Localized β-actin translation: coordinating cell-cell contact formation and spindle orientation to drive epithelial morphogenesis

β-actin mRNA localization is mediated through a 28-nucleotide sequence, located in the 3’ UTR region, known as the Zipcode sequence. Binding of Zipcode Binding Protein 1 (ZBP1) is required to both localize and translationally repress the β-actin transcript until the target destination is reached. Loss of ZBP1 in developing mice results in gross abnormalities in small intestinal tissue with a loss of epithelial tissue morphogenesis. Reintroduction of ZBP1 into breast cancer tissues increases polarity, decreases invasiveness, increases cell-cell adhesion, and reduces overall metastatic potential. However, to date, many of the underlying mechanisms which could lead to such outcomes are poorly understood.First, using the well characterized MDCK epithelial cell line, the role of ZBP1 in establishing an epithelial tissue was investigated. Transient knockdown of ZBP1 in MDCK cells revealed a loss of E-cadherin and F-actin association at sites of cell-cell contact. Generation of a stable knockdown of ZBP1 in MDCK cells further revealed significant reduction in protein levels of key adherens junction proteins E-cadherin and β-catenin. This was concomitantly associated with changes in the compaction, architecture, and tissue barrier function of the epithelial monolayer. Analysis of β-actin transcript localization revealed that decrease in ZBP1 expression, resulted in the mislocalization of β-actin transcripts. Further, analysis utilizing an MDCK cell expressing β-actin mRNA transcripts which have had the ZBP1 binding domain removed (3’ UTR) recapitulated the ZBP1 KD results. Thus, ZBP1 seems to impact epithelial tissue morphogenesis through controlling localized β-actin translation and regulating adherens junction formation. To further understand the consequences of mislocalized β-actin translation on epithelial tissue morphogenesis in a more physiologically relevant model, MDCK cyst cultures were used. Cysts generated either using ZBP1 KD or 3’ UTR cell cultures resulted in disorganized lateral and apical domains, contained multiple cell layers and multiple lumen, and had decreased epithelial barrier function. Investigation of this phenotype revealed that mislocalizing β-actin translation resulted in the misorientation of the mitotic spindle leading to loss of planar cell division. Further analysis of this pathway revealed aberrant localization of LGN, a master regulator of spindle orientation, to the apical domain due to defects in the Cdc42/Par/aPKC signaling complex. These results provide molecular evidence of the consequences of localized β-actin translation on the generation of epithelial tissues. Finally, pilot experiments using collective cell migration identify that mislocalization of β-actin translation resulted in the loss of coordinated epithelial collective cell migration, increased cell migration, and decrease in arginylated actin at cell-cell contact sites. Taken together, this thesis highlights the role of localized β-actin translation to coordinately regulate the adherens junction, spindle orientation, and cell-cell migration to generate an epithelial tissue.Ph.D.Includes bibliographical reference