| Budget Amount *help |
¥4,110,000 (Direct Cost: ¥3,600,000、Indirect Cost: ¥510,000)
Fiscal Year 2007: ¥2,210,000 (Direct Cost: ¥1,700,000、Indirect Cost: ¥510,000)
Fiscal Year 2006: ¥1,900,000 (Direct Cost: ¥1,900,000)
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| Research Abstract |
ADF/cofilin family of actin-regulatory proteins is a phosphorylation-regulated protein essential for actin filament dynamics in cells. In this project, the regulatory mechanism of Xenopus ADF/cofilin (XAC) during gastrulation was examined. We found a correlation between mobile abilities of blastomeres and phospho-regulation of XAC to examine the change in the amount of phosphorylated XAC in each blastoderm during gastrulation. Each blastoderm increased the phosphorylated form of XAC at the beginning of gastrulation, and in particular, mesodermal cells markedly increased its amount during gastrulation and this was further confirmed using activin-treated animal cap explants.
To examine the regulatory mechanisms of XAC by phospholylation, we injected mRNAs of delta-A-XAC (phosphorylatable but deficient in actin-binding activity, thus functions as a dominantly negative mutant in the phosphorylation pathway) or a phophatase-defective mutant of Xenopus slingshot (XSSH, an XAC-specific phospha
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tase) into Xenopus embryos. Both injections resulted in defects in gastrulation, and often generated spina bifida phenotype. The ectodermal epiboly, mesodermal convergent extension and endodermal movement were all inhibited. These results strongly suggest the significance of phospho-regulation of XAC during gastrulation movements in each blastoderm.
Finally, we found four phosphorylation sites in the tail domain of XSSH, one in the N-terminal region, two in the middle region, and one in the C-terminal region of its domain. Since XSSH phosphatase activity was not affected by phosphorylation, its phosphorylation may regulate intracellular localization of XSSH in moving blastomeres. Overexpression of the tail domain in embryos lead to severe defects in gastrulation and this ectopically expressed domain was significantly phosphorylated, suggesting that this domain functions as a dominantly negative form. Thus, the regulation of XSSH by phosphorylation is also required for Xenopus gastrulation movements. Less
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