Thin Filament Length Research Articles

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.

Localization of the Elastic Proteins in the Flight Muscle of Manduca sexta.

The flight muscle of Manduca sexta (DLM1) is an emerging model system for biophysical studies of muscle contraction. Unlike the well-studied indirect flight muscle of Lethocerus and Drosophila, the DLM1 of Manduca is a synchronous muscle, as are the vertebrate cardiac and skeletal muscles. Very little has been published regarding the ultrastructure and protein composition of this muscle. Previous studies have demonstrated that DLM1 express two projectin isoform, two kettin isoforms, and two large Salimus (Sls) isoforms. Such large Sls isoforms have not been observed in the asynchronous flight muscles of Lethocerus and Drosophila. The spatial localization of these proteins was unknown. Here, immuno-localization was used to show that the N-termini of projectin and Salimus are inserted into the Z-band. Projectin spans across the I-band, and the C-terminus is attached to the thick filament in the A-band. The C-terminus of Sls was also located in the A-band. Using confocal microscopy and experimental force-length curves, thin filament lengths were estimated as ~1.5 µm and thick filament lengths were measured as ~2.5 µm. This structural information may help provide an interpretive framework for future studies using this muscle system.

Osmotic Compression Influences Cross-Bridge Detachment Rate in Transgenic Hypertrophic Cardiomyopathy Variant Hctnt-I79N and Non-Transgenic Mouse Cardiac Muscle

Missense variant Ile79Asn in human cardiac troponin T (HcTnT-I79N) has been linked to adverse phenotypic outcomes of hypertrophic cardiomyopathy (HCM) including arrhythmia and sudden cardiac death. This study investigates cross-bridge kinetics of left ventricular papillary muscle bundles (Ca2+-sensitivity of isometric force, sinusoidal stiffness and rate of tension redevelopment (kTR)) in a (1) length-dependent activation manner and (2) under osmotic compression. Cardiac tissues were harvested from both non-transgenic wild-type (NTg) mice and transgenic mice bearing the HcTnT-I79N variant. Sarcomere lengths (1.9, 2.1 or 2.3 μm) were set at pCa 8.0 using HeNe laser diffraction. Sinusoidal stiffness (0.2% PTP length oscillation) and kTR were obtained during Ca2+ activation when force was at least 20% of maximum. Increased Ca2+-sensitivity of isometric force and maximum force were observed in NTg cardiac preparations upon an increase in SL (1.9 vs 2.1 μm, and 2.1 vs 2.3 μm). Interestingly, HcTnT-I79N exhibited increased Ca2+-sensitivity and maximum force at SL 1.9 vs 2.1 μm, while only maximum force increased upon further stretch (SL 2.1 vs 2.3 μm). This blunted effect at SL 2.1 vs 2.3 μm cannot be explained by differences in thin filament length in HcTnT-I79N vs NTg. Although HcTnT-I79N exhibited significantly lower maximum kTR, neither NTg nor HcTnT-I79N showed significant changes in maximum kTR over the entire range of SL (1.9-2.3 μm). After osmotically compressing the myofilament lattice using 3% Dextran T500 (SL 2.1 μm), (1) only NTg cardiac preparations displayed increased myofilament Ca2+-sensitivity; (2) kTR increased at most activation levels for both NTg and HcTnTI79N; (3) no changes in maximum kTR were observed; (4) both NTG and HcTnT-I79N displayed increased cross-bridge detachment rates (g); (5) HcTnT-I79N showed slower g when compared to NTG with and without Dextran. NIH-HL128683.

Design and Optimization of Tropomyosin Fragments for Tropomodulin Interaction Studies

Monomeric actin, or G-actin, polymerizes into filamentous actin, or F-actin which is an important component of cellular structures controlling many processes including vesicle trafficking, cell motility, inter and intracellular transport, and muscle contraction. Actin filaments are composed of a pointed end (slow growing) and a barbed end (fast growing). Interactions at each end enables control of the length of actin filaments. Tropomodulin (Tmod1-4) modulates actin filament length by interacting with tropomyosin (Tpm) and actin at the pointed end of the filament. Tmod binds two Tpm molecules through two distinct Tpm binding sites. Establishing the structure of each Tmod-Tpm binding interface is critical to understand the thin filament length regulation mechanism of Tmod. To facilitate NMR structure determination, we have established the optimal size of Tpm fragment necessary to form respective stable complexes. The four fragments we designed were residues 1-43, 1-33, 1-26 or 1-19 of Tpm1.8 fused with a GCN4 sequence promoting coiled-coil formation. We used NMR to determine and compare spectral effects between the optimized Tpm fragments and Tpm-binding sites of Tmod2. A significant change in spectra occurred when length of Tpm sequence changed from 19 to 26 residues and with almost no change with further increase in Tpm sequence length. Stability of the designed Tpm fragments and their affinity for Tpm-binding sites on Tmod2 were evaluated by CD spectroscopy. Our results revealed that Tpm1.8 fragments containing residues 1-19 and 1-43 were more stable with a melting temperature of 44 and 52oC, respectively, however, the 1-19 fragment was not long enough to represent the whole binding interface. The calculated dissociation constants showed strong affinity of Tmod2 for the Tpm1.8 1-43. Therefore, Tpm1.8-1-43 fragment is the most optimal for use in NMR studies to further study its interaction with Tmod.

Thin filament dysfunctions caused by mutations in tropomyosin Tpm3.12 and Tpm1.1

Tropomyosin is the major regulator of the thin filament. In striated muscle its function is to bind troponin complex and control the access of myosin heads to actin in a Ca2+-dependent manner. It also participates in the maintenance of thin filament length by regulation of tropomodulin and leiomodin, the pointed end-binding proteins. Because the size of the overlap between actin and myosin filaments affects the number of myosin heads which interact with actin, the filament length is one of the determinants of force development. Numerous point mutations in genes encoding tropomyosin lead to single amino acid substitutions along the entire length of the coiled coil that are associated with various types of cardiomyopathy and skeletal muscle disease. Specific regions of tropomyosin interact with different binding partners; therefore, the mutations affect diverse tropomyosin functions. In this review, results of studies on mutations in the genes TPM1 and TPM3, encoding Tpm1.1 and Tpm3.12, are described. The paper is particularly focused on mutation-dependent alterations in the mechanisms of actin-myosin interactions and dynamics of the thin filament at the pointed end.

Nebulin and titin modulate cross-bridge cycling and length-dependent calcium sensitivity.

Various mutations in the structural proteins nebulin and titin that are present in human disease are known to affect the contractility of striated muscle. Loss of nebulin is associated with reduced actin filament length and impairment of myosin binding to actin, whereas titin is thought to regulate muscle passive elasticity and is likely involved in length-dependent activation. Here, we sought to assess the modulation of muscle function by these sarcomeric proteins by using the computational platform muscle simulation code (MUSICO) to quantitatively separate the effects of structural changes, kinetics of cross-bridge cycling, and calcium sensitivity of the thin filaments. The simulations show that variation in thin filament length cannot by itself account for experimental observations of the contractility in nebulin-deficient muscle, but instead must be accompanied by a decreased myosin binding rate. Additionally, to match the observed calcium sensitivity, the rate of TnI detachment from actin needed to be increased. Simulations for cardiac muscle provided quantitative estimates of the effects of different titin-based passive elasticities on muscle force and activation in response to changes in sarcomere length and interfilament lattice spacing. Predicted force-pCa relations showed a decrease in both active tension and sensitivity to calcium with a decrease in passive tension and sarcomere length. We conclude that this behavior is caused by partial redistribution of the muscle load between active muscle force and titin-dependent passive force, and also by redistribution of stretch along the thin filament, which together modulate the release of TnI from actin. These data help advance understanding of how nebulin and titin mutations affect muscle function.

A new twist on tropomyosin binding to actin filaments: perspectives on thin filament function, assembly and biomechanics.

Tropomyosin, best known for its role in the steric regulation of muscle contraction, polymerizes head-to-tail to form cables localized along the length of both muscle and non-muscle actin-based thin filaments. In skeletal and cardiac muscles, tropomyosin, under the control of troponin and myosin, moves in a cooperative manner between blocked, closed and open positions on filaments, thereby masking and exposing actin-binding sites necessary for myosin crossbridge head interactions. While the coiled-coil signature of tropomyosin appears to be simple, closer inspection reveals surprising structural complexity required to perform its role in steric regulation. For example, component α-helices of coiled coils are typically zippered together along a continuous core hydrophobic stripe. Tropomyosin, however, contains a number of anomalous, functionally controversial, core amino acid residues. We argue that the atypical residues at this interface, including clusters of alanines and a charged aspartate, are required for preshaping tropomyosin to readily fit to the surface of the actin filament, but do so without compromising tropomyosin rigidity once the filament is assembled. Indeed, persistence length measurements of tropomyosin are characteristic of a semi-rigid cable, in this case conducive to cooperative movement on thin filaments. In addition, we also maintain that tropomyosin displays largely unrecognized and residue-specific torsional variance, which is involved in optimizing contacts between actin and tropomyosin on the assembled thin filament. Corresponding twist-induced stiffness may also enhance cooperative translocation of tropomyosin across actin filaments. We conclude that anomalous core residues of tropomyosin facilitate thin filament regulatory behavior in a multifaceted way.

Experimentally Varying the Number of Super-Repeats in the Neb Gene of the Mouse - Assessing the Role of Nebulin in Thin Filament Length Regulation

Nebulin is a giant, modular actin binding protein that spans along the thin filament in skeletal muscle. Nebulin consists of numerous tandem copies of 38.5 nm ‘super-repeats’ that correspond to the arrangement of troponin and tropomyosin on the thin filament. Besides being known as a thin filament stabilizer, nebulin has been speculated to act as a thin filament ruler in skeletal muscle, yet little is known about nebulin's direct influence on actin length or its mechanism of action. Here we developed two novel mouse models in which nebulin super-repeats 9-11 are deleted (NebΔSR9-11) or duplicated (NebdupSR9-11), respectively. Mice of either model are viable and develop without muscle weight deficits. Super-resolution microscopy (SR-SIM) and immunoelectron microscopy on extensor digitorum longus (EDL) muscle revealed that the N-terminus of nebulin, together with tropomodulin (Tmod) localizes at the thin filament pointed end. Furthermore, nebulin and thin filaments were found to be shorter by ∼115 nm in NebΔSR9-11 and longer by ∼115 nm in NebdupSR9-11 EDL. Compound heterozygous animals (NebΔSR9-11,dupSR9-11) express both the shorter and the longer nebulin close to a ratio of 1:1 and the difference in their lengths is ∼230 nm. Tmod localization suggests a bimodal thin filament length distribution in NebΔSR9-11,dupSR9-11 EDL muscle fibers according to the shorter and longer nebulin. Functional studies on skinned EDL fibers revealed leftward shift of the descending limb of the force-sarcomere length relationship in NebΔSR9-11 and rightward shift in NebdupSR9-11 consistent with their altered thin filament lengths. We conclude that nebulin rules the thin filaments in EDL muscle with each of nebulin's super-repeats being responsible for a quantal 38 nm thin filament length. Studies on other muscle types are currently ongoing.

Role of intrinsic disorder in muscle sarcomeres.

The role and utility of intrinsically disordered regions (IDRs) is reviewed for two groups of sarcomeric proteins, such as members of tropomodulin/leiomodin (Tmod/Lmod) protein homology group and myosin binding protein C (MyBP-C). These two types of sarcomeric proteins represent very different but strongly interdependent functions, being responsible for maintaining structure and operation of the muscle sarcomere. The role of IDRs in the formation of complexes between thin filaments and Tmods/Lmods is discussed within the framework of current understanding of the thin filament length regulation. For MyBP-C, the function of IDRs is discussed in the context of MYBP-C-dependent sarcomere contraction and actomyosin activation.

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.

Functionally Driven Modulation of Sarcomeric Structure and Membrane Systems in the Fast Muscles of a Copepod (Gaussia princeps).

Muscles of the mesopelagic copepod Gaussia princeps (Arthropoda, Crustacea, Calanoida) are responsible for repetitive movements of feeding and swimming appendages that are too fast to be followed by eye. This article provides a comparative functional and ultrastructural description of five muscles that have different contraction speeds and are located within different anatomical sites. All are very fast, as indicated by a thick:thin filament ratio of 3:1 and sarcomere lengths that vary between 1 and 3 μm. Measured lengths of thin and thick filaments indicate classification of the muscles into three distinct groups (short, medium, and long) and predict a difference in speed of up to threefold between fibers with the shortest and longest sarcomeres. Indeed, the kicking movement of the posterior legs (with the shortest sarcomere length) is approximately threefold faster than the simultaneous back-folding of the antennae (with the longest length). Thus, a specific relationship between speed of movement and sarcomere length is established, and we can use the latter to predict the former. Regulatory systems of contraction (sarcoplasmic reticulum [SR] and transverse [T] tubules) match the different contractile properties, varying in frequency of distribution and overall content in parallel to sarcomere variations. All muscles from appendages and body musculature show a unique disposition of contractile material, SR, and T tubules found only in copepod muscles; muscle filaments are grouped in large supermyofibrils that are riddled with frequent cylindrical shafts containing SR and T tubules. This arrangement insures a high spatial frequency of regulatory components. Anat Rec, 301:2164-2176, 2018. © 2018 Wiley Periodicals, Inc.

Nebulin increases thin filament stiffness and force per cross-bridge in slow-twitch soleus muscle fibers.

Nebulin (Neb) is associated with the thin filament in skeletal muscle cells, but its functions are not well understood. For this goal, we study skinned slow-twitch soleus muscle fibers from wild-type (Neb+) and conditional Neb knockout (Neb-) mice. We characterize cross-bridge (CB) kinetics and the elementary steps of the CB cycle by sinusoidal analysis during full Ca2+ activation and observe that Neb increases active tension 1.9-fold, active stiffness 2.7-fold, and rigor stiffness 3.0-fold. The ratio of stiffness during activation and rigor states is 62% in Neb+ fibers and 68% in Neb- fibers. These are approximately proportionate to the number of strongly attached CBs during activation. Because the thin filament length is 15% shorter in Neb- fibers than in Neb+ fibers, the increase in force per CB in the presence of Neb is ∼1.5 fold. The equilibrium constant of the CB detachment step (K 2), its rate (k 2), and the rate of the reverse force generation step (k -4) are larger in Neb+ fibers than in Neb- fibers. The rates of the force generation step (k 4) and the reversal detachment step (k -2) change in the opposite direction. These effects can be explained by Le Chatelier's principle: Increased CB strain promotes less force-generating state(s) and/or detached state(s). Further, when CB distributions among the six states are calculated, there is no significant difference in the number of strongly attached CBs between fibers with and without Neb. These results demonstrate that Neb increases force per CB. We also confirm that force is generated by isomerization of actomyosin (AM) from the AM.ADP.Pi state (ADP, adenosine diphophate; Pi, phosphate) to the AM*ADP.Pi state, where the same force is maintained after Pi release to result in the AM*ADP state. We propose that Neb changes the actin (and myosin) conformation for better ionic and hydrophobic/stereospecific AM interaction, and that the effect of Neb is similar to that of tropomyosin.

Cardiac-specific knockout of Lmod2 results in a severe reduction in myofilament force production and rapid cardiac failure

Leiomodin-2 (Lmod2) is a striated muscle-specific actin binding protein that is implicated in assembly of thin filaments. The necessity of Lmod2 in the adult mouse and role it plays in the mechanics of contraction are unknown. To answer these questions, we generated cardiac-specific conditional Lmod2 knockout mice (cKO). These mice die within a week of induction of the knockout with severe left ventricular systolic dysfunction and little change in cardiac morphology. Cardiac trabeculae isolated from cKO mice have a significant decrease in maximum force production and a blunting of myofilament length-dependent activation. Thin filaments are non-uniform and substantially reduced in length in cKO hearts, affecting the functional overlap of the thick and thin filaments. Remarkably, we also found that Lmod2 levels are directly linked to thin filament length and cardiac function in vivo, with a low amount (<20%) of Lmod2 necessary to maintain cardiac function. Thus, Lmod2 plays an essential role in maintaining proper cardiac thin filament length in adult mice, which in turn is necessary for proper generation of contractile force. Dysregulation of thin filament length in the absence of Lmod2 contributes to heart failure.

Nebulin Stiffens the Thin Filament and Augments Crossbridge Interaction - An X-Ray Diffraction Study on Intact Muscle

Nebulin, the giant sarcomeric protein is thought to play a role in thin filament length regulation but little is known about nebulin's influence on the thin filament structure in skeletal muscle. In the present work we compared the ultrastructure of relaxed and contracting intact soleus muscle dissected from conditional nebulin-knockout (cKO) and control (Ctrl) mice by using small-angle X-ray diffraction. Although the spacing of the actin subunit repeats (27.3 Å meridional reflection) was equal in both groups in relaxed muscles, during contraction the normalized percent change is more than the double in cKO (0.0054%/kPa) compared to that of the control (0.0020%/kPa). The myofibrillar area was determined by a detailed cross-sectional ultrastructural analysis thereby making the stiffness calculation of single thin filaments possible. The stiffness for 1-μm-long actin was calculated to be 38.7 pN/nm in Ctrl muscle and 14.8 pN/nm in cKO soleus. Furthermore, significantly shorter left-handed thin filament helical pitch (59 Å meridional reflection) was found in the absence of nebulin in passive muscles (58.88 Å in cKO vs. 59.14 Å in Ctrl); the shorter 59 Å helical pitch was maintained during contraction. Through the equatorial 1,0 and 1,1 intensities we found significantly less mass attached to the thin filaments in passive cKO muscles and inefficient myosin mass transfer during their contraction. According to our findings, nebulin plays a role in the passive state of muscle by fine-adjusting the 59 Å helical pitch of the thin filaments thereby modulating the binding sites of regulatory proteins along the long pitch groove of actin. Further, nebulin stiffens the thin filament which is critically important for skeletal muscle force development, explaining why the absence of the nebulin in nemaline myopathy patients leads to muscle weakness.

Dysfunctional sarcomere contractility contributes to muscle weakness in ACTA1-related nemaline myopathy (NEM3).

Nemaline myopathy (NM) is one of the most common congenital nondystrophic myopathies and is characterized by muscle weakness, often from birth. Mutations in ACTA1 are a frequent cause of NM (ie, NEM3). ACTA1 encodes alpha-actin 1, the main constituent of the sarcomeric thin filament. The mechanisms by which mutations in ACTA1 contribute to muscle weakness in NEM3 are incompletely understood. We hypothesized that sarcomeric dysfunction contributes to muscle weakness in NEM3 patients. To test this hypothesis, we performed contractility measurements in individual muscle fibers and myofibrils obtained from muscle biopsies of 14 NEM3 patients with different ACTA1 mutations. To identify the structural basis for impaired contractility, low angle X-ray diffraction and stimulated emission-depletion microscopy were applied. Our findings reveal that muscle fibers of NEM3 patients display a reduced maximal force-generating capacity, which is caused by dysfunctional sarcomere contractility in the majority of patients, as revealed by contractility measurements in myofibrils. Low angle X-ray diffraction and stimulated emission-depletion microscopy indicate that dysfunctional sarcomere contractility in NEM3 patients involves a lower number of myosin heads binding to actin during muscle activation. This lower number is not the result of reduced thin filament length. Interestingly, the calcium sensitivity of force is unaffected in some patients, but decreased in others. Dysfunctional sarcomere contractility is an important contributor to muscle weakness in the majority of NEM3 patients. This information is crucial for patient stratification in future clinical trials. Ann Neurol 2018;83:269-282.

Structural destabilization of tropomyosin induced by the cardiomyopathy-linked mutation R21H.

The missense mutation R21H in striated muscle tropomyosin is associated with hypertrophic cardiomyopathy, a genetic cardiac disease and a leading cause of sudden cardiac death in young people. Tropomyosin adopts conformation of a coiled coil which is critical for regulation of muscle contraction. In this study, we investigated the effects of the R21H mutation on the coiled-coil structure of tropomyosin and its interactions with its binding partners, tropomodulin and leiomodin. Using circular dichroism and isothermal titration calorimetry, we found that the mutation profoundly destabilized the structural integrity of αTM1a1-28 Zip, a chimeric peptide containing the first 28 residues of tropomyosin. The mutated αTM1a1-28 Zip was still able to interact with tropomodulin and leiomodin. However, the mutation resulted in a ∼30-fold decrease of αTM1a1-28 Zip's binding affinity to leiomodin. We used a crystal structure of αTM1a1-28 Zip that we solved at 1.5 Å resolution to study the mutation's effect in silico by means of molecular dynamics simulation. The simulation data indicated that while the mutation disrupted αTM1a1-28 Zip's coiled-coil structure, most notably from residue Ala18 to residue His31, it may not affect the N-terminal end of tropomyosin. The drastic decrease of αTM1a1-28 Zip's affinity to leiomodin caused by the mutation may lead to changes in the dynamics at the pointed end of thin filaments. Therefore, the R21H mutation is likely interfering with the regulation of the normal thin filament length essential for proper muscle contraction.

HSPB7 is indispensable for heart development by modulating actin filament assembly

Small heat shock protein HSPB7 is highly expressed in the heart. Several mutations within HSPB7 are associated with dilated cardiomyopathy and heart failure in human patients. However, the precise role of HSPB7 in the heart is still unclear. In this study, we generated global as well as cardiac-specific HSPB7 KO mouse models and found that loss of HSPB7 globally or specifically in cardiomyocytes resulted in embryonic lethality before embryonic day 12.5. Using biochemical and cell culture assays, we identified HSPB7 as an actin filament length regulator that repressed actin polymerization by binding to monomeric actin. Consistent with HSPB7's inhibitory effects on actin polymerization, HSPB7 KO mice had longer actin/thin filaments and developed abnormal actin filament bundles within sarcomeres that interconnected Z lines and were cross-linked by α-actinin. In addition, loss of HSPB7 resulted in up-regulation of Lmod2 expression and mislocalization of Tmod1. Furthermore, crossing HSPB7 null mice into an Lmod2 null background rescued the elongated thin filament phenotype of HSPB7 KOs, but double KO mice still exhibited formation of abnormal actin bundles and early embryonic lethality. These in vivo findings indicated that abnormal actin bundles, not elongated thin filament length, were the cause of embryonic lethality in HSPB7 KOs. Our findings showed an unsuspected and critical role for a specific small heat shock protein in directly modulating actin thin filament length in cardiac muscle by binding monomeric actin and limiting its availability for polymerization.

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.

Thin Filament Pointed Ends Redistribute in Response to Altered Thick Filaments

Striated muscle generates contractile force and shortens as polar actin (thin) filaments slide past bipolar myosin (thick) filaments. Maximal force is able to be produced when thin filaments completely overlap the motor domains (heads) which occurs when the thin filament slow-growing (pointed) ends extend to the central (∼0.2 micrometer) thick filament bare zone devoid of myosin heads. The assembly of thin and thick filaments into regular contractile units (sarcomeres) is complex and highly-regulated, yet also robust; allowing diverse vertebrate and invertebrate animals to produce a variety of sarcomeric architectures with specific physiological contractile properties. Maximal thin-thick filament overlap in vivo is highly conserved among all types of striated muscle examined and highlights its importance. How thin-thick filament overlap is specified and maintained in different muscles is uncertain, but it manifests as the uniform and precise specification of thin filament lengths and involves the exchange of actin subunits at pointed ends and dynamic pointed-end capping by tropomodulin (Tmod). Here, we use CRISPR-Cas9 gene editing and fluorescence microscopy to investigate whether interactions between myosin heads and pointed ends are necessary for specifying thin filament lengths in C. elegans obliquely striated body wall muscle. To determine the position of thin filament pointed ends, we inserted the green fluorescent protein (GFP) coding sequence in-frame with the C. elegans Tmd1 gene to generate a GFP-Tmod fusion protein. Homozygous GFP-Tmod worms are indistinguishable from wildtype worms demonstrating that the GFP fusion is completely functional. In body wall muscle, GFP-Tmod appears as regular striations that co-localize with myosin thick filaments. GFP-Tmod striations appear as single bands (singlets) or closely spaced double bands (doublets) in living worms confirming that thin-thick filament overlap is complete. In contrast, GFP-Tmod striations appear as more broadly-spaced doublets in headless-myoA transgenic worms that were designed to have an expanded (∼2 micrometer) thick filament bare zone. Our results show that thin filament pointed ends redistribute their location in response to changing the size of the thick filament bare zone. The response of pointed ends suggests that interactions with myosin heads stabilize pointed ends, increasing thin filament lengths until they fully overlap with thick filaments and permit optimal force generation.

Titin and Nebulin in Thick and Thin Filament Length Regulation.

In this review we discuss the history and the current state of ideas related to the mechanism of size regulation of the thick (myosin) and thin (actin) filaments in vertebrate striated muscles. Various hypotheses have been considered during of more than half century of research, recently mostly involving titin and nebulin acting as templates or 'molecular rulers', terminating exact assembly. These two giant, single-polypeptide, filamentous proteins are bound in situ along the thick and thin filaments, respectively, with an almost perfect match in the respective lengths and structural periodicities. However, evidence still questions the possibility that the proteins function as templates, or scaffolds, on which the thin and thick filaments could be assembled. In addition, the progress in muscle research during the last decades highlighted a number of other factors that could potentially be involved in the mechanism of length regulation: molecular chaperones that may guide folding and assembly of actin and myosin; capping proteins that can influence the rates of assembly-disassembly of the myofilaments; Ca2+ transients that can activate or deactivate protein interactions, etc. The entire mechanism of sarcomere assembly appears complex and highly dynamic. This mechanism is also capable of producing filaments of about the correct size without titin and nebulin. What then is the role of these proteins? Evidence points to titin and nebulin stabilizing structures of the respective filaments. This stabilizing effect, based on linear proteins of a fixed size, implies that titin and nebulin are indeed molecular rulers of the filaments. Although the proteins may not function as templates in the assembly of the filaments, they measure and stabilize exactly the same size of the functionally important for the muscles segments in each of the respective filaments.