Deciphering the role of the talin1/paxillin/kindlin3 complex in regulating integrin activity and function in hematopoietic cells and mice

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Integrins are a family of ubiquitously expressed cell adhesion receptors. To bind tightly to their ligands, they require an activation step followed by their assembly into complex adhesion structures. Both processes require the binding of talin and kindlin to the cytoplasmic domain of integrins. While the mechanism of talin-induced activation of integrins appears to be understood, the molecular role of kindlins is still largely unknown. In particular, how talin and kindlin cooperate to regulate integrin activity and function remains one of the greatest mysteries in the field of integrin research. We have recently provided evidence for an indirect interaction via the adapter protein paxillin, which serves as a bridge between the two proteins. We hypothesize that the talin/paxillin/kindlin complex orchestrates activated integrins into micro-clusters that allow immediate tight binding to their multivalent ligands. While our recent in vitro studies provided unequivocal evidence for the existence of this trimeric complex, it is now important to investigate the significance of the talin-paxillin and paxillin-kindlin interactions under more physiological conditions and cell culture systems and to provide unequivocal evidence for its relevance in vivo. Our study will focus on the role of this complex in the regulation of leukocyte integrins, as these cells are particularly dependent on the rapid formation of stable adhesions. In the proposal presented here, we use genetically engineered Hoxb8 progenitor cell lines capable of differentiating in vitro into neutrophil-like cells and macrophages that functionally resemble their primary counterparts. Using CRISPR/Cas9/12-mediated gene editing, we have generated Hoxb8 cell clones carrying mutations at the paxillin-binding sites of the endogenous talin1 and kindlin3 proteins. Our preliminary studies show that disruption of the talin-paxillin interaction significantly reduces integrin-mediated adhesion of neutrophils under static conditions and under flow. In comprehensive cell biological and biochemical studies using these and other novel Hoxb8 cell lines, we will study the importance of the talin/paxillin/kindlin3 complex in regulating leukocyte integrins. In particular, we aim to understand its role in leukocyte adhesion and signal transduction, integrin activation and micro-cluster formation followed by the assembly of complex adhesion structures, as well as its effects on cell migration. In addition, we aim to use biochemical methods and mass spectrometry to gain mechanistic insights into the initiation, formation, and regulation of the talin/paxillin/kindlin3 complex during the leukocyte adhesion cascade. In addition, we have already succeeded in generating talin1 mouse mutants with a mutated paxillin binding site in the talin1-rod domain. Using these animals, we will study the different steps of the leukocyte adhesion cascade ex vivo and in vivo under homeostatic and inflammatory conditions.