This proposal will focus on determinants localizing actin mRNA to the leading edge of fibroblasts, where it is proposed to be important for cell motility. Starting from the PI s recent discoveries that there are sequences within the 3' untranslated region of beta-actin mRNA that localize it to the leading edge and the identification of a protein that binds specifically to this domain (termed a zip code protein ZBP), a series of experiments are now proposed to test directly whether ZBP is a trans-acting factor essential for this specific mRNA localization. ZBP function will be tested by expressing truncated proteins as dominant inhibitors of actin RNA localization, through the use of sense and anti-sense inhibition of actin RNA localization, and through microinjection of antibodies against ZBP. To test whether beta actin RNA localization leads to a preferential enrichment of beta actin at the leading edge, the distribution of tagged beta actin (either GFP or epitope-tagged) will be monitored simultaneously with the mRNA distribution. To test whether the beta actin 3' untranslated region or localized actin RNA itself is important for determining cell polarity, an attempt will be made to isolate a cell line in which both BETA actin alleles have been disrupted. If this can be produced, then experiments will be undertaken to identify what portion of the beta actin mRNA is necessary to restore cell polarity with an initial focus on whether it is the beta isoform itself or sequences within the 3' untranslated region of it mRNA.
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