Regulation of actin during cell migration

描述（由申请人提供）：细胞迁移驱动诸如组织形态发生、免疫反应和癌症转移等关键生物学事件。我们最近的数据令人惊讶地表明，在迁移过程中，细胞前缘的形成和功能严重依赖于精氨酸化，这是一种相对未被探索的翻译后修饰。此外，在基于细胞的表型拯救实验中，我们已经表明，在缺乏精氨酸化酶ATE1的小鼠胚胎成纤维细胞中，实验中精氨酸化的-肌动蛋白可以在很大程度上恢复细胞前沿功能。在我们正在进行的研究中，我们确定了-肌动蛋白的n端精氨酸化如何促进板足的形成和定向细胞迁移，我们已经做出了新的观察，即在迁移过程中，精氨酸化的-肌动蛋白的一个突出亚群被靶向到细胞前沿。此外，我们的数据表明-肌动蛋白精氨酸化是由其mRNA序列而不是其蛋白质结构选择性调节的。在氨基酸水平上与β肌动蛋白99%相同的γ肌动蛋白，其mRNA序列差异为13%。我们已经证明，这种差异是翻译速度更快的直接原因

DESCRIPTION (provided by applicant): Cell migration drives such key biological events as tissue morphogenesis, immune response, and cancer metastases. Our recent data surprisingly show that the formation and function of the cell leading edge during migration critically depends on arginylation, a relatively unexplored posttranslational modification. Moreover, in cell-based phenotype rescue experiments, we have shown that experimentally arginylated beta actin can largely restore cell leading edge function in mouse embryonic fibroblasts lacking the arginylation enzyme ATE1. In our ongoing studies to determine how N-terminal arginylation of beta actin contributes to lamellipodia formation and directed cell migration, we have made the novel observations that a prominent subset of arginylated beta actin is targeted to the cell leading edge during migration. Moreover, our data demonstrate that beta actin arginylation is selectively regulated by its mRNA sequence rather than its protein structure. In support, gamma actin, which is 99% identical to beta actin at the amino acid level, differs by 13% in its mRNA sequence. We have shown that this difference is directly responsible for faster translation rate of beta actin and leads to its selective arginylation through a novel mechanism coupled to protein ubiquitination. It is also known that zipcode-mediated beta actin mRNA targeting regulates its leading edge localization and, like arginylation, is essential for directional cell migration. We hypothesize that mRNA-mediated regulation of N-terminal arginylation of beta actin uniquely regulates actin function during cell migration by facilitating actin polymerization at the cell leading edge. In this proposal, we will test this hypothesis through three specific aims that will: (1) test the hypothesis that beta actin arginylation facilitates actin polymerization at the cell leading edge; (2) test the hypothesis that beta actin function is uniquely regulated by coding and noncoding regions of its mRNA; and (3) test the hypothesis that this regulation is coupled to modulation of intracellular arginylation activity during cell migration. These experiments will address a novel regulatory mechanism controlling cell polarization and motility through modulating actin's properties and mRNA structure and will ultimately enable a new level of targeted functional studies of cell migration during essential physiological events.