Talin-mediated force transmission and talin rod domain unfolding independently regulate adhesion signaling

Abstract

Talin protein is one of the key components in integrin-mediated adhesion complexes. Talins transmit mechanical forces between β-integrin and actin, and regulate adhesion complex composition and signaling through the force-regulated unfolding of talin rod domain. Using modified talin proteins, we demonstrate that these functions contribute to different cellular processes and can be dissected. The transmission of mechanical forces regulates adhesion complex composition and phosphotyrosine signaling even in the absence of the mechanically regulated talin rod subdomains. However, the presence of the rod subdomains and their mechanical activation are required for the reinforcement of the adhesion complex, cell polarization and migration. Talin rod domain unfolding was also found to be essential for the generation of cellular signaling anisotropy, since both insufficient and excess activity of the rod domain severely inhibited cell polarization. Utilizing proteomics tools, we identified adhesome components that are recruited and activated either in a talin rod-dependent manner or independently of the rod subdomains. This study clarifies the division of roles between the force-regulated unfolding of a talin protein (talin 1) and its function as a physical linker between integrins and the cytoskeleton.

Talin dissociates from RIAM and associates to vinculin sequentially in response to the actomyosin force

“…This behavior likely results from the overlap between the RIAMbinding sites and VBSs in R2 and R3 33 . R1-R2-R3 is a key mechanosensitive part of talin, since a construct, almost identical to our talin R1-R2-R3, can rescue spreading, polarization and migration of talin null cells 37 . In this region of talin, R3 has a critical role, since mutations that destabilize this bundle affect ECM sensing 38 .…”

Cell Adhesion by Integrins

“…At the same time, it is clear that also integrin adapters are an intrinsic part of the mechanosensory machinery. Talin has several cryptic binding sites, that open under mechanical stretch, and is therefore not only of interest as integrin activator, but also as mechanosensor and mechanotransducer (see above) (170,353,354). Interestingly, the mechanotransduction by talin is isoform-specific, with talin2 increasing the ability of cells to spread on 1-2 kPa substrates compared to talin1 (26).…”

“…Talin and kindlin activate integrins (= change the extracellular conformation) and increase their affinity for ligands in a process of inside-out activation involving interaction of these adapter proteins with the b-integrin cytoplasmic tail. Both talin and kindlin are required for integrin activation and clustering, but appear to differentially contribute to mechanosensing (talin) and signaling (kindlin)(354,437). An important part of integrin activation are the unclasping of the aand b-subunit at the level of the transmembrane and cytoplasmic tails as well as the physical connection to the actin cytoskeleton.…”

Topological features of integrin adhesion complexes revealed by multiplexed proximity biotinylation

“…BioID has been used to probe the structure of labile and membrane-associated complexes that are difficult to study using more traditional techniques, including nuclear pore complexes, the centrosome, and cell-cell contacts (Van Itallie et al, 2013;Gupta et al, 2015;Kim et al, 2014). BioID has also been used to examine the proximity interactomes of individual IAC-associated proteins and has revealed a number of potential new adhesome candidates (Dong et al, 2016;Mekhdjian et al, 2017;Rahikainen et al, 2019). For example, KANK2 was identified as a paxillin-and kindlin-2-proximal protein in U2OS cells and was shown to localize to IACs (Dong et al, 2016).…”