Phosphorylation of Claspin is triggered by the nucleocytoplasmic ratio at the Xenopus laevis midblastula transition.
At the Xenopus midblastula transition (MBT), cell cycles lengthen, and checkpoints that respond to damaged or unreplicated DNA are established. The MBT is triggered by a critical nucleocytoplasmic (N/C) ratio; however, the molecular basis for its initiation remains unknown. In egg extracts, activation of Chk1 checkpoint kinase requires the adaptor protein Claspin, which recruits Chk1 for phosphorylation by ATR. At the MBT in embryos, Chk1 is transiently activated to lengthen the cell cycle. We show that Xenopus Claspin is phosphorylated at the MBT at both DNA replication checkpoint-dependent and -independent sites. Further, in egg extracts, Claspin phosphorylation depends on a threshold N/C ratio, but occurs even when ATR is inhibited. Not all phosphorylation that occurs at the MBT is reproduced in egg extracts. Our results identify Claspin as the most upstream molecule in the signaling pathway that responds to the N/C ratio and indicate that Claspin may also respond to an independent timer to trigger the MBT and activation of cell cycle checkpoints.
PubMed ID: 21396931
Article link: Dev Biol.
Grant support: GM076112 NIGMS NIH HHS
Genes referenced: actl6a atr cdk1 chek1 clspn
Antibodies referenced: Clspn Ab1 Clspn Ab2
Article Images: [+] show captions
|Fig. 1. Claspin is phosphorylated at theMBT. (A) Temporal expression of ClaspinmRNA. RT-PCRwas performed on RNA isolated frommature oocytes and stagedembryos (Nieuwkoop and Faber, 1956) using primers specific for Claspin and actin as a control. (B) Spatial expression of Claspin protein. Whole-mount immunohistochemistry was performed with embryos at indicated stages using anti-Claspin serum(a) and preimmune serumas a control (f).An and Veg denote a viewfromanimal and vegetal poles, respectively.Unlabeled views are from the animal pole. (C) Temporal expression of Claspin protein. Western blotting with anti-Claspin, anti-phospho Chk1, anti-Cdk1 or anti-phospho Cdk1 (Y15) antibodies was performed on extracts from mature oocytes and staged embryos (first row), or in amore detailed time course, fromearly blastula (stage 7) to early gastrula (stage 10) embryos at indicated times after fertilization (second through fifth rows). Proteins equivalent to those fromhalf an embryo were analyzed. (D) Fertilized eggs (stage 1) and late blastula embryos (stage 9) were collected. Someembryoswere treatedwith hydroxyurea (HU) fromthe 2-cell stage andwere collected at the late blastula stage. The embryo extractswere subject to immunoprecipitationwith an anti-Claspin antiserum. Immunoprecipitates were treated without (−) or with (+) lambda phosphatase. Embryo extracts (left panel) and immunoprecipitates (right panel) were subjected toWestern blotting with an anti-Claspin antibody. -=unphosphorylated Claspin, and P=phosphorylated Claspin.|
|clspn (claspin) gene expression in Xenopus laevis embryos, NF stage 7, as assayed by immunohistochemistry. Animal view|
|clspn (claspin) gene expression in Xenopus laevis embryos, NF stage 8, as assayed by immunohistochemistry. Animal view.|
|clspn (claspin) gene expression in Xenopus laevis embryos, NF stage 9, as assayed by immunohistochemistry. Animal view.|
|clspn (claspin) gene expression in Xenopus laevis embryos, NF stage 7, as assayed by immunohistochemistry. Animal (An) and Vegetal (Veg) views.|