Irregular patterns of actin and myosin filaments in human skeletal muscle cells

Summary The ultrastructural study of cross sections of normal skeletal muscle cells showed the existence of irregular patterns of actin filaments in connection with the hexagonal pattern of the myosin filaments. The actin filaments surrounding each myosin filament vary in number from 6 to 11. The most frequent relationship is 9 to 1, followed by 10 to 1 and 8 to 1. The hexagonal pattern of actin filaments was observed only in the 6 to 1 arrays; as the actin filaments increase in number, they tend to form different polygons or circles around the myosin filaments. All described patterns may occur in each sarcomere. The actin to myosin filament ratio varies from 3 to 4 within each individual myofibril. The described variability of the actin filaments arrays leads to several difficulties in an explanation of the mechanism of muscular contraction.Director, Chief of Section, Histology. Profesor Agregado de Embriología e HistologíaProfesor Adjunto de Embriología e HistologíaResidente de Anatomía Patol'ogica de la Ciudad Sanitaria La Paz     

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.     

Helical reconstruction of frozen-hydrated scallop myosin filaments.

Native myosin filaments from scallop striated muscle that have been rapidly frozen in relaxing solutions appear to be well preserved in vitreous ice. Electron micrographs of samples at -177 degrees C were recorded with an electron dose of 10 e/A2 at 1.5 microns defocus. After filament images were straightened by spline-fitting, several transforms showed well-defined layer-lines arising from the helical structure of the filament. A set of 17 near-meridional layer-lines has been collected and corrected for background and for phase and amplitude contrast functions. Preliminary helical reconstructions from this still incomplete data set reveal aspects of structure that were not apparent from earlier analysis of negatively stained filaments from scallop muscle. Individual pear-shaped myosin heads now appear to be well resolved from each other and from the filament backbone. The two heads of each myosin molecule appear to be splayed apart axially. The reconstructions also reveal that the filament backbone has a polygonal shape in cross-section, and that it appears to contain seven peripherally located subfilaments.     

The dynamics of the interaction between myosin subfragment 1 and pyrene-labelled thin filaments, from rabbit skeletal muscle

We have used actin labelled in Cys-374 with N-(1-pyrenyl)iodoacetamide to monitor the dynamics and equilibria of the interaction between myosin subfragment 1 and the actin-troponin-tropomyosin complex in the presence of calcium. These results are compared with those obtained for pure actin and myosin subfragment 1. The sensitivity of this fluorescent label allowed us to measure the binding affinity of myosin subfragment 1 for actin directly by fluorescence titration. The affinity of subfragment 1 for actin is increased sixfold by troponin-tropomyosin in the presence of calcium. Kinetic studies of the interaction of subfragment 1 and actin have revealed an isomerization of the actin-subfragment 1 complex from a state in which actin is weakly bound (Ka = 5.9 X 10(4) M-1) to a more tightly bound complex (Ka = 1.7 X 10(7) M-1) (Coates, Criddle & Geeves (1985) Biochem. J. 232, 351). Results in the presence of troponin-tropomyosin show the same isomerization. The sixfold increase in affinity of subfragment 1 for actin is shown to be due to a decrease in the rate of dissociation of actin from the weakly bound complex.     

Fluorescence resonance energy transfer between points on actin and the C-terminal region of tropomyosin in skeletal muscle thin filaments

Fluorescence resonance energy transfer between points on tropomyosin (positions 87 and 190) and actin (Gln-41, Lys-61, Cys-374, and the ATP-binding site) showed no positional change of tropomyosin relative to actin on the thin filament in response to changes in Ca2+ concentration (Miki et al. (1998) J. Biochem. 123, 1104-1111). This is consistent with recent electron cryo-microscopy analysis, which showed that the C-terminal one-third of tropomyosin shifted significantly towards the outer domain of actin, while the N-terminal half of tropomyosin shifted only a little (Narita et al. (2001) J. Mol. Biol. 308, 241-261). In order to detect any significant positional change of the C-terminal region of tropomyosin relative to actin, we generated mutant tropomyosin molecules with a unique cysteine residue at position 237, 245, 247, or 252 in the C-terminal region. The energy donor probe was attached to these positions on tropomyosin and the acceptor probe was attached to Cys-374 or Gln-41 of actin. These probe-labeled mutant tropomyosin molecules retain the ability to regulate the acto-S1 ATPase activity in conjunction with troponin and Ca2+. Fluorescence resonance energy transfer between these points of tropomyosin and actin showed a high transfer efficiency, which should be very sensitive to changes in distance between probes attached to actin and tropomyosin. However, the transfer efficiency did not change appreciably upon removal of Ca2+ ions, suggesting that the C-terminal region of tropomyosin did not shift significantly relative to actin on the reconstituted thin filament in response to the change of Ca2+ concentration.     

Variable patterns of actin filaments in mammalian skeletal muscle

Summary The ultrastructural organization of myofilaments in skeletal muscle was studied in four mammalian species (mouse, rat, hamster, goat). In all these species, myofibrils showing irregularly distributed arrays of a variable number of actin filaments (from 6 to 11) were observed. The proportion of such myofibrils and the predominant patterns of actin filaments varied from one species to another. These results are in agreement with those previously reported for human skeletal muscle.     

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.     

Phosphorylation of myosin light chain modulates the in vitro movement of fibrils composed of actin and myosin filaments from skeletal muscle

In vitro movement of fibrils composed of actin and myosin filaments purified from skeletal muscle was observed by dark field microscopy during superprecipitation at low ionic strengths at room temperature. The movement was activated by phosphorylation of light chain (LC2) of myosin. The activity of the movement was evaluated in terms of the spreading of the area where the fibrils were moving. Adenosine triphosphatase activity of actomyosin was also enhanced by phosphorylation of LC2 and was correlated with the activity of the in vitro movement.     

Three-dimensional image analysis of the complex of thin filaments and myosin molecules from skeletal muscle. V. Assignment of actin in the actin-tropomyosin-myosin subfragment-1 complex

To assign the actin molecule in the three-dimensional image of the actin-tropomyosin-myosin subfragment-1 (actin-TM-S1) complex, the three-dimensional image of the actin-tropomyosin complex was correlated to that of actin-TM-S1. To assess the similarity of two structures in a quantitative manner, we used a normalized cross-correlation function ("similarity function"). The calculation of similarity indicated that domain A and domain B defined in (1, 2) correspond to actin-tropomyosin. This assignment indicates that one S1 molecule strongly interacts with only one actin molecule, but at least two regions of S1 contribute to the binding. Comparison of the reconstituted models of thin filaments with those of decorated thin filaments suggested a change in the shape of the actin molecule.     

Extra actin filaments at the periphery of skeletal muscle myofibrils.

Myofibrils isolated from a variety of vertebrate muscle fibers have a set of peripheral filaments associated with the periphery of the Z line free to move away from the surface of the myofibril. Decoration with myosin subfragment 1 shows that these are actin filaments.     

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.     

Activation of smooth muscle myosin Mg2+-ATPase by native thin filaments and actin/tropomyosin

Application of the myosin competition test (Lehman, W., and Szent-Gy?rgyi, A. G. (1975) J. Gen. Physiol. 66, 1-30) to chicken gizzard actomyosin indicated that this smooth muscle contains a thin filament-linked regulatory mechanism. Chicken gizzard thin filaments, isolated as described previously (Marston, S. B., and Lehman, W. (1985) Biochem. J. 231, 517-522), consisted almost exclusively of actin, tropomyosin, caldesmon, and an unidentified 32-kilodalton polypeptide in molar ratios of 1:1/6:1/26:1/17, respectively. When reconstituted with phosphorylated gizzard myosin, these thin filaments conferred Ca2+ sensitivity (67.8 +/- 2.1%; n = 5) on the myosin Mg2+-ATPase. On the other hand, no Ca2+ sensitivity of the myosin Mg2+-ATPase was observed when purified gizzard actin or actin plus tropomyosin was reconstituted with phosphorylated gizzard myosin. Native thin filaments were rendered essentially free of caldesmon and the 32-kilodalton polypeptide by extraction with 25 mM MgCl2. When reconstituted with phosphorylated gizzard myosin, caldesmon-free thin filaments and native thin filaments exhibited approximately the same Ca2+ sensitivity (45.1 and 42.7%, respectively). The observed Ca2+ sensitivity appears, therefore, not to be due to caldesmon. Only trace amounts of two Ca2+-binding proteins could be detected in native thin filaments. These were identified as calmodulin (present at a molar ratio to actin of 1:733) and the 20-kilodalton light chain of myosin (present at a molar ratio to actin of 1:270). The Ca2+ sensitivity observed in an in vitro system reconstituted from gizzard thin filaments and either skeletal myosin or phosphorylated gizzard myosin is due, therefore, to calmodulin and/or an unidentified minor protein component of the thin filaments which may be an actin-binding protein involved in regulating actin filament structure in a Ca2+-dependent manner.     

X-ray diffraction measurements of the extensibility of actin and myosin filaments in contracting muscle.

We have used a small angle scattering system assembled on the high flux multipole wiggler beam line at CHESS (Cornell) to make very accurate spacing measurements of certain meridional and layer-line reflections from contracting muscles. During isometric contraction, the actin 27.3 A reflection increases in spacing from its resting value by approximately 0.3%, and other actin reflections, including the 59 and 51 A off-meridional reflections, show corresponding changes in spacing. When tension is augmented or diminished by applying moderate speed length changes to a contracting muscle, changes in spacing in the range of 0.19-0.24% (when scaled to full isometric tension) can be seen. The larger difference between the resting and isometric spacings suggests either nonlinearity at low tension levels or the presence of a component related to activation itself. Myosin filaments also show similar increases in axial period during slow stretch, in addition to the well known larger change associated with activation. An actin spacing change of 0.25-0.3% can also be measured during a 2 ms time frame immediately after a quick release, showing that the elastic behavior is rapid. These observations of filament extensions totaling 2-3 nm per half-sarcomere may necessitate some significant revision of the interpretation of a number of mechanical experiments in muscle, in which it has usually been assumed that virtually all of the elasticity resides in the cross-bridges.     

Subunit exchange between smooth muscle myosin filaments

Filaments formed from phosphorylated smooth muscle myosin are stable in the presence of MgATP, whereas dephosphorylated filaments are disassembled to a mixture of folded monomers and dimers. The stability of copolymers of phosphorylated and dephosphorylated myosin was, however, unknown. Gel filtration, sedimentation velocity, and pelleting assays were used to show that MgATP could dissociate dephosphorylated myosin from copolymers containing either rod and myosin or dephosphorylated and phosphorylated myosin. Copolymers were typically formed by dialyzing monomeric mixtures into filament-forming buffer but, unexpectedly, could also be formed within minutes of mixing preformed rod and myosin minifilaments. This result suggested that molecules can rapidly and extensively exchange between filaments, presumably via the monomeric pool of myosin in equilibrium with polymer. An exchange of molecules between filaments was demonstrated directly by electron microscopy using gold-labeled streptavidin or antibody to detect the exchanged species. By this approach it was shown that smooth muscle myosin filaments, like other macromolecular assemblies, are dynamic structures that can readily alter their composition in response to changing solvent conditions. Moreover, because folded monomeric myosin is unable to polymerize, these experiments suggest a mechanism for the disassembly of the filament by MgATP.     

Skeletal muscle myosin subfragment-1 induces bundle formation by actin filaments

As is well known, the light scattering intensity of F-actin solutions increases immediately upon formation of the rigor complex with subfragment-1 (S-1). We have found that after the initial rise in scattering, there is a further gradual increase in scattering (we call it "super-opalescence"). Fluorescence and electron microscopic observations of acto-S-1 solutions showed that super-opalescence results from formation of actin filament bundles once S-1 binds to F-actin. The actin bundles possessed transverse stripes with a periodicity of about 350 A, which suggested that in the bundles actin filaments are arranged in parallel register. The rate of the initial process of bundle formation (i.e. side-by-side dimerization) could be approximately estimated by measuring the initial rate of super-opalescence (V0). V0 had a maximum (V0m) at a molar ratio of S-1 to actin of 1;6-1;7, regardless of the actin concentration, pH (6-8.5), Mg2+ concentration (up to 5 mM), or ionic strength (up to 0.3 M KC1). Lower pH, higher Mg2+ concentration, and higher ionic strength increased V0m; V0 was proportional to the square of the actin concentration, regardless of the solution conditions.     

Temperature dependence of speed of actin filaments propelled by slow and fast skeletal myosin isoforms.

It was shown that the temperature sensitivity of shortening velocity of skeletal muscles is higher at temperatures below physiological (10-25 degrees C) than at temperatures closer to physiological (25-35 degrees C) and is higher in slow than fast muscles. However, because intact muscles invariably express several myosin isoforms, they are not the ideal model to compare the temperature sensitivity of slow and fast myosin isoforms. Moreover, temperature sensitivity of intact muscles and single muscle fibers cannot be unequivocally attributed to a modulation of myosin function itself, as in such specimen myosin works in the structure of the sarcomere together with other myofibrillar proteins. We have used an in vitro motility assay approach in which the impact of temperature on velocity can be studied at a molecular level, as in such assays acto-myosin interaction occurs in the absence of sarcomere structure and of the other myofibrillar proteins. Moreover, the temperature modulation of velocity could be studied in pure myosin isoforms (rat type 1, 2A, and 2B and rabbit type 1 and 2X) that could be extracted from single fibers and in a wide range of temperatures (10-35 degrees C) because isolated myosin is stable up to physiological temperature. The data show that, at the molecular level, the temperature sensitivity is higher at lower (10-25 degrees C) than at higher (25-35 degrees C) temperatures, consistent with experiments on isolated muscles. However, slow myosin isoforms did not show a higher temperature sensitivity than fast isoforms, contrary to what was observed in intact slow and fast muscles.     

X-ray diffraction evidence for the extensibility of actin and myosin filaments during muscle contraction.

To clarify the extensibility of thin actin and thick myosin filaments in muscle, we examined the spacings of actin and myosin filament-based reflections in x-ray diffraction patterns at high resolution during isometric contraction of frog skeletal muscles and steady lengthening of the active muscles using synchrotron radiation as an intense x-ray source and a storage phosphor plate as a high sensitivity, high resolution area detector. Spacing of the actin meridional reflection at approximately 1/2.7 nm-1, which corresponds to the axial rise per actin subunit in the thin filament, increased about 0.25% during isometric contraction of muscles at full overlap length of thick and thin filaments. The changes in muscles stretched to approximately half overlap of the filaments, when they were scaled linearly up to the full isometric tension, gave an increase of approximately 0.3%. Conversely, the spacing decreased by approximately 0.1% upon activation of muscles at nonoverlap length. Slow stretching of a contracting muscle increased tension and increased this spacing over the isometric contraction value. Scaled up to a 100% tension increase, this corresponds to a approximately 0.26% additional change, consistent with that of the initial isometric contraction. Taken together, the extensibility of the actin filament amounts to 3-4 nm of elongation when a muscle switches from relaxation to maximum isometric contraction. Axial spacings of the layer-line reflections at approximately 1/5.1 nm-1 and approximately 1/5.9 nm-1 corresponding to the pitches of the right- and left-handed genetic helices of the actin filament, showed similar changes to that of the meridional reflection during isometric contraction of muscles at full overlap. The spacing changes of these reflections, which also depend on the mechanical load on the muscle, indicate that elongation is accompanied by slight changes of the actin helical structure possibly because of the axial force exerted by the actomyosin cross-bridges. Additional small spacing changes of the myosin meridional reflections during length changes applied to contracting muscles represented an increase of approximately 0.26% (scaled up to a 100% tension increase) in the myosin periodicity, suggesting that such spacing changes correspond to a tension-related extension of the myosin filaments. Elongation of the myosin filament backbone amounts to approximately 2.1 nm per half sarcomere. The results indicate that a large part (approximately 70%) of the sarcomere compliance of an active muscle is caused by the extensibility of the actin and myosin filaments; 42% of the compliance resides in the actin filaments, and 27% of it is in the myosin filaments.     

Interactions between actin filaments and between actin filaments and membranes in quick-frozen and deeply etched hair cells of the chick ear

Replicas of the apical surface of hair cells of the inner ear (vestibular organ) were examined after quick freezing and rotary shadowing. With this technique we illustrate two previously undescribed ways in which the actin filaments in the stereocilia and in the cuticular plate are attached to the plasma membrane. First, in each stereocilium there are threadlike connectors running from the actin filament bundle to the limiting membrane. Second, many of the actin filaments in the cuticular plate are connected to the apical cell membrane by tiny branched connecting units like a "crow's foot." Where these "feet" contact the membrane there is a small swelling. These branched "feet" extend mainly from the ends of the actin filaments but some connect the lateral surfaces of the actin filaments as well. Actin filaments in the cuticular plate are also connected to each other by finer filaments, 3 nm in thickness and 74 +/- 14 nm in length. Interestingly, these 3-nm filaments (which measure 4 nm in replicas) connect actin filaments not only of the same polarity but of opposite polarities as documented by examining replicas of the cuticular plate which had been decorated with subfragment 1 (S1) of myosin. At the apicolateral margins of the cell we find two populations of actin filaments, one just beneath the tight junction as a network, the other at the level of the zonula adherens as a ring. The latter which is quite substantial is composed of actin filaments that run parallel to each other; adjacent filaments often show opposite polarities, as evidenced by S1 decoration. The filaments making up this ring are connected together by the 3-nm connectors. Because of the polarity of the filaments this ring may be a "contractile" ring; the implications of this is discussed.     

Differential regulation of actin and myosin isoenzyme synthesis in functionally overloaded skeletal muscle.

Overload hypertrophy of the chicken anterior latissimus dorsi muscle is accompanied by a replacement of one myosin isoenzyme (slow myosin-1, SM1) by another (slow myosin-2, SM2). To investigate the molecular mechanisms by which these changes occur, we measured the fractional synthesis rates (ks) in vivo of individual myosin-heavy-chain isoenzymes, total actin and total protein during the first 72 h of muscle growth. Although the ks of total protein and actin were doubled at 24 h, the ks for SM1 and SM2 were depressed. However, the ks of both isomyosins were nearly tripled by 72 h. Despite the increase in muscle size observed at 72 h, the amount of SM1 was reduced by half, indicating increased degradation of SM1. Results of translation of polyribosomes in vitro paralleled the results obtained in vivo. The proportion of total polyadenylylated mRNA in total RNA was increased at 48 and 72 h, but unchanged at 24 h despite the increase in protein synthesis at 24 h. Nuclease-protection analyses indicate that the level of specific SM1 and SM2 mRNAs change in a reciprocal fashion during overload. We conclude that gene-specific and temporal differences exist in the regulatory mechanisms that control overload-induced muscle growth.     

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.