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

肌动蛋白调节蛋白ADF/cofilin家族是一种磷酸化调节蛋白，对细胞内肌动蛋白细丝的动态变化是必不可少的。在本项目中，研究了非洲爪哇ADF/cofilin(XAC)在原肠发育过程中的调节机制。我们发现卵裂球的运动能力与XAC的磷酸化调节之间存在相关性，以检测原肠发育过程中每个胚芽中XAC磷酸化的数量的变化。为了研究Xac的磷酸化调节机制，我们注入了DeltaA-A-Xac的…(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主导作用的负突变体)或非洲爪哇磷酸酶缺陷突变体(XSSH，一种Xac特异的磷酸…更多的Tase)进入非洲爪哇胚胎。两次注射都导致原肠发育缺陷，并经常产生脊柱裂表型。外胚层表皮、中胚层收敛延伸和内胚层运动均受到抑制。最后，我们在XSSH的尾部区域发现了4个磷酸化位点，1个在其结构域的N-末端，2个在中间区域，1个在其结构域的C-末端。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在运动的卵裂球中的细胞内定位。Tail结构域在胚胎中的过度表达会导致原肠发育的严重缺陷，并且这个异位表达的结构域被显著地磷酸化，这表明该结构域的功能主要是负的。因此，通过磷酸化来调节XSSH也是非洲爪哇原肠运动所必需的。较少

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