Abstract
Cells adhere to the surrounding tissue and probe its mechanical properties by forming cell-matrix adhesions. Talin is a critical adhesion protein and participates in the transmission of mechanical signals between extracellular matrix and cell cytoskeleton. Force induced unfolding of talin rod subdomains has been proposed to act as a cellular mechanosensor, but so far evidence linking their mechanical stability and cellular response has been lacking. Here, by utilizing computationally designed mutations, we demonstrate that stepwise destabilization of the talin rod R3 subdomain decreases cellular traction force generation, which affects talin and vinculin dynamics in cell-matrix adhesions and results in the formation of talin-rich but unstable adhesions. We observed a connection between talin stability and the rate of cell migration and also found that talin destabilization affects the usage of different integrin subtypes and sensing of extracellular matrix proteins. Experiments with truncated forms of talin confirm the mechanosensory role of the talin R3 subdomain and exclude the possibility that the observed effects are caused by the release of talin head-rod autoinhibition. In conclusion, this study provides evidence into how the controlled talin rod domain unfolding acts as a key regulator of adhesion structure and function and consequently controls central cellular processes such as cell migration and substrate sensing.
Highlights
Cell-matrix adhesions are large and dynamic membrane spanning protein complexes that physically anchor animal cells to their environment
We aimed to further destabilize the R3 subdomain by mutating conserved isoleucine and leucine residues within the hydrophobic core of the helix bundle to a small polar amino acid, serine. This addition of hydrophilic residues into the core of the helix bundle makes its tertiary structure thermodynamically less favorable, and facilitates its unfolding when the helix bundle is subjected to mechanical stretching
The mechanical properties of the talin rod domain have been proposed to be an important factor in the regulation of mechanosensing and mechanosignalling
Summary
Cell-matrix adhesions are large and dynamic membrane spanning protein complexes that physically anchor animal cells to their environment. The head domain (47 kDa) contains binding sites for multiple adhesion proteins and its binding to the β-integrin tail is one of the first steps in the formation of nascent cell-matrix adhesions. The gradual force-induced exposure of the talin VBSs creates a system where higher force causes more rod subdomains to unfold, exposing more VBSs. Vinculin accumulation is known to mechanically strengthen the adhesion, and to initiate downstream signaling cascades. Vinculin accumulation is known to mechanically strengthen the adhesion, and to initiate downstream signaling cascades Such multi-step unfolding of the talin rod domain has been suggested to create a force buffer that can smooth out sudden fluctuations in the cellular traction forces[9]. If the force-induced unfolding of the talin R3 subdomain is a key step in adhesion maturation, mechanically stabilizing or destabilizing mutations should affect cellular mechanosensing and mechanosignaling. This experiment does not give any indication about whether destabilization of talin R3 subdomain would result in altered mechanosignaling and changes in cell phenotype
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