? 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. ? ?
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