A nemaline myopathy-linked mutation inhibits the actin-regulatory functions of tropomodulin and leiomodin.

Actin is a highly expressed protein in eukaryotic cells and is essential for numerous cellular processes. In particular, efficient striated muscle contraction is dependent upon the precise regulation of actin-based thin filament structure and function. Alterations in the lengths of actin-thin filaments can lead to the development of myopathies. Leiomodins and tropomodulins are members of an actin-binding protein family that fine-tune thin filament lengths, and their dysfunction is implicated in muscle diseases. An Lmod3 mutation [G326R] was previously identified in patients with nemaline myopathy (NM), a severe skeletal muscle disorder; this residue is conserved among Lmod and Tmod isoforms and resides within their homologous leucine-rich repeat (LRR) domain. We mutated this glycine to arginine in Lmod and Tmod to determine the physiological function of this residue and domain. This G-to-R substitution disrupts Lmod and Tmod's LRR domain structure, altering their binding interface with actin and destroying their abilities to regulate thin filament lengths. Additionally, this mutation renders Lmod3 nonfunctional in vivo. We found that one single amino acid is essential for folding of Lmod and Tmod LRR domains, and thus is essential for the opposing actin-regulatory functions of Lmod (filament elongation) and Tmod (filament shortening), revealing a mechanism underlying the development of NM.

Leiomodin creates a leaky cap at the pointed end of actin-thin filaments.

Improper lengths of actin-thin filaments are associated with altered contractile activity and lethal myopathies. Leiomodin, a member of the tropomodulin family of proteins, is critical in thin filament assembly and maintenance; however, its role is under dispute. Using nuclear magnetic resonance data and molecular dynamics simulations, we generated the first atomic structural model of the binding interface between the tropomyosin-binding site of cardiac leiomodin and the N-terminus of striated muscle tropomyosin. Our structural data indicate that the leiomodin/tropomyosin complex only forms at the pointed end of thin filaments, where the tropomyosin N-terminus is not blocked by an adjacent tropomyosin protomer. This discovery provides evidence supporting the debated mechanism where leiomodin and tropomodulin regulate thin filament lengths by competing for thin filament binding. Data from experiments performed in cardiomyocytes provide additional support for the competition model; specifically, expression of a leiomodin mutant that is unable to interact with tropomyosin fails to displace tropomodulin at thin filament pointed ends and fails to elongate thin filaments. Together with previous structural and biochemical data, we now propose a molecular mechanism of actin polymerization at the pointed end in the presence of bound leiomodin. In the proposed model, the N-terminal actin-binding site of leiomodin can act as a "swinging gate" allowing limited actin polymerization, thus making leiomodin a leaky pointed-end cap. Results presented in this work answer long-standing questions about the role of leiomodin in thin filament length regulation and maintenance.

Mechanosensitive Gene Regulation by Myocardin-Related Transcription Factors Is Required for Cardiomyocyte Integrity in Load-Induced Ventricular Hypertrophy.

BACKGROUND: Hypertrophic cardiomyocyte growth and dysfunction accompany various forms of heart disease. The mechanisms responsible for transcriptional changes that affect cardiac physiology and the transition to heart failure are not well understood. The intercalated disc (ID) is a specialized intercellular junction coupling cardiomyocyte force transmission and propagation of electrical activity. The ID is gaining attention as a mechanosensitive signaling hub and hotspot for causative mutations in cardiomyopathy. METHODS: Transmission electron microscopy, confocal microscopy, and single-molecule localization microscopy were used to examine changes in ID structure and protein localization in the murine and human heart. We conducted detailed cardiac functional assessment and transcriptional profiling of mice lacking myocardin-related transcription factor (MRTF)-A and MRTF-B specifically in adult cardiomyocytes to evaluate the role of mechanosensitive regulation of gene expression in load-induced ventricular remodeling. RESULTS: We found that MRTFs localize to IDs in the healthy human heart and accumulate in the nucleus in heart failure. Although mice lacking MRTFs in adult cardiomyocytes display normal cardiac physiology at baseline, pressure overload leads to rapid heart failure characterized by sarcomere disarray, ID disintegration, chamber dilation and wall thinning, cardiac functional decline, and partially penetrant acute lethality. Transcriptional profiling reveals a program of actin cytoskeleton and cardiomyocyte adhesion genes driven by MRTFs during pressure overload. Indeed, conspicuous remodeling of gap junctions at IDs identified by single-molecule localization microscopy may partially stem from a reduction in Mapre1 expression, which we show is a direct mechanosensitive MRTF target. CONCLUSIONS: Our study describes a novel paradigm in which MRTFs control an acute mechanosensitive signaling circuit that coordinates cross-talk between the actin and microtubule cytoskeleton and maintains ID integrity and cardiomyocyte homeostasis in heart disease.

Characterizing interaction forces between actin and proteins of the tropomodulin family reveals the presence of the N-terminal actin-binding site in leiomodin.

Tropomodulin family of proteins includes several isoforms of tropomodulins (Tmod) and leiomodins (Lmod). These proteins can sequester actin monomers or nucleate actin polymerization. Although it is known that their actin-binding properties are isoform-dependent, knowledge on how they vary in strengths of interactions with G-actin is missing. While it is confirmed in many studies that Tmods have two actin-binding sites, information on number and location of actin-binding sites in Lmod2 is controversial. We used atomic force microscopy to study interactions between G-actin and proteins of the tropomodulin family. Unbinding forces between G-actin and Tmod1, Tmod2, Tmod3, or Lmod2 were quantified. Our results indicated that Tmod1 and Tmod3 had unimodal force distributions, Tmod2 had a bimodal distribution and Lmod2 had a trimodal distribution. The number of force distributions correlates with the proteins' abilities to sequester actin or to nucleate actin polymerization. We assigned specific unbinding forces to the individual actin-binding sites of Tmod2 and Lmod2 using mutations that destroy actin-binding sites of Tmod2 and truncated Lmod2. Our results confirm the existence of the N-terminal actin-binding site in Lmod2. Altogether, our data demonstrate how the differences between the number and the strength of actin-binding sites of Tmod or Lmod translate to their functional abilities.

Localization of the binding interface between leiomodin-2 and α-tropomyosin.

The development of some familial dilated cardiomyopathies (DCM) correlates with the presence of mutations in proteins that regulate the organization and function of thin filaments in cardiac muscle cells. Harmful effects of some mutations might be caused by disruption of yet uncharacterized protein-protein interactions. We used nuclear magnetic resonance spectroscopy to localize the region of striated muscle α-tropomyosin (Tpm1.1) that interacts with leiomodin-2 (Lmod2), a member of tropomodulin (Tmod) family of actin-binding proteins. We found that 21 N-terminal residues of Tpm1.1 are involved in interactions with residues 7-41 of Lmod2. The K15N mutation in Tpm1.1, known to be associated with familial DCM, is located within the newly identified Lmod2 binding site of Tpm1.1. We studied the effect of this mutation on binding Lmod2 and Tmod1. The mutation reduced binding affinity for both Lmod2 and Tmod1, which are responsible for correct lengths of thin filaments. The effect of the K15N mutation on Tpm1.1 binding to Lmod2 and Tmod1 provides a molecular rationale for the development of familial DCM.

The cardiomyopathy-associated K15N mutation in tropomyosin alters actin filament pointed end dynamics.

Correct assembly of thin filaments composed of actin and actin-binding proteins is of crucial importance for properly functioning muscle cells. Tropomyosin (Tpm) mediates the binding of tropomodulin (Tmod) and leiomodin (Lmod) at the slow-growing, or pointed, ends of the thin filaments. Together these proteins regulate thin filament lengths and actin dynamics in cardiac muscle. The K15N mutation in the TPM1 gene is associated with familial dilated cardiomyopathy (DCM) but the effect of this mutation on Tpm's function is unknown. In this study, we introduced the K15N mutation in striated muscle α-Tpm (Tpm1.1) and investigated its interaction with actin, Tmod and Lmod. The mutation caused a ∼3-fold decrease in the affinity of Tpm1.1 for actin. The binding of Lmod and Tmod to Tpm1.1-covered actin filaments also decreased in the presence of the K15N mutation. Furthermore, the K15N mutation in Tpm1.1 disrupted the inhibition of actin polymerization and affected the competition between Tmod1 and Lmod2 for binding at the pointed ends. Our data demonstrate that the K15N mutation alters pointed end dynamics by affecting molecular interactions between Tpm1.1, Lmod2 and Tmod1.

Tropomyosin-binding properties modulate competition between tropomodulin isoforms.

The formation and fine-tuning of cytoskeleton in cells are governed by proteins that influence actin filament dynamics. Tropomodulin (Tmod) regulates the length of actin filaments by capping the pointed ends in a tropomyosin (TM)-dependent manner. Tmod1, Tmod2 and Tmod3 are associated with the cytoskeleton of non-muscle cells and their expression has distinct consequences on cell morphology. To understand the molecular basis of differences in the function and localization of Tmod isoforms in a cell, we compared the actin filament-binding abilities of Tmod1, Tmod2 and Tmod3 in the presence of Tpm3.1, a non-muscle TM isoform. Tmod3 displayed preferential binding to actin filaments when competing with other isoforms. Mutating the second or both TM-binding sites of Tmod3 destroyed its preferential binding. Our findings clarify how Tmod1, Tmod2 and Tmod3 compete for binding actin filaments. Different binding mechanisms and strengths of Tmod isoforms for Tpm3.1 contribute to their divergent functional capabilities.

The Effects of Noncellulosic Compounds on the Nanoscale Interaction Forces Measured between Carbohydrate-Binding Module and Lignocellulosic Biomass.

The lack of fundamental understanding of the types of forces that govern how cellulose-degrading enzymes interact with cellulosic and noncellulosic components of lignocellulosic surfaces limits the design of new strategies for efficient conversion of biomass to bioethanol. In a step to improve our fundamental understanding of such interactions, nanoscale forces acting between a model cellulase-a carbohydrate-binding module (CBM) of cellobiohydrolase I (CBH I)-and a set of lignocellulosic substrates with controlled composition were measured using atomic force microscopy (AFM). The three model substrates investigated were kraft (KP), sulfite (SP), and organosolv (OPP) pulped substrates. These substrates varied in their surface lignin coverage, lignin type, and xylan and acetone extractives' content. Our results indicated that the overall adhesion forces of biomass to CBM increased linearly with surface lignin coverage with kraft lignin showing the highest forces among lignin types investigated. When the overall adhesion forces were decoupled into specific and nonspecific component forces via the Poisson statistical model, hydrophobic and Lifshitz-van der Waals (LW) forces dominated the binding forces of CBM to kraft lignin, whereas permanent dipole-dipole interactions and electrostatic forces facilitated the interactions of lignosulfonates to CBM. Xylan and acetone extractives' content increased the attractive forces between CBM and lignin-free substrates, most likely through hydrogen bonding forces. When the substrates treated differently were compared, it was found that both the differences in specific and nonspecific forces between lignin-containing and lignin-free substrates were the least for OPP. Therefore, cellulase enzymes represented by CBM would weakly bind to organosolv lignin. This will facilitate an easy enzyme recovery compared to other substrates treated with kraft or sulfite pulping. Our results also suggest that altering the surface hydrophobicity and the surface energy of lignin that facilitates the LW forces should be a priori to avoid nonproductive binding of cellulase to kraft lignin.

Alteration of tropomyosin-binding properties of tropomodulin-1 affects its capping ability and localization in skeletal myocytes.

Tropomodulin (Tmod) is an actin-capping protein that binds to the two tropomyosins (TM) at the pointed end of the actin filament to prevent further actin polymerization and depolymerization. Therefore, understanding the role of Tmod is very important when studying actin filament dependent processes such as muscle contraction and intracellular transport. The capping ability of Tmod is highly influenced by TM and is 1000-fold greater in the presence of TM. There are four Tmod isoforms (Tmod1-4), three of which, Tmod1, Tmod3, and Tmod4, are expressed in skeletal muscles. The affinity of Tmod1 to skeletal striated TM (stTM) is higher than that of Tmod3 and Tmod4 to stTM. In this study, we tested mutations in the TM-binding sites of Tmod1, using circular dichroism (CD) and prediction analysis (PONDR). The mutations R11K, D12N, and Q144K were chosen because they decreased the affinity of Tmod1 to stTM, making it similar to that of affinity of Tmod3 and Tmod4 to stTM. Significant reduction of inhibition of actin pointed-end polymerization in the presence of stTM was shown for Tmod1 (R11K/D12N/Q144K) as compared with WT Tmod1. When GFP-Tmod1 and mutants were expressed in primary chicken skeletal myocytes, decreased assembly of Tmod1 mutants was revealed. This indicates a direct correlation between TM-binding and the actin-capping abilities of Tmod. Our data confirmed the hypothesis that assembly of Tmod at the pointed-end of the actin filament depends on its TM-binding affinity.

Recessive TMOD1 mutation causes childhood cardiomyopathy.

Familial cardiomyopathy in pediatric stages is a poorly understood presentation of heart disease in children that is attributed to pathogenic mutations. Through exome sequencing, we report a homozygous variant in tropomodulin 1 (TMOD1; c.565C>T, p.R189W) in three individuals from two unrelated families with childhood-onset dilated and restrictive cardiomyopathy. To decipher the mechanism of pathogenicity of the R189W mutation in TMOD1, we utilized a wide array of methods, including protein analyses, biochemistry and cultured cardiomyocytes. Structural modeling revealed potential defects in the local folding of TMOD1R189W and its affinity for actin. Cardiomyocytes expressing GFP-TMOD1R189W demonstrated longer thin filaments than GFP-TMOD1wt-expressing cells, resulting in compromised filament length regulation. Furthermore, TMOD1R189W showed weakened activity in capping actin filament pointed ends, providing direct evidence for the variant's effect on actin filament length regulation. Our data indicate that the p.R189W variant in TMOD1 has altered biochemical properties and reveals a unique mechanism for childhood-onset cardiomyopathy.

Tropomodulins and tropomyosins: working as a team.

Actin filaments are major components of the cytoskeleton in eukaryotic cells and are involved in vital cellular functions such as cell motility and muscle contraction. Tmod and TM are crucial constituents of the actin filament network, making their presence indispensable in living cells. Tropomyosin (TM) is an alpha-helical, coiled coil protein that covers the grooves of actin filaments and stabilizes them. Actin filament length is optimized by tropomodulin (Tmod), which caps the slow growing (pointed end) of thin filaments to inhibit polymerization or depolymerization. Tmod consists of two structurally distinct regions: the N-terminal and the C-terminal domains. The N-terminal domain contains two TM-binding sites and one TM-dependent actin-binding site, whereas the C-terminal domain contains a TM-independent actin-binding site. Tmod binds to two TM molecules and at least one actin molecule during capping. The interaction of Tmod with TM is a key regulatory factor for actin filament organization. The binding efficacy of Tmod to TM is isoform-dependent. The affinities of Tmod/TM binding influence the proper localization and capping efficiency of Tmod at the pointed end of actin filaments in cells. Here we describe how a small difference in the sequence of the TM-binding sites of Tmod may result in dramatic change in localization of Tmod in muscle cells or morphology of non-muscle cells. We also suggest most promising directions to study and elucidate the role of Tmod-TM interaction in formation and maintenance of sarcomeric and cytoskeletal structure.

CAP2 is a regulator of actin pointed end dynamics and myofibrillogenesis in cardiac muscle.

The precise assembly of actin-based thin filaments is crucial for muscle contraction. Dysregulation of actin dynamics at thin filament pointed ends results in skeletal and cardiac myopathies. Here, we discovered adenylyl cyclase-associated protein 2 (CAP2) as a unique component of thin filament pointed ends in cardiac muscle. CAP2 has critical functions in cardiomyocytes as it depolymerizes and inhibits actin incorporation into thin filaments. Strikingly distinct from other pointed-end proteins, CAP2's function is not enhanced but inhibited by tropomyosin and it does not directly control thin filament lengths. Furthermore, CAP2 plays an essential role in cardiomyocyte maturation by modulating pre-sarcomeric actin assembly and regulating α-actin composition in mature thin filaments. Identification of CAP2's multifunctional roles provides missing links in our understanding of how thin filament architecture is regulated in striated muscle and it reveals there are additional factors, beyond Tmod1 and Lmod2, that modulate actin dynamics at thin filament pointed ends.

Redefining actin dynamics of the pointed-end complex in striated muscle.

Striated muscle is intricately designed to provide efficient and powerful muscle contractions. Recently, a long sought-after missing component of the thin filament pointed-end machinery was discovered: cyclase-associated protein 2 (CAP2). CAP2 was identified as a crucial contributor to actin polymerization, striated muscle development, and severe muscle disease when mutated.

Effects of cardiomyopathy-linked mutations K15N and R21H in tropomyosin on thin-filament regulation and pointed-end dynamics.

Missense mutations K15N and R21H in striated muscle tropomyosin are linked to dilated cardiomyopathy (DCM) and hypertrophic cardiomyopathy (HCM), respectively. Tropomyosin, together with the troponin complex, regulates muscle contraction and, along with tropomodulin and leiomodin, controls the uniform thin-filament lengths crucial for normal sarcomere structure and function. We used Förster resonance energy transfer to study effects of the tropomyosin mutations on the structure and kinetics of the cardiac troponin core domain associated with the Ca2+-dependent regulation of cardiac thin filaments. We found that the K15N mutation desensitizes thin filaments to Ca2+ and slows the kinetics of structural changes in troponin induced by Ca2+ dissociation from troponin, while the R21H mutation has almost no effect on these parameters. Expression of the K15N mutant in cardiomyocytes decreases leiomodin's thin-filament pointed-end assembly but does not affect tropomodulin's assembly at the pointed end. Our in vitro assays show that the R21H mutation causes a twofold decrease in tropomyosin's affinity for F-actin and affects leiomodin's function. We suggest that the K15N mutation causes DCM by altering Ca2+-dependent thin-filament regulation and that one of the possible HCM-causing mechanisms by the R21H mutation is through alteration of leiomodin's function.

Tropomodulin's Actin-Binding Abilities Are Required to Modulate Dendrite Development.

There are many unanswered questions about the roles of the actin pointed end capping and actin nucleation by tropomodulins (Tmod) in regulating neural morphology. Previous studies indicate that Tmod1 and Tmod2 regulate morphology of the dendritic arbor and spines. Tmod3, which is expressed in the brain, had only a minor influence on morphology. Although these studies established a defined role of Tmod in regulating dendritic and synaptic morphology, the mechanisms by which Tmods exert these effects are unknown. Here, we overexpressed a series of mutated forms of Tmod1 and Tmod2 with disrupted actin-binding sites in hippocampal neurons and found that Tmod1 and Tmod2 require both of their actin-binding sites to regulate dendritic morphology and dendritic spine shape. Proximity ligation assays (PLAs) indicate that these mutations impact the interaction of Tmod1 and Tmod2 with tropomyosins Tpm3.1 and Tpm3.2. This impact on Tmod/Tpm interaction may contribute to the morphological changes observed. Finally, we use molecular dynamics simulations (MDS) to characterize the structural changes, caused by mutations in the C-terminal helix of the leucine-rich repeat (LRR) domain of Tmod1 and Tmod2 alone and when bound onto actin monomers. Our results expand our understanding of how neurons utilize the different Tmod isoforms in development.

Tropomodulin isoforms utilize specific binding functions to modulate dendrite development.

Tropomodulins (Tmods) cap F-actin pointed ends and have altered expression in the brain in neurological diseases. The function of Tmods in neurons has been poorly studied and their role in neurological diseases is entirely unknown. In this article, we show that Tmod1 and Tmod2, but not Tmod3, are positive regulators of dendritic complexity and dendritic spine morphology. Tmod1 increases dendritic branching distal from the cell body and the number of filopodia/thin spines. Tmod2 increases dendritic branching proximal to the cell body and the number of mature dendritic spines. Tmods utilize two actin-binding sites and two tropomyosin (Tpm)-binding sites to cap F-actin. Overexpression of Tmods with disrupted Tpm-binding sites indicates that Tmod1 and Tmod2 differentially utilize their Tpm- and actin-binding sites to affect morphology. Disruption of Tmod1's Tpm-binding sites abolished the overexpression phenotype. In contrast, overexpression of the mutated Tmod2 caused the same phenotype as wild type overexpression. Proximity ligation assays indicate that the mutated Tmods are shuttled similarly to wild type Tmods. Our data begins to uncover the roles of Tmods in neural development and the mechanism by which Tmods alter neural morphology. These observations in combination with altered Tmod expression found in several neurological diseases also suggest that dysregulation of Tmod expression may be involved in the pathology of these diseases. © 2016 Wiley Periodicals, Inc.