Regulation of actin during cell migration

描述（由申请人提供）：细胞迁移驱动组织形态发生、免疫应答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，在迁移过程中的细胞前缘的形成和功能，关键取决于乙酰化，一个相对未开发的翻译后修饰。此外，在基于细胞的表型拯救实验中，我们已经表明，实验性β-肌动蛋白可以在很大程度上恢复缺乏β-肌动蛋白化酶ATE 1的小鼠胚胎成纤维细胞的细胞前缘功能。在我们正在进行的研究，以确定如何N-末端的β肌动蛋白的β-actin有助于板状伪足的形成和定向细胞迁移，我们已经作出了新的观察，一个突出的亚组的β-actin是针对细胞的前沿迁移过程中。此外，我们的数据表明，β-肌动蛋白β化选择性地调节其mRNA序列，而不是其蛋白质结构。作为支持，γ肌动蛋白在氨基酸水平上与β肌动蛋白有99%的相同性，但在mRNA序列上有13%的差异。我们已经证明，这种差异是直接负责更快的翻译速度， β肌动蛋白，并导致其通过一种新的机制耦合到蛋白质泛素化的选择性去乙酰化。还已知zipcode介导的β肌动蛋白mRNA靶向调节其前沿定位，并且与β-淀粉样化一样，对于定向细胞迁移是必需的。我们假设，mRNA介导的调节β肌动蛋白的N-末端乙酰化独特地调节肌动蛋白功能，在细胞迁移过程中，通过促进肌动蛋白聚合在细胞的前沿。在本提案中，我们将通过三个具体目标来检验这一假设，这些目标将： (1)检验β-肌动蛋白β化促进细胞前沿肌动蛋白聚合的假设;（2）检验β-肌动蛋白功能受其mRNA编码区和非编码区独特调控的假设;（3）检验这种调控与细胞迁移过程中细胞内β-肌动蛋白β化活性的调节偶联的假设。这些实验将解决一种新的调节机制，通过调节肌动蛋白的特性和mRNA结构来控制细胞的极化和运动，并最终使在基本生理事件期间细胞迁移的靶向功能研究达到一个新的水平。

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.