α-Actinin/titin interaction: A dynamic and mechanically stable cluster of bonds in the muscle Z-disk

Abstract

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...

Identifying cardiac actinin interactomes reveals sarcomere crosstalk with RNA-binding proteins

“…1d). STRING confirmed identification of previously-known direct actinin interaction partners localized to the Z-disk including TTN 10 , TCAP 10 and myozenin 2 (MYOZ2) 27 (Fig. 1i), which also belonged to a sarcomere cluster including numerous other sarcomere components determined by Gene-Ontology (GO) enrichment analysis.…”

“…Here, we combined BioID with a two-stage sarcomere assembly model (i.e. Z-body and Z-disk) and confirmed previous Z-disk proteins including TTN 10 , TCAP 27 , LDB3 44 , MYOZ2 27 and CSRP3 33 . CSRP3 was the most-enriched actinin proximity partner with sarcomere assembly, which suggests that CSRP3 may regulate Z-disk organization.…”

“…In the cardiac sarcomere, alpha actinin-2 (referred henceforth as actinin) is the major structural component of the Z-disk, where it is essential for sarcomere assembly and function 8 . Actinin regulates sarcomere assembly by providing a scaffold for protein-protein interactions (PPIs) such as with titin (TTN) and actin through CaM and AB domains, respectively 8–10 . Secondary to these interactions, the thin and thick filament sarcomere systems are organized to promote twitch contraction.…”

Actininopathy: A new muscular dystrophy caused by ACTN2 dominant mutations

“…It connects the antiparallel actin filaments to the sarcomeres and is an anchor for other Z‐disk proteins. Through a number of interactions, it is involved in signaling and mechanosensation …”

“…In striated muscles, CAMD binds a specific hydrophobic alpha‐actinin–binding motif located in titin z‐repeats . The alpha‐actinin interaction with titin and actin is highly dynamic and likely to be regulated by phospholipids as well as by complex intramolecular mechanisms . The stable anchoring of the sarcomeric backbone titin within the Z‐disk is provided by its interaction with alpha‐actinin …”