Formin-like 2 associates with the alpha-catenin/E-cadherin complex to control junctional actin dynamics and epithelialization

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De novo formation of adherens junctions between single epithelial cells requires coordinated, spatial actin dynamics for specialized local force generation. The mechanisms steering nascent actin polymerization for cell-cell adhesion initiation and maintenance are only poorly understood. Here we investigate actin assembly by formins during daughter cell-cell adhesion formation in the human breast epithelial cell line MCF10A in a 3D environment. We identify formin-like 2 (FMNL2) to be required for actin assembly and turnover at newly formed cell-cell contacts as well as for de novo adherens junction formation. In long-term 3D cultures, the suppression of FMNL2 results in severely disrupted epithelial lumen formation. We find that FMNL2 localizes to cell-cell contacts in a regulated manner and FMNL2 forms a complex with alpha-catenin and E-Cadherin, which is strongly promoted by Rac1 activity. Photoactivation of an optogenetically controlled LOV-Rac1 rapidly drives FMNL2 to the junctional zone for localized actin assembly, while photobleaching experiments reveal that FMNL2 rescues Rac1 deficiency on junctional actin turnover. Thus, FMNL2 appears to be a critical factor for nascent junctional actin assembly and de novo adheres junction formation in human 3D epithelial cultures. Consequently, these findings require a detailed understanding of the mechanistic relationship between formin-induced actin polymerization and cell-cell adhesion. The aim of this proposal is to examine in depth, how FMNL2 is integrated into the AJ complex and whether this interaction participates in the regulation of FMNL2 using a combination of 3D cell culture, biochemistry, the development of optogenetic tools as well as live cell imaging techniques. These studies shall provide a deeper understanding on the roles of formins at cell-cell contacts within an intact human epithelium.