Mechanical stability of αT-catenin and its activation by force for vinculin binding

Abstract

αT (Testes)-catenin, a critical factor regulating cell–cell adhesion in the heart, directly couples the cadherin-catenin complex to the actin cytoskeleton at the intercalated disk (ICD), a unique cell–cell junction that couples cardiomyocytes. Loss of αT-catenin in mice reduces plakophilin2 and connexin 43 recruitment to the ICD. Since αT-catenin is subjected to mechanical stretch during actomyosin contraction in cardiomyocytes, its activity could be regulated by mechanical force. To provide insight in how force regulates αT-catenin function, we investigated the mechanical stability of the putative, force-sensing middle (M) domain of αT-catenin and determined how force impacts vinculin binding to αT-catenin. We show that 1) physiological levels of force, <15 pN, are sufficient to unfold the three M domains; 2) the M1 domain that harbors the vinculin-binding site is unfolded at ∼6 pN; and 3) unfolding of the M1 domain is necessary for high-affinity vinculin binding. In addition, we quantified the binding kinetics and affinity of vinculin to the mechanically exposed binding site in M1 and observed that αT-catenin binds vinculin with low nanomolar affinity. These results provide important new insights into the mechanosensing properties of αT-catenin and how αT-catenin regulates cell–cell adhesion at the cardiomyocyte ICD.

Distinct autoinhibitory mechanisms regulate vinculin binding by αT-catenin and αE-catenin

“…aT-catenin N-terminus regulates vinculin binding-Recent in vitro single molecule stretching experiments revealed that force is required to expose the vinculin binding site in aT-catenin M1-M3 (47). However, our ITC results with the aT-catenin M1-M3 fragment indicated that tension was not required to release M1.…”

“…aT-catenin M-region binds vinculin-The aE-catenin M-region is autoinhibited with respect to vinculin binding: mechanical force is required to break an internal salt bridge network within M1-M3 to reveal the vinculin binding site in M1 (25,30). The aT-catenin M-region also recruits vinculin and force is required to unfurl M1 to promote high affinity binding (47). However, our previous proteolysis experiments of full-length aT-catenin revealed that the M2-M3 region existed in a more open, proteasesensitive state (38).…”

“…However, our ITC results with the aT-catenin M1-M3 fragment indicated that tension was not required to release M1. In the stretching experiments, the aTcatenin M1-M3 fragment was flanked by a pair of titin I27 immunoglobulin-like domains and the N-terminus of the fusion protein was tethered to a substrate (47). We questioned if Nterminal associations with M1-M3 stabilize M1 and regulate vinculin binding.…”

“…F-actin binding is also not allosterically regulated, as the b-catenin/aTcatenin complex binds to F-actin with the same affinity as aT-catenin monomer (38). Single molecule pulling experiments have shown the aT-catenin M-region to be mechanoresponsive as it unfurls in a force range similar to aEcatenin (47).…”

Implementing Optogenetic Modulation in Mechanotransduction

“…In addition, many of the structural domains in a forcetransmission pathway undergo dynamic unfolding and refolding over a force range from several to tens of piconewtons at physiological pulling or loading rates [15][16][17][18]. This process makes them an effective force buffer to maintain the tension in the force-transmission pathway at a similar range [15,17].…”