Dataset Information

Caldesmon controls stress fiber force-balance through dynamic cross-linking of myosin II and actin-tropomyosin filaments.

ABSTRACT: Contractile actomyosin bundles are key force-producing and mechanosensing elements in muscle and non-muscle tissues. Whereas the organization of muscle myofibrils and mechanism regulating their contractility are relatively well-established, the principles by which myosin-II activity and force-balance are regulated in non-muscle cells have remained elusive. We show that Caldesmon, an important component of smooth muscle and non-muscle cell actomyosin bundles, is an elongated protein that functions as a dynamic cross-linker between myosin-II and tropomyosin-actin filaments. Depletion of Caldesmon results in aberrant lateral movement of myosin-II filaments along actin bundles, leading to irregular myosin distribution within stress fibers. This manifests as defects in stress fiber network organization and contractility, and accompanied problems in cell morphogenesis, migration, invasion, and mechanosensing. These results identify Caldesmon as critical factor that ensures regular myosin-II spacing within non-muscle cell actomyosin bundles, and reveal how stress fiber networks are controlled through dynamic cross-linking of tropomyosin-actin and myosin filaments.

SUBMITTER: Kokate SB

PROVIDER: S-EPMC9561149 | biostudies-literature | 2022 Oct

REPOSITORIES: biostudies-literature

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Caldesmon controls stress fiber force-balance through dynamic cross-linking of myosin II and actin-tropomyosin filaments.

Kokate Shrikant B SB Ciuba Katarzyna K Tran Vivien D VD Kumari Reena R Tojkander Sari S Engel Ulrike U Kogan Konstantin K Kumar Sanjay S Lappalainen Pekka P

Nature communications 20221013 1

Contractile actomyosin bundles are key force-producing and mechanosensing elements in muscle and non-muscle tissues. Whereas the organization of muscle myofibrils and mechanism regulating their contractility are relatively well-established, the principles by which myosin-II activity and force-balance are regulated in non-muscle cells have remained elusive. We show that Caldesmon, an important component of smooth muscle and non-muscle cell actomyosin bundles, is an elongated protein that function ...[more]

PMID: 36229430

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Project description:The motor protein myosin drives muscle and nonmuscle motility by binding to and moving along actin of thin filaments. Myosin binding to actin also modulates interactions of the regulatory protein, tropomyosin, on thin filaments, and conversely tropomyosin affects myosin binding to actin. Insight into this reciprocity will facilitate a molecular level elucidation of tropomyosin regulation of myosin interaction with actin in muscle contraction, and in turn, promote better understanding of nonmuscle cell motility. Indeed, experimental approaches such as fiber diffraction, cryoelectron microscopy, and three-dimensional reconstruction have long been used to define regulatory interaction of tropomyosin and myosin on actin at a structural level. However, their limited resolution has not proven sufficient to determine tropomyosin and myosin contacts at an atomic-level and thus to fully substantiate possible functional contributions. To overcome this deficiency, we have followed a hybrid approach by performing new cryogenic electron microscopy reconstruction of myosin-S1-decorated F-actin-tropomyosin together with atomic scale protein-protein docking of tropomyosin to the EM models. Here, cryo-EM data were derived from filaments reconstituted with α1-actin, cardiac αα-tropomyosin, and masseter muscle β-myosin complexes; masseter myosin, which shares sequence identity with β-cardiac myosin-heavy chain, was used because of its stability in vitro. The data were used to build an atomic model of the tropomyosin cable that fits onto the actin filament between the tip of the myosin head and a cleft on the innermost edge of actin subunits. The docking and atomic scale fitting showed multiple discrete interactions of myosin loop 4 and acidic residues on successive 39-42 residue-long tropomyosin pseudorepeats. The contacts between S1 and tropomyosin on actin appear to compete with and displace ones normally found between actin and tropomyosin on myosin-free thin filaments in relaxed muscle, thus restructuring the filament during myosin-induced activation.

Project description:Tropomyosins (Tpms) are rod-shaped proteins that interact head-to-tail to form a continuous polymer along both sides of most cellular actin filaments. Head-to-tail interaction between adjacent Tpm molecules and the formation of an overlap complex between them leads to the assembly of actin filaments with one type of Tpm isoform in time and space. Variations in the affinity of tropomyosin isoforms for different actin structures are proposed as a potential sorting mechanism. However, the detailed mechanisms of the spatio-temporal sorting of Tpms remain elusive. In this study, we investigated the early intermediates during actin-tropomyosin filament assembly, using a skeletal/cardiac Tpm isoform (Tpm1.1) and a cytoskeletal isoform (Tpm1.6) that differ only in the last 27 amino acids. We investigated how the muscle isoform Tpm1.1 and the cytoskeletal isoform Tpm1.6 nucleate domains on the actin filament, and tested whether (1) recruitment is affected by the actin isoform (muscle vs. cytoskeletal) and (2) whether there is specificity in recruiting the same isoform to a domain at these early stages. To address these questions, actin filaments were exposed to low concentrations of fluorescent tropomyosins in solution. The filaments were immobilized onto glass coverslips and the pattern of decoration was visualized by TIRF microscopy. We show that at the early assembly stage, tropomyosins formed multiple distinct fluorescent domains (here termed "cluster") on the actin filaments. An automated image analysis algorithm was developed and validated to identify clusters and estimate the number of tropomyosins in each cluster. The analysis showed that tropomyosin isoform sorting onto an actin filament is unlikely to be driven by a preference for nucleating on the corresponding muscle or cytoskeletal actin isoforms, but rather is facilitated by a higher probability of incorporating the same tropomyosin isoforms into an early assembly intermediate. We showed that the 27 amino acids at the end of each tropomyosin seem to provide enough molecular information for the attachment of the same tropomyosin isoforms adjacent to each other on an actin filament. This results in the formation of homogeneous clusters composed of the same isoform rather than clusters with mixed isoforms.

Dataset Information

Caldesmon controls stress fiber force-balance through dynamic cross-linking of myosin II and actin-tropomyosin filaments.

Publications

Caldesmon controls stress fiber force-balance through dynamic cross-linking of myosin II and actin-tropomyosin filaments.

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