Mechanotransduction in talin through the interaction of the R8 domain with DLC1.

The mechanical unfolding of proteins is a cellular mechanism for force transduction with potentially broad implications in cell fate. Despite this, the mechanism by which protein unfolding elicits differential downstream signalling pathways remains poorly understood. Here, we used protein engineering, atomic force microscopy, and biophysical tools to delineate how protein unfolding controls cell mechanics. Deleted in liver cancer 1 (DLC1) is a negative regulator of Ras homolog family member A (RhoA) and cell contractility that regulates cell behaviour when localised to focal adhesions bound to folded talin. Using a talin mutant resistant to force-induced unfolding of R8 domain, we show that talin unfolding determines DLC1 downstream signalling and, consequently, cell mechanics. We propose that this new mechanism of mechanotransduction may have implications for a wide variety of associated cellular processes.

Mechanical unfolding reveals stable 3-helix intermediates in talin and α-catenin.

Mechanical stability is a key feature in the regulation of structural scaffolding proteins and their functions. Despite the abundance of α-helical structures among the human proteome and their undisputed importance in health and disease, the fundamental principles of their behavior under mechanical load are poorly understood. Talin and α-catenin are two key molecules in focal adhesions and adherens junctions, respectively. In this study, we used a combination of atomistic steered molecular dynamics (SMD) simulations, polyprotein engineering, and single-molecule atomic force microscopy (smAFM) to investigate unfolding of these proteins. SMD simulations revealed that talin rod α-helix bundles as well as α-catenin α-helix domains unfold through stable 3-helix intermediates. While the 5-helix bundles were found to be mechanically stable, a second stable conformation corresponding to the 3-helix state was revealed. Mechanically weaker 4-helix bundles easily unfolded into a stable 3-helix conformation. The results of smAFM experiments were in agreement with the findings of the computational simulations. The disulfide clamp mutants, designed to protect the stable state, support the 3-helix intermediate model in both experimental and computational setups. As a result, multiple discrete unfolding intermediate states in the talin and α-catenin unfolding pathway were discovered. Better understanding of the mechanical unfolding mechanism of α-helix proteins is a key step towards comprehensive models describing the mechanoregulation of proteins.

Adhesive ligand tether length affects the size and length of focal adhesions and influences cell spreading and attachment.

Cells are known to respond to physical cues from their microenvironment such as matrix rigidity. Discrete adhesive ligands within flexible strands of fibronectin connect cell surface integrins to the broader extracellular matrix and are thought to mediate mechanosensing through the cytoskeleton-integrin-ECM linkage. We set out to determine if adhesive ligand tether length is another physical cue that cells can sense. Substrates were covalently modified with adhesive arginylglycylaspartic acid (RGD) ligands coupled with short (9.5 nm), medium (38.2 nm) and long (318 nm) length inert polyethylene glycol tethers. The size and length of focal adhesions of human foreskin fibroblasts gradually decreased from short to long tethers. Furthermore, we found cell adhesion varies in a linker length dependent manner with a remarkable 75% reduction in the density of cells on the surface and a 50% reduction in cell area between the shortest and longest linkers. We also report the interplay between RGD ligand concentration and tether length in determining cellular spread area. Our findings show that without varying substrate rigidity or ligand density, tether length alone can modulate cellular behaviour.

All Subdomains of the Talin Rod Are Mechanically Vulnerable and May Contribute To Cellular Mechanosensing.

Although the relevance of mechanotransduction in cell signaling is currently appreciated, the mechanisms that drive this process remain largely unknown. Mechanical unfolding of proteins may trigger distinct downstream signals in cells, providing a mechanism for cellular mechanotransduction. Force-induced unfolding of talin, a prominent focal adhesion protein, has been demonstrated previously for a small portion of its rod domain. Here, using single-molecule atomic force microscopy (smAFM), we show that the entire talin rod can be unfolded by mechanical extension, over a physiological range of forces between 10 and 40 pN. We also demonstrate, through a combination of smAFM and steered molecular dynamics, that the different bundles within the talin rod exhibit a distinct hierarchy of mechanical stability. These results provide a mechanism by which different force conditions within the cell control a graduated unfolding of the talin rod. Mechanical unfolding of the rod subdomains, and the subsequent effect on talin's binding interactions, would allow for a finely tuned cellular response to internally or externally applied forces.