Force transmission via dynamic stretching of Talin as revealed by live-cell single-molecule imagingSawako Yamashiro (Kyoto University)

Focal adhesions (FAs) are responsible for transmitting intracellular forces to the extracellular matrix. It is widely recognized that Talin is an essential FA protein that links actin filaments (F-actin) to integrins. However, we previously revealed that the actin network constantly moves at ~20 nm/sec over FAs as a single-unit by direct single-molecule observation of F-actin movement in Xenopus XTC cells (Yamashiro et al. MBoC 25:1010, 2014). How can FA molecules simultaneously maintain the connection to moving F-actin and transmit forces to integrins? In this study, we address this issue by performing Single-Molecule Speckle (SiMS) microscopy which elucidates true functions and kinetics of individual Talin molecules in live cells. Our SiMS data show that the majority of Talin molecules are bound only to either the moving F-actin network or the substrate whereas a small portion of Talin is liked to both structures via elastic transient clutch in FAs. By reconstituting Talin knockdown cells with Talin chimeric mutants, in which the Talin rod subdomains are replaced with the stretchable β-spectrin repeats, we show that the stretchable property is critical for force transmission. We carried out simulations to test whether unfolding of the Talin rod subdomains increases work at FAs. Our findings provide new insights into coupling and force transmission of the retrograde actin flow to the extracellular matrix.