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Integrins are heterodimeric transmembrane receptors that were key to the dawn of metazoa. In multicellular animals they are widely expressed in all organs and tissue. There, they provide a link between the extracellular matrix and the cell’s actin cytoskeleton. They are used as physical anchorage points that fix the cells to the underlying substrate and mediate bidirectional signaling across the cell membrane. Integrins are thus necessary to respond to biophysical and biochemical changes in the extracellular environment. They govern cell differentiation, proliferation, survival, cell migration, embryonic development, immune defense as well as thrombus formation. Since their discovery in the early 80’s, their ubiquitous expression and their crucial role in both health and disease made them one of the most intensely investigated proteins of the human body. Upon ECM engagement integrins recruit a large, multimeric protein complex, called focal adhesions (FAs). FAs make up a complex network, consisting of several hundreds of proteins and thousands of protein-protein interactions. This complex is also referred to as the integrin adhesome. At the core of these complexes are a number of scaffolding and adaptor proteins. One of them is the “über-linker” paxillin, which mediates a vast number of protein interactions and regulates the controlled assembly and disassembly of FAs as well as actin cytoskeleton remodeling for efficient cell migration. The immense complexity of this network, its dynamic nature but also the redundancy that exists between multiple protein isoforms, makes it notoriously hard to study those structures. In the first two chapters we present novel tools to reduce the complexity and redundancies of the system. These tools are then applied in chapter III to reveal a novel feature of the adaptor protein paxillin. In the first study, a novel approach for the microscopic evaluation of protein-protein interactions at integrin clusters is presented. This approach, called Opa protein triggered integrin clustering (OPTIC), uses bacteria as multivalent ligands to cluster chimeric integrin receptors and induce minimal versions of the focal adhesion complex. These clusters solely depend on the short cytoplasmic tail of integrins and are independent of endogenous integrin ligands. They don’t rely on force generation and recruit only a small fraction of the integrin adhesome. Due to their drastically reduced complexity and their easy manipulability, these structures can be used to investigate protein-protein interactions and can be applied to verify biochemical data in a cellular context. Using integrin orthologues from a unicellular ancestor of metazoa, the OPTIC is utilized to proof the evolutionary conservation of the talin-integrin interaction. The second study uses a novel streamlined approach for the rapid disruption of multiple genes and subsequent generation of stable re-expression cell lines. The workflow is applied to simultaneously delete two members of the paxillin family, paxillin and Hic-5, which have both distinct and redundant functions. Biochemical and morphological analysis of the generated cell lines reveal a phenotype that is surprisingly different from previously described single knockout cells. The double knockout cell lines were then used to characterize an important role for paxillin and Hic-5 in host cell invasion of the opportunistic human pathogen Staphylococcus aureus. Paxillin is one of the first proteins to localize to FAs and its localization is a prerequisite for its function. FA targeting is mediated via its C-terminal LIM2 and LIM3 domains. Yet, the exact mechanism of its recruitment is not entirely clear. In the last chapter we use 3D NMR measurements to solve the structure of paxillin’s LIM2/3 domain and identify a direct interaction with the cytoplasmic tails of integrin beta1 and beta3. We apply a combination of biochemical assays and the OPTIC presented in chapter I, to determine the binding interface of both proteins. Reverse genetics verify our findings in a cellular context and reveal an important role for the LIM3-integrin beta3 interaction in FA maturation and cell spreading. published |
