Arp2/3 mRNA localization and directional cell migration

描述（由申请人提供）： 定向细胞迁移是成功伤口愈合的关键方面。损伤后不久，细胞进入伤口床并在组织重塑中发挥关键作用。广泛的研究提供了关于细胞外基质环境如何变化和成纤维细胞迁移到伤口床中的“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伤口床。为了定向运动，细胞必须变得不对称，并建立一个主导的领先突起，其中局部肌动蛋白聚合是一个标志。Arp 2/3复合物是一种由7种蛋白质组成的肌动蛋白聚合成核剂，定位于培养细胞的前板层。我们已经证明Arp 2/3复合物定位于培养的细胞和愈合的伤口中。Arp 2/3蛋白复合物的定位与其促进高度分支的肌动蛋白网络形成以用于突起的功能一致。然而，Arp 2/3蛋白复合物如何在迁移细胞中靶向是未知的。我们已经证明，所有七个亚基的Arp 2/3复合物的mRNA定位在细胞领先的突起。这是第一个证明蛋白质复合物的mRNA共定位于共同的功能位点的证据。由于mRNA的不对称分布是通过定位蛋白质合成将蛋白质靶向功能位点的重要机制，因此这一发现表明Arp 2/3蛋白复合物通过mRNA定位靶向于前导突起。我们假设Arp 2/3 mRNA定位是将Arp 2/3蛋白靶向于主要突起从而控制定向细胞迁移的机制。为了验证这一假设，我们将鉴定Arp 2（复合物的关键成员）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Arp 2 mRNA的定位：1）使用反义寡核苷酸阻断zipcode; 2）创建具有非定位3 '-UTR的Arp 2 mRNA嵌合体; 3）遗传突变或删除zipcode。为了测试Arp 2/3定位在体外和体内定向细胞迁移中的重要性，我们将研究离域Arp 2 mRNA对组织培养和愈合伤口中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，我们的实验将为调节纤维化和肿瘤转移等致病环境中细胞迁移的疗法提供新的基础。

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.