Area of Science:
- Molecular and Cellular Biology
- Developmental Biology
- Biochemistry
Background:
- The mechanistic target of rapamycin (mTOR) kinase is crucial for cell growth, proliferation, and survival, operating via mTORC1 and mTORC2 complexes.
- p70 S6 Kinase 1 (S6K1), a downstream effector of mTOR, plays a significant role in cellular processes.
- The precise role of S6K1 and its relationship with mTORC1/mTORC2 during early mouse embryogenesis remained poorly understood.
Purpose of the Study:
- To investigate the expression and activity of S6K1 in early mouse embryos.
- To elucidate the involvement of mTORC1 signaling in regulating S6K1 during the initial stages of mouse development.
- To determine the impact of mTORC1 inhibition on cell cycle progression and mitotic events in one-cell mouse embryos.
Main Methods:
- Quantification of total S6K1 and its phosphorylation at Thr389 in different phases of one-cell mouse embryos.
- Treatment with rapamycin to inhibit mTOR activity and assess its effects on M-phase promoting factor (MPF) and mitotic cleavage.
- Genetic manipulation using siRNA to down-regulate mTOR and raptor, followed by immunofluorescence staining for phospho-S6K1 (Thr389).
Main Results:
- S6K1 exhibited activity throughout the cell cycle, with heightened activity observed during the G2 and M phases.
- Rapamycin treatment led to decreased MPF activity and delayed the first mitotic cleavage.
- Down-regulation of mTOR and raptor significantly reduced S6K1 phosphorylation at Thr389, and rapamycin/raptor shRNA decreased phospho-S6K1 immunofluorescence.
Conclusions:
- mTORC1 signaling, through the regulation of S6K1, appears to play a critical role in controlling MPF activity.
- This regulation by mTORC1 and S6K1 is important for the progression of early mouse embryonic development.
- The findings suggest a novel mechanism by which mTORC1 influences cell cycle control during embryogenesis.
