Molecular mechanism of phospho-regulation of cofilin, an actin-regulatory protein, in the gastrulation movement

ADF/cofilin家族肌动蛋白调节蛋白是一种磷酸化调节蛋白，对细胞内肌动蛋白丝动力学至关重要。在这个项目中，非洲爪蟾ADF/cofilin（XAC）在原肠胚形成过程中的调节机制进行了研究。我们发现了卵裂球的移动的能力和XAC的磷酸化调节之间的相关性，以检查在原肠胚形成期间每个胚盘中磷酸化XAC的量的变化。每个胚盘在原肠胚形成开始时增加XAC的磷酸化形式，特别是中胚层细胞在原肠胚形成期间显著增加其量，并且使用激活素处理的动物帽外植体进一步证实。（可磷酸化但缺乏肌动蛋白结合活性，因此在磷酸化途径中起显性负突变体的作用）或非洲爪蟾弹弓的磷酸酶缺陷型突变体（XSSH，XAC特异性磷酸酶 关于我们 tase）注入非洲爪蟾胚胎中。两种注射都导致原肠胚形成缺陷，并且经常产生脊柱裂表型。外胚叶外生、中胚叶会聚延伸和内胚叶运动均受到抑制。最后，我们在XSSH的尾部结构域中发现了4个磷酸化位点，一个位于N端区域，两个位于中间区域，一个位于C端区域。由于XSSH磷酸酶活性不受磷酸化的影响，其磷酸化可能调节XSSH在运动卵裂球中的细胞内定位。尾部结构域在胚胎中的过度表达导致原肠胚形成的严重缺陷，并且该异位表达的结构域被显著磷酸化，表明该结构域作为显性负性形式起作用。因此，通过磷酸化调节XSSH也是非洲爪蟾原肠胚形成运动所必需的。少

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 … More 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