Arp2/3 mRNA localization and directional cell migration

描述(由申请人提供)： 细胞的定向迁移是创面成功愈合的关键。损伤后不久，细胞进入创面床，在组织重塑中发挥关键作用。广泛的研究已经提供了关于细胞外基质环境如何变化以及成纤维细胞迁移到创面床的“先决条件”的有价值的信息。然而，目前尚不清楚细胞内机制如何调节细胞定向迁移到创面床。为了定向移动，细胞必须变得不对称，并建立一个主导的突起，其中局部肌动蛋白聚合是一个标志。Arp2/3复合体是一种肌动蛋白聚合核因子，由7种蛋白质组成，定位于培养细胞的前板。我们已经证明，Arp2/3复合体定位于培养的细胞和愈合的伤口中。Arp2/3蛋白复合体的定位与其促进形成高度分支的肌动蛋白网络以促进突起的功能一致。然而，目前尚不清楚Arp2/3蛋白复合体是如何在迁移细胞中靶向的。我们已经证明，Arp2/3复合体的所有七个亚基的mRNAs都定位于细胞引导的突起。这是第一个证明蛋白质复合体的mRNAs共定位于一个共同的功能位点的证据。由于mRNA的不对称分布是通过局部蛋白质合成将蛋白质靶向功能部位的重要机制，这一发现表明Arp2/3蛋白复合体通过mRNA定位靶向领先的突起。我们假设Arp2/3mRNA定位是一种将Arp2/3蛋白定位到主要突起从而控制细胞定向迁移的机制。为了验证这一假设，我们将确定Arp2(复合体的关键成员)中的mRNA定位信号序列(邮政编码)。我们将使用三种不同的方法来破坏Arp2 mRNA的定位：1)使用反义寡核苷酸来阻断邮政编码；2)用非定位的3‘-UTR创建Arp2 mRNA嵌合体；以及3)基因突变或缺失邮政编码。为了测试Arp2/3定位在体外和体内定向细胞迁移中的重要性，我们将研究去定位Arp2 mRNA在组织培养和伤口愈合中对成纤维细胞迁移行为的影响。通过阐明细胞调节其定向运动的基本机制，我们的实验将为治疗在纤维化和肿瘤转移等致病环境中调节细胞迁移提供新的基础。

DESCRIPTION (provided by applicant): Directional cell migration is a critical aspect of successful wound healing. Soon after injury, cells move into the wound bed and play key roles in tissue remodeling. Extensive studies have provided valuable information about how the extracellular matrix environment changes and "pre-conditions" fibroblasts migrating into the wound bed. However, it is poorly understood how the intracellular mechanisms regulate the cells migrating directionally to the wound bed. To move directionally, a cell must become asymmetric and establish a dominant leading protrusion in which localized actin polymerization is a hallmark. The Arp2/3 complex, an actin polymerization nucleator consisting of seven proteins, is localized to the leading lamella in cultured cells. We have demonstrated that the Arp2/3 complex is localized in cells in culture and in healing wounds. The localization of the Arp2/3 protein complex is consistent with its function to promote formation of highly branched actin network for protrusion. However, it is unknown how the Arp2/3 protein complex is targeted in migrating cells. We have demonstrated that mRNAs for all the seven subunits of the Arp2/3 complex are localized at cell leading protrusion. This is the first evidence that mRNAs for a protein complex are co-localized to a common site of function. Because asymmetric distribution of mRNA is an important mechanism to target protein to the site of function through localized protein synthesis, this finding suggests that the Arp2/3 protein complex is targeted to the leading protrusion by mRNA localization. We hypothesize that Arp2/3 mRNA localization is a mechanism to target the Arp2/3 protein to the leading protrusion thereby governing directional cell migration. To test this hypothesis, we will identify the mRNA localization signaling sequence (the zipcode) in Arp2 (a key member of the complex). We will use three different approaches to disrupt the localization of Arp2 mRNA : 1) using antisense oligonucleotides to block the zipcode; 2) creating Arp2 mRNA chimera with non-localizing 3'-UTR; and 3) genetically mutating or deleting the zipcode. To test the importance of Arp2/3 localization in directional cell migration in vitro and in vivo, we will study the impact of delocalizing Arp2 mRNA on fibroblast migration behavior in tissue culture and in healing wounds. By elucidating the fundamental mechanisms with which cells regulate their directional motility, our experiments will provide a new basis for therapies to modulate cell migration in pathogenic settings such as fibrosis and tumor metastasis.