LASP1 as a functional linker protein between cadherin and actin dynamics at epithelial adherens junctions

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Epithelial cadherin (E-cadherin) mediates epithelial adhesion and connects via the beta-catenin/alpha-catenin complex to the actin cytoskeleton that in turn contributes to adherence junction organization and dynamics. Epithelia display an organ-specific Phenotype that is beyond tight junctions largely determine by adherens junctions. We postulate that the supramolecular organization of adherens junctions has a significant impact on the organization and function of AJs. Similarly, we suspect that the expression of mesenchymal cadherins such as VE-cadherin and cadherin-11 during EMT alters the supramolecular organization of AJs, thereby contributing to an altered cell-junction dynamics of carcinomas. Recently we found a novel mechanism of junction regulation in endothelium, which is based on actin-driven junction-associated intermittent lamellipodia (JAIL). JAIL drive VE-cadherin dynamics and thus controls adhesion features, cell migration and monolayer integrity. Similarily, here we show for the first time that epithelial cell lines and human breast carcinoma cell lines display comparable JAIL-like structures. Furthermore, AJ organization and dynamics is controlled by actin-binding and cadherin regulating proteins together with signaling molecules involving acto-myosin mediated contractility. In this context, we identified the LIM and SH3 protein 1 (LASP1), an actin binding protein, as a component of epithelial adherens junctions, and found it overexpressed in dedifferentiated breast cancer MDA cell lines as well as in chronically inflamed tissue, situations in which alterations in cell-to-cell adhesion plays a central role. Furthermore, we identified LASP1 as a new binding partner for β-catenin, which localizes at epithelial adherens junctions and interestingly in JAIL-like plasma membrane protrusion. Mechanistically, we propose LASP1 as a novel adherens junction associated protein in epithelium, which in concerted interaction with actin and b-catenin contributes to junction organization and dynamics. To test the impact of LASP1 and ectopically expressed cadherins on adherens junction dynamics, we will investigate different differentiated MDCK cell lines and human breast cancer cell lines that ectopic express cadherin-11 and VE-cadherin. Fluorescence tagged adherens junction and cytoskeletal proteins will be expressed and fluorescence-based life cell imaging performed to quantitatively monitor cadherin and actin dynamics. Data will be complemented by superresolution microscopy to characterize the supramolecular organization of adherens junctions in a cadherin and LASP1 dependent manner along with established cell- and molecular biology techniques. We plan to use a CHO expression system for confirmation of particular interactions. We expect to establish a molecular mechanistic model that helps to explain the differentiation dependent control of epithelial adherens junction dynamics in physiology, and in EMT and tumor development.