Stable anchoring of titin within the muscle Z-disk is essential for preserving muscle integrity during passive stretching. One of the main candidates for anchoring titin in the Z-disk is the actin crosslinker α-actinin. The calmodulin-like domain of α-actinin binds to the Z-repeats of titin. However, the mechanical and kinetic properties of this important interaction are still unknown. Here, we use a dual-beam optical tweezers assay to study the mechanics of this interaction at the single-molecule level. A single interaction of α-actinin and titin turns out to be surprisingly weak if force is applied. Depending on the direction of force application, the unbinding forces can more than triple. Our results suggest a model where multiple α-actinin/Z-repeat interactions cooperate to ensure long-term stable titin anchoring while allowing the individual components to exchange dynamically.M uscle is the tissue that is constantly subjected to high mechanical loads. Whereas thick and thin filaments are responsible for active force production, the passive elasticity of muscle is dominated by titin/connectin filaments (1). Hence, under passive stretching conditions the integrity of muscle relies on titin's being firmly anchored within the sarcomere, preventing the interdigitated muscle filaments from falling apart (Fig. 1A). Whereas titin is firmly attached to thick filaments in the A-band and the M-line (2-6), it is much less clear how stable anchoring is achieved in the Z-disk, where adjacent sarcomeres overlap. The superstable titin/telethonin interaction within the Z-disk was considered important for titin anchoring (7-9), but knockout mutants later showed that it is not essential for muscle integrity (10-12). Apart from a direct interaction between actin filaments and titin at the Z-disk edge (13), the most prominent candidate for the anchoring of titin within the Z-disk is its interaction with α-actinin (Fig. 1B) (6,12,14).Four isoforms of human α-actinin have been identified: the calcium-insensitive muscle isoforms 2 and 3, which cross-link actin filaments in sarcomere-delimiting Z-disk complexes, and calcium-sensitive nonmuscle isoforms 1 and 4. α-Actinin is an antiparallel homodimer whose most prominent task is crosslinking actin filaments of neighboring sarcomeres in the Z-disk ( Fig. 1B; reviewed in ref. 14). In each subunit, a flexible region called the neck separates the actin binding domain (ABD) from four spectrin-like repeats (SR) forming the rod region (Fig. 1B and Fig. S1). The rod regions of the two subunits interact and provide a rigid spacer between the actin filaments. At the other end of each subunit a calmodulin-like domain (CaMD) formed by two pairs of EF-hands (EF1-2 and EF3-4) is able to bind a Z-disk region of titin formed by the so-called Z-repeats (15-17). The current model for α-actinin 2 dynamic regulation suggests that EF3-4 hands of one subunit bind to the neck region of the juxtaposed subunit, thus not being available for the interaction with titin Z-repeats (Fig. S1) (18, 19). Upon activ...

Integrin-mediated adhesion is essential for metazoan life. Integrin binding to ligand requires an activation step prior to binding ligand that depends on direct binding of talin and kindlin to the β-integrin cytoplasmic tail and the transmission of force from the actomyosin via talin to the integrin–ligand bonds. However, the affinity of talin for integrin tails is low. It is therefore still unclear how such low-affinity bonds are reinforced to transmit forces up to 10 to 40 pN. In this study, we use single-molecule force spectroscopy by optical tweezers to investigate the mechanical stability of the talin•integrin bond in the presence and absence of kindlin. While talin and integrin alone form a weak and highly dynamic slip bond, the addition of kindlin-2 induces a force-independent, ideal talin•integrin bond, which relies on the steric proximity of and the intervening amino acid sequences between the talin- and kindlin-binding sites in the β-integrin tail. Our findings show how kindlin cooperates with talin to enable transmission of high forces required to stabilize cell adhesion.

Integrins are large heterodimeric proteins that play an important role in force transduction across the cell membrane. In order to bind the extracellular matrix integrins have to be in an activated open form. The activation of integrins requires the binding of the talin N-terminal F3 domain to the integrin b-tail and the association with the contractile actomyosin cytoskeleton. Despite its critical role in force transduction, the affinity between integrin b-tail and talin-F3 domain is surprisingly weak for all isoform pairs (K d >10 mM). This raises the question how such a weak bond resists the actomyosin pulling forces and ensures a stable connection across the cell membrane. To address this question we designed fusion constructs between the talin-F3 domain (but also entire talin head domain) and the b1-integrin cytoplasmic tail. Using a dual-beam optical tweezer setup we probed the mechanics of the talinintegrin tail bond at the single molecule level. Our results confirm the dynamic character of the talin-integrin tail bond, which shows an unbinding-rate of z 50/s. The binding energy of the talin-integrin tail bond is weak and ranges around 4-6 k B T, and the bond is mainly in an open state at forces higher than 5 pN. Interestingly, we discovered that the talin-integrin tail bond is stabilized by the presence of kindlin, which is also a FERM-domain protein that synergizes with talin to activate integrins. When measuring the talin-integrin tail interaction with kindlin in solution, the talin-integrin bound states become longer. Surprisingly, the length of the bound states is not influenced by force. This kindlin-induced stabilization effect might be of crucial importance for integrin activation, clustering and development of adhesion site assemblies.