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Electron cryomicroscopy reveals the three-dimensional structure of F-actin at a resolution of 3.7 Å in complex with tropomyosin at a resolution of 6.5 Å; the stabilizing interactions and the effects of disease-causing mutants are also investigated. The interaction of F-actin and tropomyosin (Rausner SS) Filamentous actin (F-actin) — a main component of the cytoskeleton — is the major protein of thin filaments in the muscle. The binding of the motor protein myosin to F-actin is mediated by another protein called tropomyosin, which also binds to F-actin in smooth muscle and in non-muscle cells, stabilizing and regulating these filaments. Using electron cryomicroscopy, Stefan Raunser and colleagues have obtained the first high-resolution, three-dimensional structure of F-actin in complex with tropomyosin. The structure reveals the interactions that stabilize the F-actin and sheds light on the possible effect of prominent disease-causing mutations. Comparison of the F-actin structure with the crystal structure of monomeric (G)-actin reveals conformational changes associated with filament formation. Filamentous actin (F-actin) is the major protein of muscle thin filaments, and actin microfilaments are the main component of the eukaryotic cytoskeleton. Mutations in different actin isoforms lead to early-onset autosomal dominant non-syndromic hearing loss 1 , familial thoracic aortic aneurysms and dissections 2 , and multiple variations of myopathies 3 . In striated muscle fibres, the binding of myosin motors to actin filaments is mainly regulated by tropomyosin and troponin 4 , 5 . Tropomyosin also binds to F-actin in smooth muscle and in non-muscle cells and stabilizes and regulates the filaments there in the absence of troponin 6 . Although crystal structures for monomeric actin (G-actin) are available 7 , a high-resolution structure of F-actin is still missing, hampering our understanding of how disease-causing mutations affect the function of thin muscle filaments and microfilaments. Here we report the three-dimensional structure of F-actin at a resolution of 3.7 Å in complex with tropomyosin at a resolution of 6.5 Å, determined by electron cryomicroscopy. The structure reveals that the D-loop is ordered and acts as a central region for hydrophobic and electrostatic interactions that stabilize the F-actin filament. We clearly identify map density corresponding to ADP and Mg 2+ and explain the possible effect of prominent disease-causing mutants. A comparison of F-actin with G-actin reveals the conformational changes during filament formation and identifies the D-loop as their key mediator. We also confirm that negatively charged tropomyosin interacts with a positively charged groove on F-actin. Comparison of the position of tropomyosin in F-actin–tropomyosin with its position in our previously determined F-actin–tropomyosin–myosin structure 8 reveals a myosin-induced transition of tropomyosin. Our results allow us to understand the role of individual mutations in the genesis of actin- and tropomyosin-related diseases and will serve as a strong foundation for the targeted development of drugs.

Background Tropomyosin is a major allergen in crustaceans, including mud crab species, but its molecular and allergenic properties in Scylla olivacea are not well known. Thus, this study aimed to produce the recombinant tropomyosin protein from S. olivacea and subsequently investigate its IgE reactivity. Methods and Results The tropomyosin gene was cloned and expressed in the Escherichia coli system, followed by SDS-PAGE and immunoblotting test to identify the allergenic potential of the recombinant protein. The 855-base pair of tropomyosin gene produced was found to be 99.18% homologous to Scylla serrata . Its 284 amino acids matched the tropomyosin of crustaceans, arachnids, insects, and Klebsiella pneumoniae , ranging from 79.03 to 95.77%. The tropomyosin contained 89.44% alpha-helix folding with a tertiary structure of two-chain alpha-helical coiled-coil structures comprising a homodimer heptad chain. IPTG-induced histidine tagged-recombinant tropomyosin was purified at the size of 42 kDa and confirmed as tropomyosin using anti-tropomyosin monoclonal antibodies. The IgE binding of recombinant tropomyosin protein was reactive in 90.9% (20/22) of the sera from crab-allergic patients. Conclusions This study has successfully produced an allergenic recombinant tropomyosin from S. olivacea. This recombinant tropomyosin may be used as a specific allergen for the diagnosis of allergy.

The actin cytoskeleton is one of the most important players in cell motility, adhesion, division, and functioning. The regulation of specific microfilament formation largely determines cellular functions. The main actin-binding protein in animal cells is tropomyosin (Tpm). The unique structural and functional diversity of microfilaments is achieved through the diversity of Tpm isoforms. In our work, we studied the properties of the cytoplasmic isoforms Tpm1.8 and Tpm1.9. The results showed that these isoforms are highly thermostable and differ in the stability of their central and C-terminal fragments. The properties of these isoforms were largely determined by the 6th exons. Thus, the strength of the end-to-end interactions, as well as the affinity of the Tpm molecule for F-actin, differed between the Tpm1.8 and Tpm1.9 isoforms. They were determined by whether an alternative internal exon, 6a or 6b, was included in the Tpm isoform structure. The strong interactions of the Tpm1.8 and Tpm1.9 isoforms with F-actin led to the formation of rigid actin filaments, the stiffness of which was measured using an optical trap. It is quite possible that the structural and functional features of the Tpm isoforms largely determine the appearance of these isoforms in the rigid actin structures of the cell cortex.

Pediatric dilated cardiomyopathy (DCM) is a rare heart muscle disorder leading to the enlargement of all chambers and systolic dysfunction. We identified a novel de novo variant, c.88A>G (p.Lys30Glu, K30E), in the TPM1 gene encoding the major cardiac muscle tropomyosin (Tpm) isoform, Tpm1.1. The variant was found in a proband with DCM and left ventricular non-compaction who progressed to terminal heart failure at the age of 3 years and 8 months. To study the properties of the mutant protein, we produced recombinant K30E Tpm and used various biochemical and biophysical methods to compare its properties with those of WT Tpm. The K30E substitution decreased the thermal stability of Tpm and its complex with actin and significantly reduced the sliding velocity of the regulated thin filaments over a surface covered by ovine cardiac myosin in an in vitro motility assay across the entire physiological range of Ca2+ concentration. Our molecular dynamics simulations suggest that the charge reversal of the 30th residue of Tpm alters the actin monomer to which it is bound. We hypothesize that this rearrangement of the actin–Tpm interaction may hinder the transition of a myosin head attached to a nearby actin from a weakly to a strongly bound, force-generating state, thereby reducing myocardial contractility. The impaired myosin interaction with regulated actin filaments and the decreased thermal stability of the actin–Tpm complex at a near physiological temperature likely contribute to the pathogenicity of the variant and its causative role in progressive DCM.

Key Points Neurotrophins bind to several combinations of cell surface receptors to regulate neuronal survival, function and plasticity. The p75 neurotrophin receptor has numerous functions and is not just a 'death' receptor; under some circumstances it may counteract neurodegenerative signalling. Potential factors that limit the application of neurotrophins as neurological therapeutics include their limited stability, poor central nervous system (CNS) bioavailability, binding to multiple (rather than individual) neurotrophin receptors and mechanism-based side effects. Studies using synthetic oligopeptides have demonstrated the feasibility of creating small molecules that can act as ligands for neurotrophin receptors. Small-molecule ligands can be targeted to specific neurotrophin receptors to modulate signalling. Ligands have been developed that mimic, partially mimic or inhibit the actions of neurotrophins and, importantly, achieve effects that are distinct from those of neurotrophins. Small-molecule modulation of neurotrophin receptor signalling can correct or counteract the deleterious intracellular signalling patterns that exist in various neuropathological states. The administration of small-molecule ligands to several in vivo neurological disease models can correct neuropathological and behavioural abnormalities. Small-molecule ligands are in early stages of clinical development. Although neurotrophins could provide benefit in neurological diseases, their therapeutic application is limited by poor pharmacological properties and undesirable pleiotropic actions. Here, Longo and Massa highlight recent progress in the targeting of individual neurotrophin receptors using small-molecule ligands in an effort to overcome these limitations. Neurotrophins and their receptors modulate multiple signalling pathways to regulate neuronal survival and to maintain axonal and dendritic networks and synaptic plasticity. Neurotrophins have potential for the treatment of neurological diseases. However, their therapeutic application has been limited owing to their poor plasma stability, restricted nervous system penetration and, importantly, the pleiotropic actions that derive from their concomitant binding to multiple receptors. One strategy to overcome these limitations is to target individual neurotrophin receptors — such as tropomyosin receptor kinase A (TRKA), TRKB, TRKC, the p75 neurotrophin receptor or sortilin — with small-molecule ligands. Such small molecules might also modulate various aspects of these signalling pathways in ways that are distinct from the programmes triggered by native neurotrophins. By departing from conventional neurotrophin signalling, these ligands might provide novel therapeutic options for a broad range of neurological indications.

Background In many types of tumors, the expression patterns of actin-binding proteins –fascin-1 and various isoforms of tropomyosin – are altered. Fascin-1 is an actin-bundling protein that promotes cancer cell motility, whereas tropomyosin functions as a tumor and metastasis suppressor. However, the mechanisms by which tropomyosin isoforms regulate fascin-1 remain poorly understood. This study aimed to investigate the reciprocal effects of fascin-1 and tropomyosin isoforms on their interactions with actin and on the formation of actin bundles. Methods Recombinant fascin-1 and the cytoskeletal tropomyosin isoforms encoded by TPM2 (Tpm2.1, Tpm2.3, and Tpm2.4) were expressed in BL21-DE3 cells and purified. High-speed centrifugation was employed to assess the actin affinities of fascin-1 and the Tpm2 isoforms. Actin filament bundling was analyzed using low-speed centrifugation and fluorescence microscopy. A pull-down assay was performed to examine direct interactions between fascin-1 and the Tpm2 isoforms. Confocal microscopy was used to analyze the localization of fascin-1 in the metastatic SAOS-2 LM5 cell line overexpressing Tpm2 isoforms. Results Among the three recombinant, acetylated Tpm2 isoforms, Tpm2.4 exhibited the highest affinity for F-actin. All Tpm2 isoforms strongly inhibited fascin-1-mediated actin bundling at low fascin-1 concentrations, with bundling restored only at substantially higher fascin-1 levels. The resulting actin bundles contained both Tpm2 and fascin-1; however, the number of filaments per bundle was reduced in the presence of any Tpm2 isoform. Fascin-1’s affinity for actin was decreased in the presence of Tpm2 isoforms, and increased Tpm2 occupancy on actin filaments partially displaced fascin-1. In contrast, fascin-1 binding did not affect the affinity of Tpm2 isoforms for actin. Pull-down assays revealed that Tpm2 isoforms can directly interact with fascin-1, with Tpm2.4 showing the highest affinity. The inhibitory effect of Tpm2 on fascin-1–actin interactions was further supported by cellular data, which showed that overexpression of cytoplasmic Tpm2.1, Tpm2.3, or Tpm2.4 in SAOS-2 LM5 cells reduced fascin co-localization with actin. Conclusion Cytoplasmic Tpm2 isoforms regulate actin bundling activity of fascin-1 by organizing protein composition in the bundles, a mechanism that may contribute to the suppression of metastatic phenotype in cancer cells.

Contraction of striated muscles is driven by cyclic interactions of myosin head projecting from the thick filament with actin filament and is regulated by Ca 2+ released from sarcoplasmic reticulum. Muscle thin filament consists of actin, tropomyosin and troponin, and Ca 2+ binding to troponin triggers conformational changes of troponin and tropomyosin to allow actin-myosin interactions. However, the structural changes involved in this regulatory mechanism remain unknown. Here we report the structures of human cardiac muscle thin filament in the absence and presence of Ca 2+ by electron cryomicroscopy. Molecular models in the two states built based on available crystal structures reveal the structures of a C-terminal region of troponin I and an N-terminal region of troponin T in complex with the head-to-tail junction of tropomyosin together with the troponin core on actin filament. Structural changes of the thin filament upon Ca 2+ binding now reveal the mechanism of Ca 2+ regulation of muscle contraction. The contraction of cardiac and skeletal muscles is regulated by Ca 2+ released from the sarcoplasmic reticulum in muscle cells. Here the authors provide molecular insights into Ca 2+ regulation of muscle contraction by determining the cryo-EM structures of the human cardiac muscle thin filament in the absence and presence of Ca 2+ .

Tropomyosin is an actin-binding protein (ABP) which protects actin filaments from cofilin-mediated disassembly. Distinct tropomyosin isoforms have long been hypothesized to differentially sort to subcellular actin networks and impart distinct functionalities. Nevertheless, a mechanistic understanding of the interplay between Tpm isoforms and their functional contributions to actin dynamics has been lacking. In this study, we present and characterize mNeonGreen-Tpm fusion proteins that exhibit good functionality in cells as a sole copy, surpassing limitations of existing probes and enabling real-time dynamic tracking of Tpm-actin filaments in vivo . Using these functional Tpm fusion proteins, we find that S. cerevisiae Tpm isoforms, Tpm1 and Tpm2, colocalize on actin cables and indiscriminately bind to actin filaments nucleated by either formin isoform - Bnr1 and Bni1 in vivo , in contrast to the long-held paradigm of Tpm-formin pairing . We show that cellular Tpm levels regulate endocytosis by affecting the balance between linear and branched actin networks in yeast cells. Finally, we discover that Tpm2 can protect and organize functional actin cables in the absence of Tpm1. Overall, our work supports a concentration-dependent and formin isoform independent model of Tpm isoform binding to F-actin and demonstrates for the first time, the functional redundancy of the paralog Tpm2 in actin cable maintenance in S. cerevisiae.

By stabilizing actin filaments and recruiting non-muscle myosin II, the closely related tropomyosin (Tpm) isoforms Tpm3.1 and Tpm3.2 support actin-dependent processes including membrane dynamics, cell migration, and cytokinesis. Actin dynamics are essential for B cell function, but the roles of Tpm3.1 and 3.2 (collectively termed Tpm3.1/3.2) in B cells have not been explored. Moreover, new treatments are needed to limit the growth and dissemination of diffuse large B-cell lymphoma (DLBCL), the most prevalent B-cell malignancy. To test whether Tpm3.1/3.2 is essential for B-cell actin dynamics and could be a target for treating DLBCL, we employed ATM-3507, a compound that selectively interferes with Tpm3.1/3.2 function. We show that ATM-3507 treatment inhibited B-cell receptor-induced formation of the peripheral ring of branched actin that drives cell spreading and also prevented the formation of actomyosin arcs at the inner face of the peripheral actin ring. Tpm3.1/3.2 localizes to these structures during B-cell spreading. Treating DLBCL cell lines with ATM-3507 inhibited cell growth and caused the cells to accumulate in the G2/M phase of the cell cycle. Furthermore, ATM-3507 markedly reduced CXCL12-stimulated chemotaxis and integrin-dependent motility of DLBCL cell lines on fibronectin. Tpm3.1/3.2 orchestrates key actin-driven processes in B cells, and drugs that target Tpm3.1/3.2 may be useful adjuncts for treating DLBCL.

Myosin-binding protein C (MyBP-C) is an accessory protein of striated muscle thick filaments and a modulator of cardiac muscle contraction. Defects in the cardiac isoform, cMyBP-C, cause heart disease. cMyBP-C includes 11 Ig- and fibronectin-like domains and a cMyBP-C-specific motif. In vitro studies show that in addition to binding to the thick filament via its C-terminal region, cMyBP-C can also interact with actin via its N-terminal domains, modulating thin filament motility. Structural observations of F-actin decorated with N-terminal fragments of cMyBP-C suggest that cMyBP-C binds to actin close to the low Ca ²⁺ binding site of tropomyosin. This suggests that cMyBP-C might modulate thin filament activity by interfering with tropomyosin regulatory movements on actin. To determine directly whether cMyBP-C binding affects tropomyosin position, we have used electron microscopy and in vitro motility assays to study the structural and functional effects of N-terminal fragments binding to thin filaments. 3D reconstructions suggest that under low Ca ²⁺ conditions, cMyBP-C displaces tropomyosin toward its high Ca ²⁺ position, and that this movement corresponds to thin filament activation in the motility assay. At high Ca ²⁺, cMyBP-C had little effect on tropomyosin position and caused slowing of thin filament sliding. Unexpectedly, a shorter N-terminal fragment did not displace tropomyosin or activate the thin filament at low Ca ²⁺ but slowed thin filament sliding as much as the larger fragments. These results suggest that cMyBP-C may both modulate thin filament activity, by physically displacing tropomyosin from its low Ca ²⁺ position on actin, and govern contractile speed by an independent molecular mechanism.