Cellular senescence is a phenotype characterized by cessation of cell division, which can be caused by exhaustive replication or environmental stress. It is involved in age-related pathophysiological conditions and affects both the cellular cytoskeleton and the prime cellular mechanosensors, focal adhesion complexes. While the size of focal adhesions increases during senescence, it is unknown if and how this is accompanied by a remodeling of the internal focal adhesion structure. Our study uses metal-induced energy transfer to study the axial dimension of focal adhesion proteins from oxidative-stress-induced senescent cells with nanometer precision, and compares these to unstressed cells. We influenced cytoskeletal tension and the functioning of mechanosensitive ion channels using drugs and studied the combined effect of senescence and drug intervention on the focal adhesion structure. We found that H2O2-induced restructuring of the focal adhesion complex indicates a loss of tension and altered talin complexation. Mass spectroscopy-based proteomics confirmed the differential regulation of several cytoskeletal proteins induced by H2O2 treatment.
Bibliographical noteFunding Information:
Molecular graphics and analyses were performed using UCSF ChimeraX, developed by the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco, with support from the National Institutes of Health R01-GM129325 and the Office of Cyber Infrastructure and Computational Biology, National Institute of Allergy and Infectious Diseases. To generate structural models of the talin-vinculin-actin complex, we started from an elongated talin model obtained by Benjamin Gould, the full-length vinculin structure (pdb code 1tr2;) with added missing loop residues using Modeller using standard settings, and an electron microscopy-based model of the complex of the vinculin tail and F-actin (pdb-code 3jbi). The orientation of the membrane relative to the talin FERM domain was based on the structure of an integrin b/talin complex (PDB code 3g9w) and membrane prediction from the OPM database. The relative orientation of the vinculin head and talin was based on the experimental structure of the VBS3/talin complex (PDB code 1rkc). We superimposed matching domains using the matchmaker function of ChimeraX and adapted the flexible linkers between the domains using the loop optimization routine implemented in Modeller. We then interactively positioned the respective domains of talin, vinculin, and actin at the measured distance from the membrane plane, and positioned the rod domains manually between the ABS and IBS. The relative orientation of IBS and the membrane is unknown because of the lack of an experimental structure for the IBS2/integrin complex. Therefore, we positioned rod 11 manually close to the membrane plane to analyze the scaling behavior. Afterwards, all loops and, if needed, rod domains were treated as flexible loops in Modeller to obtain the final models. Acknowledgements
© 2023, The Author(s).