Elastic filaments in situ in cardiac muscle: deep-etch replica analysis in combination with selective removal of actin and myosin filaments

To clarify the full picture of the connectin (titin) filament network in situ, we selectively removed actin and myosin filaments from cardiac muscle fibers by gelsolin and potassium acetate treatment, respectively, and observed the residual elastic filament network by deep-etch replica electron microscopy. In the A bands, elastic filaments of uniform diameter (6-7 nm) projecting from the M line ran parallel, and extended into the I bands. At the junction line in the I bands, which may correspond to the N2 line in skeletal muscle, individual elastic filaments branched into two or more thinner strands, which repeatedly joined and branched to reach the Z line. Considering that cardiac muscle lacks nebulin, it is very likely that these elastic filaments were composed predominantly of connectin molecules; indeed, anti-connectin monoclonal antibody specifically stained these elastic filaments. Further, striations of approximately 4 nm, characteristic of isolated connectin molecules, were also observed in the elastic filaments. Taking recent analyses of the structure of isolated connectin molecules into consideration, we concluded that individual connectin molecules stretched between the M and Z lines and that each elastic filament consisted of laterally-associated connectin molecules. Close comparison of these images with the replica images of intact and S1-decorated sarcomeres led us to conclude that, in intact sarcomeres, the elastic filaments were laterally associated with myosin and actin filaments in the A and I bands, respectively. Interestingly, it was shown that the elastic property of connectin filaments was not restricted by their lateral association with actin filaments in intact sarcomeres. Finally, we have proposed a new structural model of the cardiac muscle sarcomere that includes connectin filaments.     

Connectin filaments link thick filaments and Z lines in frog skeletal muscle as revealed by immunoelectron microscopy

In an earlier study connectin, an elastic protein of striated muscle, was found to be associated with "gap filaments" originating from the thick filaments in the myofibril, but it was not clear whether it extends to Z lines or not (Maruyama, K., H. Sawada, S. Kimura, K. Ohashi, H. Higuchi, and Y. Umazume, 1984, J. Cell Biol., 99:1391-1397). In the present immunoelectron microscopic study using polyclonal antibodies against native connectin, we have concluded that the connectin structures are directly linked to Z lines from the thick (myosin) filaments in myofibrils of skinned fibers of frog skeletal muscle. There were five distinct antibody-binding stripes in each half of the A band and two stripes in the A-I junction region. Deposits of antibodies were recognized in I bands and Z lines. We suggest that connectin filaments run alongside the thick filaments, starting from a region approximately 0.15 micron from the center of the A band.     

Partial activation of thin filaments in resting frog skeletal muscle fibers

X-ray patterns from frog skeletal muscles at rest show a series of relatively weak meridional reflections which may be indexed as the 5, 7, 9, 11 and 13 orders of the repeat period of about 2 x 385 A. According to the model of the thin filaments structure, suggested by V. V. Lednev and G. M. Frank (1977), this period is specific for the activated or "switched on" state of the actin--containing filaments. At the same time, according to the generally accepted model (suggested in 1972), the axial repeat period of the thin filament structure is approximately equal to 385 A and does not depend on the functional state of the muscle. The existence of the repeat period of about 2 x 385 A in the thin filaments of a resting muscle suggests that even at rest the thin filaments of vertebrate skeletal muscle are not completely inhibited. It may be suggested that partial activation of the thin filaments in a resting muscle is the result of formation of long life rigorlike crossbridges, the existence of which was postulated by D. K. Hill in 1968 on the basis of his studies on resting tension in the frog skeletal muscle.     

Compliance of thin filaments in skinned fibers of rabbit skeletal muscle.

The mechanical compliance (reciprocal of stiffness) of thin filaments was estimated from the relative compliance of single, skinned muscle fibers in rigor at sarcomere lengths between 1.8 and 2.4 micron. The compliance of the fibers was calculated as the ratio of sarcomere length change to tension change during imposition of repetitive cycles of small stretches and releases. Fiber compliance decreased as the sarcomere length was decreased below 2.4 micron. The compliance of the thin filaments could be estimated from this decrement because in this range of lengths overlap between the thick and thin filaments is complete and all of the myosin heads bind to the thin filament in rigor. Thus, the compliance of the overlap region of the sarcomere is constant as length is changed and the decrease in fiber compliance is due to decrease of the nonoverlap length of the thin filaments (the I band). The compliance value obtained for the thin filaments implies that at 2.4-microns sarcomere length, the thin filaments contribute approximately 55% of the total sarcomere compliance. Considering that the sarcomeres are approximately 1.25-fold more compliant in active isometric contractions than in rigor, the thin filaments contribute approximately 44% to sarcomere compliance during isometric contraction.     

Elastic behavior of connectin filaments during thick filament movement in activated skeletal muscle

Connectin (also called titin) is a huge, striated muscle protein that binds to thick filaments and links them to the Z-disc. Using an mAb that binds to connectin in the I-band region of the molecule, we studied the behavior of connectin in both relaxed and activated skinned rabbit psoas fibers by immunoelectron microscopy. In relaxed fibers, antibody binding is visualized as two extra striations per sarcomere arranged symmetrically about the M-line. These striations move away from both the nearest Z-disc and the thick filaments when the sarcomere is stretched, confirming the elastic behavior of connectin within the I- band of relaxed sarcomeres as previously observed by several investigators. When the fiber is activated, thick filaments in sarcomeres shorter than 2.8 microns tend to move from the center to the side of the sarcomere. This translocation of thick filaments within the sarcomere is accompanied by movement of the antibody label in the same direction. In that half-sarcomere in which the thick filaments move away from the Z-disc, the spacings between the Z-disc and the antibody and between the antibody and the thick filaments both increase. Conversely, on the side of the sarcomere in which the thick filaments move nearer to the Z-line, these spacings decrease. Regardless of whether I-band spacing is varied by stretch of a relaxed sarcomere or by active sliding of thick filaments within a sarcomere of constant length, the spacings between the Z-line and the antibody and between the antibody and the thick filaments increase with I-band length identically. These results indicate that the connectin filaments remain bound to the thick filaments in active fibers, and that the elastic properties of connectin are unaltered by calcium ions and cross-bridge activity.     

Binding of myosin cross-bridges to thin filaments of rabbit skeletal muscle.

Cross-linking of myosin subfragment 1 (S1) with a molar excess of actin in vitro reveals the presence of an actin-S1-actin complex. It is absolutely essential that actin be present in molar excess over S1 so that the decoration of F-actin with S1 be incomplete. However, the excess of actin may not be available in the overlap zone of sarcomeres of skeletal muscle. We therefore found it necessary to test for the presence of the actin-S1-actin complex in vivo. Myofibrils from rabbit skeletal muscle were reacted with zero-length cross-linker, the products were resolved by polyacrylamide gel electrophoresis and analyzed by Western blots using antibodies against actin and against heavy and light chains of myosin. The cross-linking produced the evidence of formation of actin-S1-actin complex.     

Clinical and genetic heterogeneity in nemaline myopathy--a disease of skeletal muscle thin filaments

The term nemaline myopathy (NM) encompasses a heterogeneous group of disorders of primary skeletal muscle weakness characterized by the presence of nemaline rods in muscles of affected individuals. Disease severity is variable and unpredictable, with prognosis ranging from neonatal death to almost normal motor function. Recent advances in the identification of NM disease genes demonstrate that NM is a disease of the skeletal muscle sarcomere and, in particular, of the thin filaments. These findings are starting to alter the approach that neurologists and geneticists take to diagnosing and counseling patients with NM, and could lead to insights into specific directed therapies in the future.     

Organization of filaments underneath the plasma membrane of developing chicken skeletal muscle cells in vitro revealed by the freeze-dry and rotary replica method

Summary Cytoskeletal organization and its association with plasma membranes in embryonic chick skeletal muscle cells in vitro was studied by the freeze-drying and rotary-shadowing method of physically ruptured cells. The cytoskeletal filaments underlying the plasma membranes were sparse in myogenic cells at the stage when cells exhibited great lipid fluidity in plasma membranes (fusion competent mononucleated myoblasts and recently fused young myotubes). Myotubes at more advanced stages of development possessed a highly interconnected dense filamentous network just underneath the cell membrane. This subsarcolemmal network was composed predominantly of 8–10 nm filaments; they were identified as actin filaments because of their decoration with myosin subfragment-1. Fine fibrils having a diameter of 3–5 nm were found on the protoplasmic surface of the plasmalemma at both the early and advanced stages of development. They were associated with the subsarcolemmal cytoskeletal filaments. Short 2–5 nm cross-linking filaments were occasionally seen between filaments in the subsarcolemmal network. We conclude that, although the subsarcolemmal cytoskeletal network contains many actin filaments, this domain appears to play some role in preserving the cell shape in the form of the membrane skeleton rather than membrane mobility.     

Ca(2+)-induced movement of tropomyosin in skeletal muscle thin filaments observed by multi-site FRET

To obtain information on Ca(2+)-induced tropomyosin (Tm) movement in Ca(2+)-regulated muscle thin filaments, frequency-domain fluorescence energy transfer data were collected between 5-(2-iodoacetyl-amino-ethyl-amino)naphthalene-1-sulfonic acid at Cys-190 of Tm and phalloidin-tetramethylrhodamine B isothiocyanate bound to F-actin. Two models were used to fit the experimental data: an atomic coordinate (AC) model coupled with a search algorithm that varies the position and orientation of Tm on F-actin, and a double Gaussian distance distribution (DD) model. The AC model showed that little or no change in transfer efficiency is to be expected between different sites on F-actin and Tm if Ca(2+) causes azimuthal movement of Tm of the magnitude suggested by structural data (C. Xu, R. Craig, L. Tobacman, R. Horowitz, and W. Lehman. 1999. Biophys. J. 77:985-992). However, Ca(2+) produced a small but significant change in our phase/modulation versus frequency data, showing that changes in lifetime decay can be detected even when a change of the steady-state transfer efficiency is very small. A change in Tm azimuthal position of 17 on the actin filament obtained with the AC model indicates that solution data are in reasonable agreement with EM image reconstruction data. In addition, the data indicate that Tm also appears to rotate about its axis, resulting in a rolling motion over the F-actin surface. The DD model showed that the distance from one of the two chains of Tm to F-actin was mainly affected, further verifying that Ca(2+) causes Tm to roll over the F-actin surface. The width of the distance distributions indicated that the position of Tm in absence and in presence of Ca(2+) is well defined with appreciable local flexibility.     

Tropomyosin positions in regulated thin filaments revealed by cryoelectron microscopy.

Past attempts to detect tropomyosin in electron micrograph images of frozen-hydrated troponin-regulated thin filaments under relaxing conditions have not been successful. This raised the possibility that tropomyosin may be disordered on filaments in the off-state, a possibility at odds with the steric blocking model of muscle regulation. By using cryoelectron microscopy and helical image reconstruction we have now resolved the location of tropomyosin in both relaxing and activating conditions. In the off-state, tropomyosin adopts a position on the outer domain of actin with a binding site virtually identical to that determined previously by negative staining, although at a radius of 3.8 nm, slightly higher than found in stained filaments. Molecular fitting to the atomic model of F-actin shows that tropomyosin is localized over sites on actin subdomain 1 required for myosin binding. Restricting access to these sites would inhibit the myosin-cross-bridge cycle, and hence contraction. Under high Ca(2+) activating conditions, tropomyosin moved azimuthally, away from its blocking position to the same site on the inner domain of actin previously determined by negative staining, also at 3.8 nm radius. These results provide strong support for operation of the steric mechanism of muscle regulation under near-native solution conditions and also validate the use of negative staining in investigations of muscle thin filament structure.     

Calcium-induced changes in the location and conformation of troponin in skeletal muscle thin filaments.

Troponin is the regulatory protein of striated muscle. Without Ca2+, the contraction of striated muscle is inhibited. Binding of Ca2+ to troponin activates contraction. The location of troponin on the thin filaments and its relation to the regulatory mechanism has been unknown, though the Ca2+-induced dislocation of tropomyosin has been studied. By binding troponin(C+I) to actin in an almost stoichiometric ratio and reconstituting actin-tropomyosin-troponin(C+I) filaments, we reconstructed the three-dimensional structure of actin-tropomyosin-troponin(C+I) with or without Ca2+ from electron cryomicrographs to about 2.5 or 3 nm resolution, respectively. Without Ca2+, the three-dimensional map reveals the extra-density region due to troponin(C+I), which extends perpendicularly to the helix axis and covers the N-terminal and C-terminal regions of actin. In the presence of Ca2+, the C-terminal region of actin became more exposed, and troponin(C+I) became V-shaped with one arm extending towards the pointed end of the actin filament. This structure can be considered to show the location of troponin(C+I) in at least one of the states of skeletal muscle thin filaments. These Ca2+-induced changes of troponin(C+I) provide a clue to the regulatory mechanism of contraction.     

Elastic filaments and giant proteins in muscle.

Striated muscle is now known to contain a third major class of filaments, additional to the thick and thin filaments. The presence of such extra filaments has seemed likely for many years, but details of their location, structure, and composition are only now becoming clear. They are composed of massively large proteins and, in contrast to thick and thin filaments, they are elastic.     

Interaction of bacterial flagellum filaments with skeletal muscle myosin

Interaction of isolated bacterial flagellum filaments (BFF) and intact flagella from E. coli MS 1350 and B. brevis G.-B.p+ with rabbit skeletal myosin was studied. BFF were shown to coprecipitate with myosin (but not with isolated myosin rod) at low ionic strength, that is, under conditions of myosin aggregation. The data of electron microscopy indicate that filaments of intact bacterial flagella interact with isolated myosin heads (myosin subfragment 1, S1), and this interaction is fully prevented by addition of Mg2+ -ATP. Addition of BFF inhibited both K+ -EDTA- and Ca2+ -ATPase activity of skeletal muscle myosin, but had no effect on its Mg2+ -ATPase activity. Monomeric flagellin did not coprecipitate with myosin and had no effect on its ATPase activities. BFF were shown to compete with F-actin in myosin binding. It is concluded that BFF interact with myosin heads and affect their ATPase activity. Thus, BFF composed of a single protein flagellin are in many respects similar to actin filaments. Common origin of actin and flagellin may be a reason for this similarity.     

Co-operative activation of skeletal muscle thin filaments by rigor crossbridges. The effect of troponin C extraction

When Ca2+ binds to troponin C (TnC), all 26 troponin-tropomyosin (Tn-Tm) complexes of a regulatory strand change in concert from the inactive to the active configuration. To see if the complexes respond similarly when they are activated by rigor crossbridges in the absence of Ca2+, we determined the slope (ns) of the bell-shaped pS/tension (pS = -log [MgATP], where S = MgATP2-) relationship between pS 5, where the tension is maximal, and pS 2.3, where fibers are fully relaxed. In control skinned rabbit psoas fibers the ns value is greater than 4; it progressively decreases with TnC extraction. This decrease in ns with TnC extraction is analogous to the decrease in the slope (Hill coefficient) of the pCa/tension (pCa = -log [Ca2+]) relationship with extraction. Complete TnC extraction reduces the maximum substrate-induced tension by only 25%; in contrast, it reduces the maximum Ca2+ induced tension to zero. The effects of TnC extraction on the slope of the pS/tension curve are explained by the assumptions that (1) extracted Tn-Tm complexes no longer change in concert with their neighbors but change independently of them, and (2) co-operative signals cannot cross extracted Tn-Tm complexes. The ns value, therefore, like the nH, is a direct function of the number of contiguous, intact, Tn-Tm complexes in a stretch of a regulatory strand. To describe qualitatively the bi-phasic pS/tension relationship, the mono-phasic pCa/tension relationship, and the effects of TnC extraction on them, we introduce a version of the concerted-transition formalism which includes two activating ligands, Ca2+ and rigor crossbridges.     

Beta-actinin: a capping protein at the pointed end of thin filaments in skeletal muscle

We examined the function of beta-actinin as a pointed end capping protein of thin filaments in skeletal muscle. An improvement in preparing beta-actinin yielded purified beta-actinin which retained its activity for more than a week. Two-dimensional gel electrophoresis showed that the two subunits, beta I and beta II, of beta-actinin are, respectively, split into two to three components (isoforms) with different isoelectric points. Polyclonal antibody was raised by injecting such purified and undenatured chicken breast muscle beta-actinin composed of several components into a rabbit. Immuno-gold labeling examination with electron microscopy of an F-actin-beta-actinin complex decorated with HMM showed that 85% of bound gold particles was on the pointed end of actin filaments, while the remaining 15% was on the barbed end. This suggests that in beta-actinin preparation pointed end and barbed end capping proteins inevitably coexist. Immunofluorescence and immunoelectron microscopy directly showed that beta-actinin is located at the pointed end of thin filaments in myofibrils; it was also suggested that a capping protein having common antigenic determinants to beta-actinin is located at Z-line. Thus, the physiological function of beta-actinin as a pointed end capping protein was examined as follows: When beta-actinin was dissociated from the pointed end of thin filaments in an I-Z-I brush by using a high salt solution, thin filaments could be disassembled at the pointed ends at concentrations of exogenous actin lower than a critical value. At a physiological ionic strength, these salt-washed thin filaments gradually shortened at a constant rate of about 45 nm/h. Both the association and dissociation of monomeric actin at the pointed end were suppressed by the rebinding of exogenous beta-actinin. The main physiological role of beta-actinin is therefore to stabilize thin filaments in the sarcomere by preventing addition and removal of actin monomers at the pointed filament end.     

Interactions between actin and myosin filaments in skeletal muscle visualized in frozen-hydrated thin sections.

For the purpose of determining net interactions between actin and myosin filaments in muscle cells, perhaps the single most informative view of the myofilament lattice is its averaged axial projection. We have studied frozen-hydrated transverse thin sections with the goal of obtaining axial projections that are not subject to the limitations of conventional thin sectioning (suspect preservation of native structure) or of equatorial x-ray diffraction analysis (lack of experimental phases). In principle, good preservation of native structure may be achieved with fast freezing, followed by low-dose electron imaging of unstained vitrified cryosections. In practice, however, cryosections undergo large-scale distortions, including irreversible compression; furthermore, phase contrast imaging results in a nonlinear relationship between the projected density of the specimen and the optical density of the micrograph. To overcome these limitations, we have devised methods of image restoration and generalized correlation averaging, and applied them to cryosections of rabbit psoas fibers in both the relaxed and rigor states. Thus visualized, myosin filaments appear thicker than actin filaments by a much smaller margin than in conventional thin sections, and particularly so for rigor muscle. This may result from a significant fraction of the myosin S1-cross-bridges averaging out in projection and thus contributing only to the baseline of projected density. Entering rigor incurs a loss of density from an annulus around the myosin filament, with a compensating accumulation of density around the actin filament. This redistribution of mass represents attachment of the fraction of cross-bridges that are visible above background. Myosin filaments in the "nonoverlap" zone appear to broaden on entering rigor, suggesting that on deprivation of ATP, cross-bridges in situ move outwards even without actin in their immediate proximity.     

Tropomodulin is associated with the free (pointed) ends of the thin filaments in rat skeletal muscle

The length and spatial organization of thin filaments in skeletal muscle sarcomeres are precisely maintained and are essential for efficient muscle contraction. While the major structural components of skeletal muscle sarcomeres have been well characterized, the mechanisms that regulate thin filament length and spatial organization are not well understood. Tropomodulin is a new, 40.6-kD tropomyosin-binding protein from the human erythrocyte membrane skeleton that binds to one end of erythrocyte tropomyosin and blocks head-to-tail association of tropomyosin molecules along actin filaments. Here we show that rat psoas skeletal muscle contains tropomodulin based on immunoreactivity, identical apparent mobility on SDS gels, and ability to bind muscle tropomyosin. Results from immunofluorescence labeling of isolated myofibrils at resting and stretched lengths using anti-erythrocyte tropomodulin antibodies indicate that tropomodulin is localized at or near the free (pointed) ends of the thin filaments; this localization is not dependent on the presence of myosin thick filaments. Immunoblotting of supernatants and pellets obtained after extraction of myosin from myofibrils also indicates that tropomodulin remains associated with the thin filaments. 1.2-1.6 copies of muscle tropomodulin are present per thin filament in myofibrils, supporting the possibility that one or two tropomodulin molecules may be associated with the two terminal tropomyosin molecules at the pointed end of each thin filament. Although a number of proteins are associated with the barbed ends of the thin filaments at the Z disc, tropomodulin is the first protein to be specifically located at or near the pointed ends of the thin filaments. We propose that tropomodulin may cap the tropomyosin polymers at the pointed end of the thin filament and play a role in regulating thin filament length.     

Structural changes of the regulatory proteins bound to the thin filaments in skeletal muscle contraction by X-ray fiber diffraction

In order to clarify the structural changes related to the regulation mechanism in skeletal muscle contraction, the intensity changes of thin filament-based reflections were investigated by X-ray fiber diffraction. The time course and extent of intensity changes of the first to third order troponin (TN)-associated meridional reflections with a basic repeat of 38.4 nm were different for each of these reflections. The intensity of the first and second thin filament layer lines changed in a reciprocal manner both during initial activation and during the force generation process. The axial spacings of the TN-meridional reflections decreased by ∼0.1% upon activation relative to the relaxing state and increased by ∼0.24% in the force generation state, in line with that of the 2.7-nm reflection. Ca²⁺-binding to TN triggered the shortening and a change in the helical symmetry of the thin filaments. Modeling of the structural changes using the intensities of the thin filament-based reflections suggested that the conformation of the globular core domain of TN altered upon activation, undergoing additional conformational changes at the tension plateau. The tail domain of TN moved together with tropomyosin during contraction. The results indicate that the structural changes of regulatory proteins bound to the actin filaments occur in two steps, the first in response to the Ca²⁺-binding and the second induced by actomyosin interaction.     

Maximal activation of skeletal muscle thin filaments requires both rigor myosin S1 and calcium

The regulation by calcium and rigor-bound myosin-S1 of the rate of acceleration of 2'-deoxy-3'-O-(N-methylanthraniloyl)ADP (mdADP) release from myosin-mdADP-P(i) by skeletal muscle thin filaments (reconstituted from actin-tropomyosin-troponin) was measured using double mixing stopped-flow fluorescence with the nucleotide substrate 2'-deoxy-3'-O-(N-methylanthraniloyl). The predominant mechanism of regulation is the acceleration of product dissociation by a factor of approximately 200 by thin filaments in the fully activated conformation (bound calcium and rigor S1) relative to the inhibited conformation (no bound calcium or rigor S1). In contrast, only 2-3-fold regulation is due to a change in actin affinity such as would be expected by "steric blocking" of the myosin binding site of the thin filament by tropomyosin. The binding of one ligand (either calcium or rigor-S1) produces partial activation of the rate of product dissociation, but the binding of both is required to maximally accelerate product dissociation to a rate similar to that obtained with F-actin in the absence of regulatory proteins. The data support an allosteric regulation model in which the binding of either calcium or rigor S1 alone to the thin filament shifts the equilibrium in favor of the active conformation, but full activation requires binding of both ligands.