NanoPlan - Deciphering the molecular construction rules of integrin-based cell adhesions.
Final Report Abstract
The ability of mammalian cells to adhere tightly to the extracellular matrix and sense the mechanical and chemical properties of the environment is critical for many developmental, homeostatic and pathophysiological processes in multicellular organisms. In both animals and humans, this function is largely mediated by a family of receptors, the integrins, which are expressed on the surface of cells, where – after activation – they bind extracellular ligands. At the same time, the integrins connect to the intracellular cytoskeleton and thus establish a mechanical linkage between cells and their extracellular environment. Interestingly, integrins are tightly regulated by intracellular proteins that assemble at the cytoplasmic domain of the receptor to form a multimolecular structure, the focal adhesion. Both the activation of integrin receptors and their association with the cytoskeleton are mediated by adaptor proteins such as talin and kindlin. However, before the start of this project, it was unclear how the intracellular adaptor proteins associate with integrins, for example to mediate receptor activation. In this project, we therefore wanted to investigate the nanoscale organization of integrin-based adhesion in cells. We pursued novel super-resolution microscopy-based approaches that should allow quantitative analyses of integrin-based adhesions on molecular scales. The ultimately established method, which goes well beyond the originally envisioned approach, combines DNA-PAINT-based single protein imaging, automated cluster detection and in silico data simulations with cell biological controls to determine absolute fractions and spatial coordinates of interacting molecular species at the molecular level. Using this new method, we were able to show for the first time that β1-integrin receptors can simultaneously associate with talin and kindlin to form a ternary adhesion complex during cell adhesion. Since the developed approach requires careful genetic engineering, which will not always be possible for many future projects, we have further developed our approach to study the nanoscale organization of adhesion proteins using a peptide-PAINT approach. This work shows that molecularlevel information can be quantified in genetically unmodified cells and even tissue samples with similar sensitivity to DNA-PAINT-based strategies. Overall, the results of the project not only provide first insights into the molecular organization underlying cell-matrix adhesions, but also provide a quantitative technology to study subcellular structures at the molecular level.
Publications
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Unforgettable force – crosstalk and memory of mechanosensitive structures. Biological Chemistry, 400(6), 687-698.
Kanoldt, Verena; Fischer, Lisa & Grashoff, Carsten
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Molecular Force Measurement with Tension Sensors. Annual Review of Biophysics, 50(1), 595-616.
Fischer, Lisa S.; Rangarajan, Srishti; Sadhanasatish, Tanmay & Grashoff, Carsten
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Peptide‐PAINT Enables Investigation of Endogenous Talin with Molecular Scale Resolution in Cells and Tissues. ChemBioChem, 22(19), 2872-2879.
Fischer, Lisa S.; Schlichthaerle, Thomas; Chrostek‐Grashoff, Anna & Grashoff, Carsten
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Quantitative single-protein imaging reveals molecular complex formation of integrin, talin, and kindlin during cell adhesion. Nature Communications, 12(1).
Fischer, Lisa S.; Klingner, Christoph; Schlichthaerle, Thomas; Strauss, Maximilian T.; Böttcher, Ralph; Fässler, Reinhard; Jungmann, Ralf & Grashoff, Carsten
