Electron Tomography of Cryofixed,Isometrically Contracting Insect Flight Muscle Reveals Novel Actin-Myosin Interactions

Background

Isometric muscle contraction, where force is generated without muscle shortening, is a molecular traffic jam in which the number of actin-attached motors is maximized and all states of motor action are trapped with consequently high heterogeneity. This heterogeneity is a major limitation to deciphering myosin conformational changes in situ.

Methodology

We used multivariate data analysis to group repeat segments in electron tomograms of isometrically contracting insect flight muscle, mechanically monitored, rapidly frozen, freeze substituted, and thin sectioned. Improved resolution reveals the helical arrangement of F-actin subunits in the thin filament enabling an atomic model to be built into the thin filament density independent of the myosin. Actin-myosin attachments can now be assigned as weak or strong by their motor domain orientation relative to actin. Myosin attachments were quantified everywhere along the thin filament including troponin. Strong binding myosin attachments are found on only four F-actin subunits, the “target zone”, situated exactly midway between successive troponin complexes. They show an axial lever arm range of 77°/12.9 nm. The lever arm azimuthal range of strong binding attachments has a highly skewed, 127° range compared with X-ray crystallographic structures. Two types of weak actin attachments are described. One type, found exclusively in the target zone, appears to represent pre-working-stroke intermediates. The other, which contacts tropomyosin rather than actin, is positioned M-ward of the target zone, i.e. the position toward which thin filaments slide during shortening.

Conclusion

We present a model for the weak to strong transition in the myosin ATPase cycle that incorporates azimuthal movements of the motor domain on actin. Stress/strain in the S2 domain may explain azimuthal lever arm changes in the strong binding attachments. The results support previous conclusions that the weak attachments preceding force generation are very different from strong binding attachments.     

Three-Dimensional Organization of Troponin on Cardiac Muscle Thin Filaments in the Relaxed State

Muscle contraction is regulated by troponin-tropomyosin, which blocks and unblocks myosin binding sites on actin. To elucidate this regulatory mechanism, the three-dimensional organization of troponin and tropomyosin on the thin filament must be determined. Although tropomyosin is well defined in electron microscopy helical reconstructions of thin filaments, troponin density is mostly lost. Here, we determined troponin organization on native relaxed cardiac muscle thin filaments by applying single particle reconstruction procedures to negatively stained specimens. Multiple reference models led to the same final structure, indicating absence of model bias in the procedure. The new reconstructions clearly showed F-actin, tropomyosin, and troponin densities. At the 25 Å resolution achieved, troponin was considerably better defined than in previous reconstructions. The troponin density closely resembled the shape of troponin crystallographic structures, facilitating detailed interpretation of the electron microscopy density map. The orientation of troponin-T and the troponin core domain established troponin polarity. Density attributable to the troponin-I mobile regulatory domain was positioned where it could hold tropomyosin in its blocking position on actin, thus suggesting the underlying structural basis of thin filament regulation. Our previous understanding of thin filament regulation had been limited to known movements of tropomyosin that sterically block and unblock myosin binding sites on actin. We now show how troponin, the Ca²⁺ sensor, may control these movements, ultimately determining whether muscle contracts or relaxes.     

Three-Dimensional Organization of Troponin on Cardiac Muscle Thin Filaments in the Relaxed State

Muscle contraction is regulated by troponin-tropomyosin, which blocks and unblocks myosin binding sites on actin. To elucidate this regulatory mechanism, the three-dimensional organization of troponin and tropomyosin on the thin filament must be determined. Although tropomyosin is well defined in electron microscopy helical reconstructions of thin filaments, troponin density is mostly lost. Here, we determined troponin organization on native relaxed cardiac muscle thin filaments by applying single particle reconstruction procedures to negatively stained specimens. Multiple reference models led to the same final structure, indicating absence of model bias in the procedure. The new reconstructions clearly showed F-actin, tropomyosin, and troponin densities. At the 25 Å resolution achieved, troponin was considerably better defined than in previous reconstructions. The troponin density closely resembled the shape of troponin crystallographic structures, facilitating detailed interpretation of the electron microscopy density map. The orientation of troponin-T and the troponin core domain established troponin polarity. Density attributable to the troponin-I mobile regulatory domain was positioned where it could hold tropomyosin in its blocking position on actin, thus suggesting the underlying structural basis of thin filament regulation. Our previous understanding of thin filament regulation had been limited to known movements of tropomyosin that sterically block and unblock myosin binding sites on actin. We now show how troponin, the Ca²⁺ sensor, may control these movements, ultimately determining whether muscle contracts or relaxes.     

Direct Measurements of Local Coupling between Myosin Molecules Are Consistent with a Model of Muscle Activation

Muscle contracts due to ATP-dependent interactions of myosin motors with thin filaments composed of the proteins actin, troponin, and tropomyosin. Contraction is initiated when calcium binds to troponin, which changes conformation and displaces tropomyosin, a filamentous protein that wraps around the actin filament, thereby exposing myosin binding sites on actin. Myosin motors interact with each other indirectly via tropomyosin, since myosin binding to actin locally displaces tropomyosin and thereby facilitates binding of nearby myosin. Defining and modeling this local coupling between myosin motors is an open problem in muscle modeling and, more broadly, a requirement to understanding the connection between muscle contraction at the molecular and macro scale. It is challenging to directly observe this coupling, and such measurements have only recently been made. Analysis of these data suggests that two myosin heads are required to activate the thin filament. This result contrasts with a theoretical model, which reproduces several indirect measurements of coupling between myosin, that assumes a single myosin head can activate the thin filament. To understand this apparent discrepancy, we incorporated the model into stochastic simulations of the experiments, which generated simulated data that were then analyzed identically to the experimental measurements. By varying a single parameter, good agreement between simulation and experiment was established. The conclusion that two myosin molecules are required to activate the thin filament arises from an assumption, made during data analysis, that the intensity of the fluorescent tags attached to myosin varies depending on experimental condition. We provide an alternative explanation that reconciles theory and experiment without assuming that the intensity of the fluorescent tags varies.     

Modulation of contractile activation in skeletal muscle by a calcium-insensitive troponin C mutant

Calcium controls the level of muscle activation via interactions with the troponin complex. Replacement of the native, skeletal calcium-binding subunit of troponin, troponin C, with mixtures of functional cardiac and mutant cardiac troponin C insensitive to calcium and permanently inactive provides a novel method to alter the number of myosin cross-bridges capable of binding to the actin filament. Extraction of skeletal troponin C and replacement with functional and mutant cardiac troponin C were used to evaluate the relationship between the extent of thin filament activation (fractional calcium binding), isometric force, and the rate of force generation in muscle fibers independent of the calcium concentration. The experiments showed a direct, linear relationship between force and the number of cross-bridges attaching to the thin filament. Further, above 35% maximal isometric activation, following partial replacement with mixtures of cardiac and mutant troponin C, the rate of force generation was independent of the number of actin sites available for cross-bridge interaction at saturating calcium concentrations. This contrasts with the marked decrease in the rate of force generation when force was reduced by decreasing the calcium concentration. The results are consistent with hypotheses proposing that calcium controls the transition between weakly and strongly bound cross-bridge states.     

Structure and Orientation of Troponin in the Thin Filament

The troponin complex on the thin filament plays a crucial role in the regulation of muscle contraction. However, the precise location of troponin relative to actin and tropomyosin remains uncertain. We have developed a method of reconstructing thin filaments using single particle analysis that does not impose the helical symmetry of actin and is independent of a starting model. We present a single particle three-dimensional reconstruction of the thin filament. Atomic models of the F-actin filament were fitted into the electron density maps and troponin and tropomyosin located. The structure provides evidence that the globular head region of troponin labels the two strands of actin with a 27.5-Å axial stagger. The density attributed to troponin appears tapered with the widest point toward the barbed end. This leads us to interpret the polarity of the troponin complex in the thin filament as reversed with respect to the widely accepted model.Regulation of actin filament function is a fundamental biological process with implications ranging from cell migration to muscle contraction. Skeletal and cardiac muscle thin filaments consist of actin and the regulatory proteins troponin and tropomyosin. Contraction is initiated by release of Ca²⁺ into the sarcomere and the consequent binding of Ca²⁺ to regulatory sites on troponin. Troponin is believed to undergo a conformational change leading to an azimuthal movement of tropomyosin, which allows myosin heads to interact with actin, hydrolyze ATP, and generate force. The molecular basis by which troponin acts to regulate muscle contraction is only partly understood. It is essential that the structure of troponin in the thin filament at high and low Ca²⁺ is determined to properly understand the mechanism of regulation.The basic structure of the thin filament was described by Ebashi in 1972 (1). In this structure each tropomyosin molecule covers seven actin monomers, and there is a 27.5-Å stagger between troponin molecules. The 7-Å tropomyosin structure (2), the atomic model of F-actin (3), and the troponin “core domain” (4) have recently been used to generate atomic models of the thin filament in low and high Ca²⁺ states (5). While the position of troponin in these models was constrained by known distance measurements between filament components, the exact arrangement of the complex on the filament has not been determined a priori. Although recently published crystal structures of partial troponin complexes (4, 6) have provided valuable insights into the arrangement of the globular head or core domain, the complex in its entirety has not been crystallized.Troponin is believed to consist of a globular core domain with an extended tail (7). The globular core contains the Ca²⁺-binding subunit (TnC),² the inhibitory subunit (TnI), and the C-terminal part (residues 156–262) of the tropomyosin-binding subunit (TnT). The extended tail consists of the N-terminal part of TnT (residues 1–155). A structural rearrangement associated with Ca²⁺ dissociation from the troponin core has been observed (4) such that the helix connecting the two domains of TnC collapses, releasing the TnI inhibitory segment. It is postulated that the TnI inhibitory segment then becomes able to bind actin, in so doing biasing tropomyosin (8). To understand properly how Ca²⁺ binding to TnC leads to movement of tropomyosin, it is necessary to determine a high resolution structure of troponin attached to the thin filament, allowing unambiguous docking of the available crystal structures and direct observation of any changes at a molecular level caused by Ca²⁺ binding.Direct visualization of the thin filament is possible using electron microscopy. Tropomyosin strands have been resolved in the low and high Ca²⁺ states confirming the movement of tropomyosin and the steric blocking model (9, 10). Until recently the actin helical repeat has been imposed in the majority of reconstructions of the thin filament causing artifacts. Helical averaging using the actin repeat spreads troponin density over every actin monomer, which prevents the detailed position and shape of the troponin complex from being found (11). It is possible to avoid this effect by applying a single particle approach. Individual filament images are divided into segments and each segment treated as a particle. Three-dimensional reconstruction may then be carried out by single particle techniques of alignment, classification (12, 13), Euler angle assignment (14–16) and exact filter back-projection (17, 18).Two forms of single particle analysis have emerged: helical single particle analysis (19), where the determined helical symmetry is applied to the final reconstruction, and non-helical single particle analysis, which treats the complex as a truly asymmetric particle. Helical single particle analysis has been used to successfully reconstruct a myosin containing invertebrate thick filament to a resolution of 25 Å (20), and non-helical single particle analysis has been applied to the vertebrate skeletal muscle thick filament allowing azimuthal perturbations of the myosin heads to be observed (21).Model-based single particle image processing methods have recently been applied to the structural analysis of the vertebrate (5, 22, 23) and the insect thin filament (24). We have deliberately avoided starting with a model and any potential model bias by using a reference-free alignment procedure. The adaptation of conventional procedures and their application to the structural study of the muscle thin filament has been documented (25).     

Study of reciprocal effects of cardiac myosin and tropomyosin isoforms on actin-myosin interaction with in vitro motility assay

Interaction of myosin with actin in striated muscle is controlled by Ca²⁺ via thin filament associated proteins: troponin and tropomyosin. In cardiac muscle there is a whole pattern of myosin and tropomyosin isoforms. The aim of the current work is to study regulatory effect of tropomyosin on sliding velocity of actin filaments in the in vitro motility assay over cardiac isomyosins. It was found that tropomyosins of different content of α- and β-chains being added to actin filament effects the sliding velocity of filaments in different ways. On the other hand the velocity of filaments with the same tropomyosins depends on both heavy and light chains isoforms of cardiac myosin.     

Troponin Revealed: Uncovering the Structure of the Thin Filament On-Off Switch in Striated Muscle

Recently, our understanding of the structural basis of troponin-tropomyosin’s Ca²⁺-triggered regulation of striated muscle contraction has advanced greatly, particularly via cryo-electron microscopy data. Compelling atomic models of troponin-tropomyosin-actin were published for both apo- and Ca²⁺-saturated states of the cardiac thin filament. Subsequent electron microscopy and computational analyses have supported and further elaborated the findings. Per cryo-electron microscopy, each troponin is highly extended and contacts both tropomyosin strands, which lie on opposite sides of the actin filament. In the apo-state characteristic of relaxed muscle, troponin and tropomyosin hinder strong myosin-actin binding in several different ways, apparently barricading the actin more substantially than does tropomyosin alone. The troponin core domain, the C-terminal third of TnI, and tropomyosin under the influence of a 64-residue helix of TnT located at the overlap of adjacent tropomyosins are all in positions that would hinder strong myosin binding to actin. In the Ca²⁺-saturated state, the TnI C-terminus dissociates from actin and binds in part to TnC; the core domain pivots significantly; the N-lobe of TnC binds specifically to actin and tropomyosin; and tropomyosin rotates partially away from myosin’s binding site on actin. At the overlap domain, Ca²⁺ causes much less tropomyosin movement, so a more inhibitory orientation persists. In the myosin-saturated state of the thin filament, there is a large additional shift in tropomyosin, with molecular interactions now identified between tropomyosin and both actin and myosin. A new era has arrived for investigation of the thin filament and for functional understandings that increasingly accommodate the recent structural results.     

Electron tomography of fast frozen, stretched rigor fibers reveals elastic distortions in the myosin crossbridges

As a first step toward freeze-trapping and 3-D modeling of the very rapid load-induced structural responses of active myosin heads, we explored the conformational range of longer lasting force-dependent changes in rigor crossbridges of insect flight muscle (IFM). Rigor IFM fibers were slam-frozen after ramp stretch (1000 ms) of 1-2% and freeze-substituted. Tomograms were calculated from tilt series of 30 nm longitudinal sections of Araldite-embedded fibers. Modified procedures of alignment and correspondence analysis grouped self-similar crossbridge forms into 16 class averages with 4.5 nm resolution, revealing actin protomers and myosin S2 segments of some crossbridges for the first time in muscle thin sections. Acto-S1 atomic models manually fitted to crossbridge density required a range of lever arm adjustments to match variably distorted rigor crossbridges. Some lever arms were unchanged compared with low tension rigor, while others were bent and displaced M-ward by up to 4.5 nm. The average displacement was 1.6 +/- 1.0 nm. "Map back" images that replaced each unaveraged 39 nm crossbridge motif by its class average showed an ordered mix of distorted and unaltered crossbridges distributed along the 116 nm repeat that reflects differences in rigor myosin head loading even before stretch.     

Conformation of the troponin core complex in the thin filaments of skeletal muscle during relaxation and active contraction

Contraction of skeletal and cardiac muscles is regulated by Ca(2+) binding to troponin in the actin-containing thin filaments, leading to an azimuthal movement of tropomyosin around the filament that uncovers the myosin binding sites on actin. Here, we use polarized fluorescence to determine the orientation of the C-terminal lobe of troponin C (TnC) in skeletal muscle cells as a step toward elucidating the molecular mechanism of troponin-mediated regulation. Assuming, as shown by X-ray crystallography, that this lobe of TnC is part of a well-defined troponin domain called the IT arm, we show that the coiled coil formed by troponin components I and T makes an angle of about 55° with the thin filament axis in relaxed muscle, in contrast with previous models based on electron microscopy in which this angle is close to 0°. The E helix of TnC makes an angle of about 45° with the thin filament axis. Both the IT coiled coil and the TnC E helix tilt by about 10° on muscle activation. By combining in situ measurements of the orientation of the IT arm and regulatory domain of troponin, which together form the troponin core complex, with published intermolecular distances between thin filament components, we derive models of thin filament structure in which the IT arm of troponin holds its regulatory domain close to the actin surface. Although the structure and function of troponin regions outside the core complex remain to be characterized, the present results provide useful constraints for molecular models of the mechanism of muscle regulation.     

Cryo-EM Structures of the Actin:Tropomyosin Filament Reveal the Mechanism for the Transition from C- to M-State

Tropomyosin (Tm) is a key factor in the molecular mechanisms that regulate the binding of myosin motors to actin filaments (F-Actins) in most eukaryotic cells. This regulation is achieved by the azimuthal repositioning of Tm along the actin (Ac):Tm:troponin (Tn) thin filament to block or expose myosin binding sites on Ac. In striated muscle, including involuntary cardiac muscle, Tm regulates muscle contraction by coupling Ca^2 + binding to Tn with myosin binding to the thin filament. In smooth muscle, the switch is the posttranslational modification of the myosin. Depending on the activation state of Tn and the binding state of myosin, Tm can occupy the blocked, closed, or open position on Ac. Using native cryogenic 3DEM (three-dimensional electron microscopy), we have directly resolved and visualized cardiac and gizzard muscle Tm on filamentous Ac in the position that corresponds to the closed state. From the 8-Å-resolution structure of the reconstituted Ac:Tm filament formed with gizzard-derived Tm, we discuss two possible mechanisms for the transition from closed to open state and describe the role Tm plays in blocking myosin tight binding in the closed-state position.     

Ca(2+)-dependent, myosin subfragment 1-induced proximity changes between actin and the inhibitory region of troponin I.

In order to help understand the spatial rearrangements of thin filament proteins during the regulation of muscle contraction, we used fluorescence resonance energy transfer (FRET) to measure Ca(2+)-dependent, myosin-induced changes in distances and fluorescence energy transfer efficiencies between actin and the inhibitory region of troponin I (TnI). We labeled the single Cys-117 of a mutant TnI with N-(iodoacetyl)-N'-(1-sulfo-5-naphthyl)ethylenediamine (IAEDANS) and Cys-374 of actin with 4-dimethylaminophenylazophenyl-4'-maleimide (DABmal). These fluorescent probes were used as donor and acceptor, respectively, for the FRET measurements. We reconstituted a troponin-tropomyosin (Tn-Tm) complex which contained the AEDANS-labeled mutant TnI, together with natural troponin T (TnT), troponin C (TnC) and tropomyosin (Tm) from rabbit fast skeletal muscle. Fluorescence titration of the AEDANS-labeled Tn-Tm complex with DABmal-labeled actin, in the presence and absence of Ca(2+), resulted in proportional, linear increases in energy transfer efficiency up to a 7:1 molar excess of actin over Tn-Tm. The distance between AEDANS on TnI Cys-117 and DABmal on actin Cys-374 increased from 37.9 A to 44.1 A when Ca(2+) bound to the regulatory sites of TnC. Titration of reconstituted thin filaments, containing AEDANS-labeled Tn-Tm and DABmal-labeled actin, with myosin subfragment 1 (S1) decreased the energy transfer efficiency, in both the presence and absence of Ca(2+). The maximum decrease occurred at well below stoichiometric levels of S1 binding to actin, showing a cooperative effect of S1 on the state of the thin filaments. S1:actin molar ratios of approximately 0.1 in the presence of Ca(2+), and approximately 0.3 in the absence of Ca(2+), were sufficient to cause a 50% reduction in normalized transfer efficiency. The distance between AEDANS on TnI Cys-117 and DABmal on actin Cys-374 increased by approximately 7 A in the presence of Ca(2+) and by approximately 2 A in the absence of Ca(2+) when S1 bound to actin. Our results suggest that TnI's interaction with actin inhibits actomyosin ATPase activity by modulating the equilibria among active and inactive states of the thin filament. Structural rearrangements caused by myosin S1 binding to the thin filament, as detected by FRET measurements, are consistent with the cooperative behavior of the thin filament proteins.     

Cooperative interactions between troponin molecules bound to the cardiac thin filament

Striated muscle thin filaments contain many troponin molecules, which contact each other indirectly via tropomyosin and actin. Such allosteric interactions between troponin molecules may be responsible for cooperative Ca2+ binding to the regulatory sites of the cardiac thin filament (Tobacman, L. S., and Sawyer, D. S. (1990) J. Biol. Chem. 265, 931-939). To test whether thin filament-bound troponin molecules interact, we studied the competitive binding of troponin and troponin T-troponin I (an inhibitory complex lacking the Ca2+ binding subunit troponin C) to actin-tropomyosin. The relative affinities of these two forms of troponin for the thin filament depended upon their relative concentrations. Under conditions where total binding was saturated, each form binds with greater apparent affinity to sites that have similar neighbors. A theoretical model for competitive binding of two ligands to interacting sites on a linear lattice was developed and fit to the data. Surprisingly, energetically unfavorable interactions occurred between adjacent troponin and troponin T-troponin I molecules not only in the presence of Ca2+, but also in the presence of [ethylenebis(oxyethylenenitrilo)]tetraacetic acid and/or myosin subfragment 1. Removal of Ca2+ strengthened the affinity of troponin for the thin filament less than 50%. These results suggest that, even in the absence of myosin, long range allosteric interactions occur between troponin molecules. The detailed involvement of tropomyosin and actin in these interactions remains to be established.     

Calpain proteolysis of free and bound forms of calponin, a troponin T-like protein in smooth muscle

Calponin, a novel homologue of troponin T, purified from chicken gizzard was found to be one of the most susceptible proteins among smooth muscle contraction-associated proteins to hydrolysis by calpain I purified from human red blood cells. The high susceptibility of calponin was comparable to that reported for troponin T. The rate of degradation of calponin, unlike caldesmon and myosin light chain kinase, was accelerated when bound to calmodulin. When calponin existed as a bound form in both reconstituted actin filament and native thin filament, the rate of proteolysis was markedly retarded, indicating close association of calponin with actin filament. These observations are compatible with the view that calponin is an integral part of the actin-linked contractile machinery in smooth muscle.     

A structural origin of latency relaxation in frog skeletal muscle

A time-resolved x-ray diffraction study at a time resolution of 0.53 ms was made to investigate the structural origin of latency relaxation (LR) in frog skeletal muscle. Intensity and spacing measurements were made on meridional reflections from the Ca-binding protein troponin and the thick filament and on layer lines from the thin filament. At 16 degrees C, the intensity and spacing of all reflections started to change at 4 ms, simultaneously with the LR. At 0 degrees C, the intensity of the troponin reflection and the layer lines from the thin filament and the spacing of the 14.3-nm myosin meridional reflection, but not the spacing of other myosin meridional reflections, began to change at approximately 15 ms, when the LR also started. Intensity of myosin-based reflections started to change later. When the muscle was stretched to non-overlap length, the intensity and spacing changes of the myosin reflections disappeared. The simultaneous spacing change of the 14.3-nm myosin meridional reflection with the LR suggests that detachment of myosin heads that are bound to actin in the resting muscle is the cause of the LR.     

Suppression of muscle hypercontraction by mutations in the myosin heavy chain gene of Drosophila melanogaster

The indirect flight muscles (IFM) of Drosophila melanogaster provide a good genetic system with which to investigate muscle function. Flight muscle contraction is regulated by both stretch and Ca(2+)-induced thin filament (actin + tropomyosin + troponin complex) activation. Some mutants in troponin-I (TnI) and troponin-T (TnT) genes cause a "hypercontraction" muscle phenotype, suggesting that this condition arises from defects in Ca(2+) regulation and actomyosin-generated tension. We have tested the hypothesis that missense mutations of the myosin heavy chain gene, Mhc, which suppress the hypercontraction of the TnI mutant held-up(2) (hdp(2)), do so by reducing actomyosin force production. Here we show that a "headless" Mhc transgenic fly construct that reduces the myosin head concentration in the muscle thick filaments acts as a dose-dependent suppressor of hypercontracting alleles of TnI, TnT, Mhc, and flightin genes. The data suggest that most, if not all, mutants causing hypercontraction require actomyosin-produced forces to do so. Whether all Mhc suppressors act simply by reducing the force production of the thick filament is discussed with respect to current models of myosin function and thin filament activation by the binding of calcium to the troponin complex.     

Interaction between troponin and myosin enhances contractile activity of myosin in cardiac muscle

Ca(2+) signaling in striated muscle cells is critically dependent upon thin filament proteins tropomyosin (Tm) and troponin (Tn) to regulate mechanical output. Using in vitro measurements of contractility, we demonstrate that even in the absence of actin and Tm, human cardiac Tn (cTn) enhances heavy meromyosin MgATPase activity by up to 2.5-fold in solution. In addition, cTn without Tm significantly increases, or superactivates sliding speed of filamentous actin (F-actin) in skeletal motility assays by at least 12%, depending upon [cTn]. cTn alone enhances skeletal heavy meromyosin's MgATPase in a concentration-dependent manner and with sub-micromolar affinity. cTn-mediated increases in myosin ATPase may be the cause of superactivation of maximum Ca(2+)-activated regulated thin filament sliding speed in motility assays relative to unregulated skeletal F-actin. To specifically relate this classical superactivation to cardiac muscle, we demonstrate the same response using motility assays where only cardiac proteins were used, where regulated cardiac thin filament sliding speeds with cardiac myosin are >50% faster than unregulated cardiac F-actin. We additionally demonstrate that the COOH-terminal mobile domain of cTnI is not required for this interaction or functional enhancement of myosin activity. Our results provide strong evidence that the interaction between cTn and myosin is responsible for enhancement of cross-bridge kinetics when myosin binds in the vicinity of Tn on thin filaments. These data imply a novel and functionally significant molecular interaction that may provide new insights into Ca(2+) activation in cardiac muscle cells.     

Electron tomography of swollen rigor fibers of insect flight muscle reveals a short and variably angled S2 domain

Subfragment 2 (S2), the segment that links the two myosin heads to the thick filament backbone, may serve as a swing-out adapter allowing crossbridge access to actin, as the elastic component of crossbridges and as part of a phosphorylation-regulated on-off switch for crossbridges in smooth muscle. Low-salt expansion increases interfilament spacing (from 52 nm to 67 nm) of rigor insect flight muscle fibers and exposes a tethering segment of S2 in many crossbridges. Docking an actoS1 atomic model into EM tomograms of swollen rigor fibers identifies in situ for the first time the location, length and angle assignable to a segment of S2. Correspondence analysis of 1831 38.7 nm crossbridge repeats grouped self-similar forms from which class averages could be computed. The full range of the variability in angles and lengths of exposed S2 was displayed by using class averages for atomic fittings of acto-S1, while S2 was modeled by fitting a length of coiled-coil to unaveraged individual repeats. This hybrid modeling shows that the average length of S2 tethers along the thick filament (except near the tapered ends) is approximately 10 nm, or 16% of S2's total length, with an angular range encompassing 90 degrees axially and 120 degrees azimuthally. The large range of S2 angles indicates that some rigor bridges produce positive force that must be balanced by others producing drag force. The short tethering segment clarifies constraints on the function of S2 in accommodating variable myosin head access to actin. We suggest that the short length of S2 may also favor intermolecular head-head interactions in IFM relaxed thick filaments.     

Myosin binding-induced cooperative activation of the thin filament in cardiac myocytes and skeletal muscle fibers.

Myosin binding-induced activation of the thin filament was examined in isolated cardiac myocytes and single slow and fast skeletal muscle fibers. The number of cross-bridge attachments was increased by stepwise lowering of the [MgATP] in the Ca(2+)-free solution bathing the preparations. The extent of thin filament activation was determined by monitoring steadystate isometric tension at each MgATP concentration. As pMgATP (where pMgATP is -log [MgATP]) was increased from 3.0 to 8.0, isometric tension increased to a peak value in the pMgATP range of 5.0-5.4. The steepness of the tension-pMgATP relationship, between the region of the curve where tension was zero and the peak tension, is hypothesized to be due to myosin-induced cooperative activation of the thin filament. Results showed that the steepness of the tension-pMgATP relationship was markedly greater in cardiac as compared with either slow or fast skeletal muscle fibers. The steeper slope in cardiac myocytes provides evidence of greater myosin binding-induced cooperative activation of the thin filament in cardiac as compared with skeletal muscle, at least under these experimental conditions of nominal free Ca2+. Cooperative activation is also evident in the tension-pCa relation, and is dependent upon thin filament molecular interactions, which require the presence of troponin C. Thus, it was determined whether myosin-based cooperative activation of the thin filament also requires troponin C. Partial extraction of troponin C reduced the steepness of the tension-pMgATP relationship, with the effect being significantly greater in cardiac than in skeletal muscle. After partial extraction of troponin C, muscle type differences in the steepness of the tension-pMgATP relationship were no longer apparent, and reconstitution with purified troponin C restored the muscle lineage differences. These results suggest that, in the absence of Ca2+, myosin-mediated activation of the thin filament is greater in cardiac than in skeletal muscle, and this apparent cooperativity requires the presence of troponin C on thin filament regulatory strands.     

The troponin tail domain promotes a conformational state of the thin filament that suppresses myosin activity

In cardiac and skeletal muscles tropomyosin binds to the actin outer domain in the absence of Ca(2+), and in this position tropomyosin inhibits muscle contraction by interfering sterically with myosin-actin binding. The globular domain of troponin is believed to produce this B-state of the thin filament (Lehman, W., Hatch, V., Korman, V. L., Rosol, M., Thomas, L. T., Maytum, R., Geeves, M. A., Van Eyk, J. E., Tobacman, L. S., and Craig, R. (2000) J. Mol. Biol. 302, 593-606) via troponin I-actin interactions that constrain the tropomyosin. The present study shows that the B-state can be promoted independently by the elongated tail region of troponin (the NH(2) terminus (TnT-(1-153)) of cardiac troponin T). In the absence of the troponin globular domain, TnT-(1-153) markedly inhibited both myosin S1-actin-tropomyosin MgATPase activity and (at low S1 concentrations) myosin S1-ADP binding to the thin filament. Similarly, TnT-(1-153) increased the concentration of heavy meromyosin required to support in vitro sliding of thin filaments. Electron microscopy and three-dimensional reconstruction of thin filaments containing TnT-(1-153) and either cardiac or skeletal muscle tropomyosin showed that tropomyosin was in the B-state in the complete absence of troponin I. All of these results indicate that portions of the troponin tail domain, and not only troponin I, contribute to the positioning of tropomyosin on the actin outer domain, thereby inhibiting muscle contraction in the absence of Ca(2+).