Regulation of actin during cell migration

描述(申请人提供)：细胞迁移驱动关键生物学事件，如组织形态发生、免疫反应和癌症转移。我们最近的数据令人惊讶地表明，细胞迁移过程中前沿的形成和功能关键取决于精氨酸化，这是一种相对未被探索的翻译后修饰。此外，在基于细胞的表型挽救实验中，我们已经证明，实验中精氨酸化的β-肌动蛋白可以在很大程度上恢复缺乏精氨酸化酶ATE1的小鼠胚胎成纤维细胞的细胞前沿功能。在我们正在进行的研究中，为了确定β肌动蛋白的N末端精氨酸化如何促进板脂形成和定向细胞迁移，我们进行了新的观察，发现精氨酸化的β肌动蛋白的一个重要子集在迁移过程中针对细胞前沿。此外，我们的数据表明，β-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地调节的。在支持方面，在氨基酸水平上与β肌动蛋白99%相同的伽马肌动蛋白，在其mRNA序列上有13%的不同。我们已经证明，这种差异直接导致了更快的翻译速度 β-肌动蛋白通过与蛋白质泛素化耦合的新机制导致其选择性精氨酸化。众所周知，Zipcode介导的β-肌动蛋白mRNA靶向调节其前沿定位，与精氨酸化一样，对于定向细胞迁移是必不可少的。我们假设，在细胞迁移过程中，通过促进细胞前沿的肌动蛋白聚合，mRNA介导的β-肌动蛋白N末端精氨酸化的调节是唯一调节肌动蛋白功能的途径。在本提案中，我们将通过三个具体目标来检验这一假设，这三个目标将： (1)检验β-肌动蛋白精氨酸化促进细胞前沿肌动蛋白聚合的假说；(2)检验β-肌动蛋白功能受其mRNA编码区和非编码区唯一调控的假说；(3)检验这种调节与细胞迁移过程中细胞内精氨酸化活性调控相耦合的假说。这些实验将解决一种新的调控机制，通过调节肌动蛋白的性质和mRNA结构来控制细胞的极化和运动，并最终实现对细胞在基本生理事件中迁移的靶向功能研究的新水平。

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.