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Abstract

Cell adhesion is essential for the formation and functional integrity of tissues and organs in multicellular organisms. In vertebrates, the epithelium is a specialized tissue that maintains a protective barrier around organs. Epithelial cells are attached to the extracellular matrix and form tight connections with each other via E-cadherin mediated multiprotein complexes called adherens junctions (AJs). In this thesis, I present a method for light-induced dissociation of dimerizer-mediated AJs. This is the first optochemical tool which allows to control the formation and disassembly of adhesion complexes inside living cells with high spatiotemporal precision. The applied dimerizers are bifunctional small molecules that combine ligands of self-labeling protein tags like Halo and SNAP tag, via a photocleavable linker. These synthetic molecules induce dimeric complexes of proteins expressed as fusion constructs with the respective self-labelling tag inside living cells. The complexes are efficiently disassembled by cleaving the dimerizers with light. To utilize photocleavable dimerizers in the context of cell adhesion, I first established a covalent-covalent binding dimerizer to ensure mechanical stability against mechanical forces acting on the induced protein complexes. I showed the potential to control the formation of adhesions complexes via retention, recruitment and complementation approaches for different target proteins. For example, I replaced the catenin binding sites on E-cadherin with a Halo tag and coexpressed them with SNAP-tagged a-catenin that was deficient of the b-catenin binding site. The dimerizer-mediated E-cadherin-a-catenin complexes could restore the epithelial phenotype of human epidermoid carcinoma cells (A431 cells) when induced in a-catenin KOs, but not in E-cadherin deficient A431D cells. I could show that the lack of AJ formation in A431D cells is associated with the failure of recruiting b-catenin. In a-catenin KOs -catenin was indirectly recruited to the dimerizer-mediated E-cadherin-a-catenin complex via lateral clustering with endogenous E-cadherin. This in turn led to the formation of AJ complexes, which are the coupling points for the contractile actomyosin network between epithelial cells. Moreover, the a-catenin KO phenotype could be restored upon light induced dissociation of the dimerizer-mediated AJs via exposure to near UV light. When using a 405 nm laser to cleave the dimerizers, I was able to target the AJs between two adjacent cells with subcellular precision or to create patterns of deactivated cell-cell adhesion in epithelial monolayers. Furthermore, I could prove the mechanical functionality of the reconstituted AJs by performing a correlation analysis of collective monolayer migration and traction force microscopy. Herein, I demonstrated the application of photocleavable dimerizers to study the cellular response to AJ assembly and disassembly at different scales of space and time. Photocleavable dimerizers present several advantages for applications in living cells. Since the proteinprotein interaction depends on external addition of the dimerizers and can be reliably abrogated by breaking the molecule, the setup offers two binary switches for the formation of AJ complexes, virtually without any basal activity. Furthermore, the system is bioorthogonal and light is a trigger that allows to dissociate the protein complexes at subcellular and subsecond resolution. The possibility to combine it with specialized imaging techniques like traction force microscopy renders it a powerful tool to study the mechanobiology of AJs and its contribution to processes in epithelial cell layers like cellular jamming and unjamming, collective migration and stress propagation. This new method will help to gain new insights in the dynamic regulation of cell adhesion in fundamental pathophysiological processes like embryonal development, wound healing or cancer metastasis.