Disassembly kinetics of thick filaments in rabbit skeletal muscle fibers. Effects of ionic strength, Ca2+ concentration, pH, temperature, and cross-bridges on the stability of thick filament structure.

The kinetics of dissociation from both ends of thick filaments in a muscle fiber was investigated by an optical diffraction method. The dissociation velocity of thick filaments at a sarcomere length of 2.75 microns increased with increasing the KCl concentration (from 60 mM to 0.5 M), increasing the pH value (from 6.2 to 8.0) or decreasing the temperature (from 25 to 5 degrees C) in the presence of 10 mM pyrophosphate and 5 mM MgCl2. Micromolar concentrations of Ca2+ suppressed the dissociation velocity markedly at shorter sarcomere lengths. The dissociation velocity, v, decreased as thick filaments became shorter, and v = -db/dt = vo exp (alpha b), where b is the length of the thick filament at time t and vo and alpha are constants. The vo value was largely dependent on the KCl concentration but the alpha value was not. The stiffness of a muscle fiber decreased nearly in proportion to the decrease of overlap between thick and thin filaments induced by the dissociation of thick filaments. This indicates that cross-bridges are uniformly distributed and contribute independently to the stiffness of a muscle fiber during the dissociation of thick filaments.     

Diffraction rings obtained from a suspension of skeletal myofibrils by laser light illumination. Study of internal structure of sarcomeres.

Diffraction rings corresponding to the first, second, and third order were obtained by laser light illumination from a suspension of rabbit glycerinated psoas myofibrils (diameter, 1-2 microns; average length of the straight region, 44 microns; average sarcomere length, 2.2-2.6 microns) of which the optical thickness was appropriately chosen. Dispersed myofibrils were nearly randomly oriented in two dimensions, so that the effects of muscle volume were minimized; these effects usually interfere significantly with a quantitative analysis of laser optical diffraction in the fiber system. The diameters of diffraction rings represented the average sarcomere length. By using this system, we confirmed the ability of the unit cell (sarcomere) structure model to explain the intensity change of diffraction lines accompanying the dissociation from both ends of thick filaments in a high salt solution. The length of an A-band estimated from the relative intensity of diffraction rings and that directly measured on phase-contrast micrographs coincided well with each other. Also, we found that myofibrils with a long sarcomere length shorten to a slack length accompanying the decrease in overlap between thick and thin filaments produced by the dissociation of thick filaments.     

Purification and characterization of myosin from the clonal rat glial cell strain C-6.

A myosin was isolated from the clonal rat glial cell strain C-6 and compared with rat skeletal muscle myosin. After cell extracts were subjected to gel filtration chromatography in the presence of KI and magnesium pyrophosphate the C-6 myosin was rapidly purified by a procedure similar to that used for skeletal muscle myosin. The C-6 myosin resembles muscle myosin both physically and enzymatically. It contains heavy chains of 200,000 daltons and two classes of light chains of 17,000 and 19,000 daltons in approximately equal molar ratios. This myosin forms bipolar thick filaments in 0.1 M KCl and binds reversibly to skeletal muscle F-actin, the binding being inhibited by MgATP. Skeletal muscle F-actin stimulates the C-6 myosin adenosine triphosphatase 2- to 3-fold in the presence of KCl and Mg2+. The action activation of muscle myosin ATPase at low ionic strength is 10-fold greater than that of C-6 myosin. Ca2+ and EDTA stimulated the ATPase activities of both enzymes. When assayed in the presence of 0.6 M KCl and 1 mM EDTA the skeletal muscle myocin ATPase demonstrates substrate saturation while the C-6 myosin enzyme activity is stimulated by ATP concentrations above 2.5 mM.     

Cooperative interactions between calcium-binding sites on glycerinated muscle fibers. The influence of cross-bridge attachment

A double isotope technique and EGTA buffers were used to measure the binding of Ca2+ to rabbit psoas muscle fibers extracted with detergent and glycerol. These experiments were designed to test the effect of rigor complex formation, determined by the degree of filament overlap, on the properties of the Ca2+-binding sites in the intact filament lattice. In the presence of 5 mM MgCl2 (no ATP), reduction of filament overlap was associated with a reduced binding of Ca2+ over the entire range of free Ca2+ concentrations (5.10(-8)-2.10(-5) M). With maximum filament overlap (sarcomere length 2.1-2.2 micrometer) the maximum bound Ca2+ was equivalent to 4 mol Ca2+/mol troponin and there was significant positive interaction between binding sites, as shown by Scatchard and Hill plots. With no filament overlap (sarcomere length 3.8-4.4 micrometer) the maximum bound Ca2+ was equivalent to 3 mumole Ca2+/mol troponin and graphical analysis indicated a single class of non-interacting sites. The data provide evidence that when cross-bridge attachments between actin and myosin filaments are formed not only does an additional Ca2+ binding site appear, but cooperative properties are imposed upon the binding sites.     

Supramolecular forms of actin from amoebae of Dictyostelium discoideum.

Actin purified from amoebae of Dictyostelium discoideum polymerizes into filaments at 24 degrees upon addition of KCl, as judged by a change in optical density at 232 nm and by electron microscopy. The rate and extent of formation of this supramolecular assembly and the optimal KCl concentrations (0.1 M) for assembly are similar to those of striated muscle actin. The apparent equilibrium constant for the monomer-polymer transition is 1.3 muM for both Dictyostelium and muscle actin. Although assembly of highly purified Dictyostelium actin monomers into individual actin filaments resembles that of muscle actin, Dictyostelium actin but not muscle actin was observed to assemble into two-dimensional nets in 10 mM CaCl2. The Dictyostelium actin also forms filament bundles which are 0.1 mum in diameter and which assemble in the presence of 5 mM MgCl2. These bundles formed from partially purified Dictyostelium actin preparations but not from highly purified preparations, suggesting that their formation may depend on the presence of another component. These actin bundles reconstituted in vitro resemble the actin-containing bundles found in situ by microscopy in many non-muscle cells.     

Changes in thick filament length in Limulus striated muscle

Here we describe the change in thick filament length in striated muscle of Limulus, the horseshoe crab. Long thick filaments (4.0 microns) are isolated from living, unstimulated Limulus striated muscle while those isolated from either electrically or K+-stimulated fibers are significantly shorter (3.1 microns) (P less than 0.001). Filaments isolated from muscle glycerinated at long sarcomere lengths are long (4.4 microns) while those isolated from muscle glycerinated at short sarcomere lengths are short (2.9 microns) and the difference is significant (P less than 0.001). Thin filaments are 2.4 microns in length. The shortening of thick filaments is related to the wide range of sarcomere lengths exhibited by Limulus telson striated muscle.     

Assembly-dependent phosphorylation of myosin and paramyosin of native thick filaments in Caenorhabditis elegans.

Phosphorylation of the thick filament proteins myosin and paramyosin was studied in Caenorhabditis elegans. We have incubated partially purified, native thick filaments with [gamma 32P] ATP in the presence of 50-750 mM NaCl, pH 6.5-8.0. Myosin heavy chain and paramyosin were phosphorylatable only upon solubilization at 450 mM and higher NaCl concentrations. Under conditions preserving native structures, no phosphorylation of these proteins occurred. The phosphorylation required Mg2+ but was unaffected by cAMP, cGMP or Ca2+. The specific inhibitor of cAMP and cGMP kinase catalytic subunits, H8, inhibits the activity. Sedimentation experiments show that the kinase may associate with but is not an intrinsic component of thick filaments. In C. elegans, phosphorylation by the thick filament associated activity of myosin and paramyosin is dependent upon the state of their assembly.     

Structural proteins in the myofilaments and regulation of contraction in vertebrate smooth muscle.

The contractile systems of vertebrate smooth and striated muscles are compared. Smooth muscles contain relatively large amounts of actin and tropomyosin organized into thin filaments, and smaller amounts of myosin in the form of thick filaments. The protein contents are consistent with observed thin:thick filament ratios of about 15-18:1 in smooth compared to 2:1 in striated muscle. The basic characteristics of both types of contractile proteins are similar; but there are a variety of quantitative differences in protein structures, enzymatic activities and filament stabilities. Biochemical and X-ray diffraction data generally support recent ultrastructural evidence concerning the organization of the myofilaments in smooth muscle, although a basic contractile unit comparable to the sarcomere in striated muscle has not been discerned. Myofilament interactions and contraction in smooth muscle are controlled by changes in the Ca2+ concentration. Recent evidence suggests the Ca2+-binding regulatory site is associated with the myosin in vertebrate smooth muscle (as in a variety of invertebrate muscles), rather than with troponin which is the regulatory protein associated with the thin filament in vertebrate striated muscle.     

Order-disorder structural transitions in synthetic filaments of fast and slow skeletal muscle myosins under relaxing and activating conditions

In the previous study (Podlubnaya et al., 1999, J. Struc. Biol. 127, 1-15) Ca2+-induced reversible structural transitions in synthetic filaments of pure fast skeletal and cardiac muscle myosins were observed under rigor conditions (-Ca2+/+Ca2+). In the present work these studies have been extended to new more order-producing conditions (presence of ATP in the absence of Ca2+) aimed at arresting the relaxed structure in synthetic filaments of both fast and slow skeletal muscle myosin. Filaments were formed from column-purified myosins (rabbit fast skeletal muscle and rabbit slow skeletal semimebranosusproprius muscle). In the presence of 0.1 mM free Ca2+, 3 mM Mg2+ and 2 mM ATP (activating conditions) these filaments had a spread structure with a random arrangement of myosin heads and subfragments 2 protruding from the filament backbone. Such a structure is indistinguishable from the filament structures observed previously for fast skeletal, cardiac (see reference cited above) and smooth (Podlubnaya et al., 1999, J. Muscle Res. Cell Motil. 20, 547-554) muscle myosins in the presence of 0.1 mM free Ca2+. In the absence of Ca2+ and in the presence of ATP (relaxing conditions) the filaments of both studied myosins revealed a compact ordered structure. The fast skeletal muscle myosin filaments exhibited an axial periodicity of about 14.5 nm and which was much more pronounced than under rigor conditions in the absence of Ca2+ (see the first reference cited). The slow skeletal muscle myosin filaments differ slightly in their appearance from those of fast muscle as they exhibit mainly an axial repeat of about 43 nm while the 14.5 nm repeat is visible only in some regions. This may be a result of a slightly different structural properties of slow skeletal muscle myosin. We conclude that, like other filaments of vertebrate myosins, slow skeletal muscle myosin filaments also undergo the Ca2+-induced structural order-disorder transitions. It is very likely that all vertebrate muscle myosins possess such a property.     

Electron Microscopic Studies of Skeletal and Cardiac Muscle of a Benthic Fish. I. Myofibrillar Structure in Resting and Contracted Muscle

SYNOPSIS. Electron microscopic studies are reported on glycerinatedskeletal and cardiac muscle of a benthic fish, Coryphaenoidesspecies. In white skeletal muscle, the sarcomeres have a restinglength of approximately 1.8 µ, with thick filaments 1.4µ and thin filaments 0.75 µ in length. These dimensionsare somewhat shorter than filament lengths of oilier vertebratemuscles, possibly due to the elfect of volume increase duringassembly of thick and thin filaments at high hydrostatic pressure.During ATP-induced contraction of Coryphaenoides muscle fromsarcomere lengths of 1.8 µ to 1.6 µ, there is acharacteristic interdigitation of thick and thin filaments,with decrease in I band length and no change in length of thickor thin filaments. However, in sarcomeres contracted to lengthsof 1.5 µ. to 1.2 µ, there is a slight shorteningof the A band, apparently due to shortening of thick filaments,that occurs despite the presence of residual I band in the samesarcomeres. There is no obvious crumpling or distortion of thickfilaments during contraction to sarcomere lengths as low as1.0 µ, but filament organization undergoes extensive disarrayat sarcomere lengths approaching 0.7 µ. Although effectsfrom heterogeneity of filament length cannot be excluded withcertainty, the present evidence does suggest that contractionot Coryphaenoides muscle from 1.6 µ to 1.0 µ sarcomerelengih is accompanied by shortening of thick filaments consequentto a structural change within the thick filament core.     

Viscoelasticity of the sarcomere matrix of skeletal muscles. The titin-myosin composite filament is a dual-stage molecular spring.

The mechanical roles of sarcomere-associated cytoskeletal lattices were investigated by studying the resting tension-sarcomere length curves of mechanically skinned rabbit psoas muscle fibers over a wide range of sarcomere strain. Correlative immunoelectron microscopy of the elastic titin filaments of the endosarcomeric lattice revealed biphasic extensibility behaviors and provided a structural interpretation of the multiphasic tension-length curves. We propose that the reversible change of contour length of the extensible segment of titin between the Z line and the end of thick filaments underlies the exponential rise of resting tension. At and beyond an elastic limit near 3.8 microns, a portion of the anchored titin segment that adheres to thick filaments is released from the distal ends of thick filament. This increase in extensible length of titin results in a net length increase in the unstrained extensible segment, thereby lowering the stiffness of the fiber, lengthening the slack sarcomere length, and shifting the yield point in postyield sarcomeres. Thus, the titin-myosin composite filament behaves as a dual-stage molecular spring, consisting of an elastic connector segment for normal response and a longer latent segment that is recruited at and beyond the elastic limit of the sarcomere. Exosarcomeric intermediate filaments contribute to resting tension only above 4.5 microns. We conclude that the interlinked endo- and exosarcomeric lattices are both viscoelastic force-bearing elements. These distinct cytoskeletal lattices appear to operate over two ranges of sarcomere strains and collectively enable myofibrils to respond viscoelastically over a broad range of sarcomere and fiber lengths.     

Direct measurement of actin polymerization rate constants by electron microscopy of actin filaments nucleated by isolated microvillus cores

We used actin filament bundles isolated from intestinal brush-border microvilli to nucleate the polymerization of pure muscle actin monomers into filaments. Growth rates were determined by electron microscopy by measuring the change in the length of the filaments as a function of time. The linear dependence of the growth rates on the actin monomer concentration provided the rate constants for monomer association and dissociation at the two ends of the growing filament. The rapidly growing ("barbed") end has higher association and dissociation rate constants than the slowly growing ("pointed") end. The values of these rate constants differ in 20 mM KCl compared with 75 mM KCl, 5 mM MgSO4. 2 microM cytochalasin B blocks growth entirely at the barbed end, apparently by reducing both association and dissociation rate constants to near zero, but inhibits growth at the pointed end to only a small extent.     

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.     

Overrelaxation of muscle models in media with high concentrations of calcium ions and exposed to MgATP in "contraction-inducing" concentrations]

Cross-striation pattern and sarcomere length in isolated myofibrils (both glycerinated and fresh) as well as isometric tension of glycerinated fibers of rabbit m. psoas are unaffected by an evaluation in ionic strength of CaCl2 up to 0.2 in the absence of ATP. An addition of MgATP (1 to 3mM) to the Ca2+ media induces the changes which have been shown to be characteristic of overrelaxation [1, 2]: A band shortening occurs followed by a complete plastification of the fibres. A tentative mechanism of the process is discussed in terms of spontaneous rearrangement of calcium myosinate packing in thick filaments that follows disrupting of rigor crossbridges with thin filaments under the action of ATP. Released calcium myosinate heads fail to form "active" bridges with actin; thick filaments undergo a conformational change resulted in their shattening due to increase in the equilibrium region of LMM tail overlap. The effects do not depend on ionic strength only: on replacing CaCl2 by KCl at equal ionic strength 0.2, an addition of ATP induces normal contraction instead of overrelaxation. A possibility is discussed that in a living muscle overrelaxation could provide a siding to prevent damage in case of emergency.     

Ultrastructural and mechanical properties of electrically inexcitable skeletal muscle fibers of the crustacean Atya lanipes.

Examination of the ultrastructure and mechanical activation of the ventro-abdominal flexor muscle of the freshwater crustacean Atya lanipes shows that the fibers are of the long sarcomere, tonic type. The fibers possess an ample and well-organized internal membrane system, with extensive regions of T/SR dyad contacts near the ends of the A bands. An orbit of 10-12 thin filaments surrounds each thick filament. The lanthanum tracer method reveals a highly regular organization of the T-system, Z-tubules, and multiple internal clefts. Tension generation responds to extracellular potassium in a concentration dependent manner and is very slow. Mechanical activation is strictly dependent on extracellular Ca2+, even though these muscle fibers do not generate Ca2+ currents when depolarized. Tension development responds to caffeine and is also dependent on extracellular Na+, suggesting that Ca2+ release from the SR and Ca2+ influx via the Na/Ca exchanger intervene in mechanical activation.     

The bundling of actin with polyethylene glycol 8000 in the presence and absence of gelsolin.

Actin filament and bundle formation occur in the cytosol under conditions of very high total macromolecular concentration. In this study we have utilized the inert molecule polyethylene glycol 8000 (PEG) as a means of simulating crowded conditions in vitro. Column-purified Ca-actin was polymerized in the absence and presence of gelsolin (to regulate mean filament lengths between 50 and 5000 mers) and PEG (2-8%) using various concentrations of KCl and/or 2 mM divalent cations. Bundling was characterized by the scattered light intensity and mean diffusion coefficients obtained from dynamic light scattering, as well as by fluorescence and phase-contrast microscopy. The minimum concentration of KCl required for bundling decreases both with increasing concentration of PEG at a fixed mean filament length, and with decreasing filament length at a fixed concentration of PEG. In the absence of divalent cation, bundling is reversible on dilution, as determined by intensity levels, diffusion coefficients, and microscopy. However, with either 2 mM Mg2+ or Ca2+ added, bundling is irreversible under conditions of higher PEG concentrations or longer filaments, indicating that osmotic pressure effects cannot fully explain actin bundling with PEG. Weaker divalent cation-binding sites on actin as well as disulfide bonds appear to be involved in the irreversible bundling.     

Interaction of myosin with thin filaments during contraction and relaxation: effect of ionic strength

The influence of ionic strength on the isometric tension, stiffness, shortening velocity and ATPase activity of glycerol-treated rabbit psoas muscle fiber in the presence and the absence of Ca2+ has been studied. When the ionic strength of an activating solution (containing Mg2+-ATP and Ca2+) was decreased by varying the KCl concentration from 120 to 5 mM at 20 degrees C, the isometric tension and stiffness increased by 30% and 50%, respectively. The ATPase activity increased 3-fold, while the shortening velocity decreased to one-fourth. At 6 degrees C, similar results were obtained. These results suggest that at low ionic strengths ATP is hydrolyzed predominantly without dissociation of myosin cross-bridges from F-actin. In the absence of Ca2+, with decreasing KCl concentration the isometric tension and stiffness developed remarkably at 20 degrees C. However, the ATPase activity and shortening velocity were very low. At low ionic strength, even in the absence of Ca2+ myosin heads are bound to thin filaments. The development of the tension and stiffness were greatly reduced at 6 degrees C or at physiological ionic strength.     

The binding of calcium to glycerinated muscle fibers in rigor. The effect of filament overlap.

The binding of Ca2+ to glycerinated rabbit psoas fibers of varying sarcomere length was measured with a double isotope technique and ethyleneglycol-bis-(beta-aminoethylether)-N,N'-tetraacetic acid buffers. Experiments were carried out under rigor conditions with fiber bundles pre-set at different lengths prior to extraction with detergent and glycerol. These experiments were designed to test whether rigor complex formation, determined by the degree of filament overlap, enhances Ca2+-receptor affinity in the intact filament lattice, as it does in reconstituted actomyosin systems. The Ca2+-receptor affinity, as indicated by the free Ca2+ concentration at half-saturation and by the slopes of Scatchard plots, was found to be relatively unaffected by variations in filament overlap. However, the maximum bound Ca2+ was significantly reduced in stretched fibers. With maximum filament overlap the bound Ca2+ was equivalent to 4 mol per mol troponin. When stretched to zero overlap the fibers bound a maximum of 3 mol Ca2+ per mol troponin. When fibers with maximum overlap were incubated in the presence of 5 mM MgATP there was a reduction in the number of Ca2+-binding sites equivalent to that caused by stretching the fibers. These findings, taken together with other data in the literature, suggest that in the intact filament lattice at least one of the Ca2+-binding sites is present only when cross-bridge attachments are formed.     

Localization of the parallel elastic components in frog skinned muscle fibers studied by the dissociation of the A- and I-bands.

Localization of the parallel elastic components (PECs) in skinned muscle fibers was investigated by analyzing the change of the resting tension, which accompanies the dissociation of the A- and I-bands. The A-band was dissociated from both ends by increasing the concentration of KCl under relaxing conditions (0.09-0.54 M KCl, 4.0 mM MgATP, 1.0 mM Mg2+, 4.0 mM EGTA, pH 6.0-9.0, 20 degrees C). At sarcomere lengths greater than or equal to 3.5 microns, the length of the A-band was estimated by comparing the intensity of the first-order optical diffraction line with the results of model calculations. These results were supported by differential-interference microscopy and sodium dodecyl sulfate gel electrophoresis. It was shown that the resting tension decreased nearly in proportion to the residual length of the A-band. At sarcomere lengths less than or equal to 4.0 microns, the resting tension after the dissociation of the A-band was lowered to less than 10% of the initial value. On the other hand, at sarcomere lengths greater than or equal to 5.0 microns the resting tension after the dissociation of the A-band still showed approximately 35% of the initial value and did not change even after the I-band was dissociated by a solution containing KI. From these results, we propose that most of the PECs contributing to resting tension bind almost uniformly to the A-band and there are also PECs connecting Z-lines.     

Stoichiometry and stability of caldesmon in native thin filaments from sheep aorta smooth muscle.

Ca2(+)-regulated native thin filaments were extracted from sheep aorta smooth muscle. The caldesmon content determined by quantitative gel electrophoresis was 0.06 caldesmon molecule/actin monomer (1 caldesmon molecule per 16.3 actin monomers). Dissociation of caldesmon and tropomyosin from the thin filament and the depolymerization of actin was measured by sedimenting diluted thin filaments. Actin critical concentration was 0.05 microM at 10.1 and 0.13 at 10.05 compared with 0.5 microM for pure F-actin. Tropomyosin was tightly bound, with half-maximal dissociation at less than 0.3 microM thin filaments (actin monomer) under all conditions. Caldesmon dissociation was independent of tropomyosin and not co-operative. The concentration of thin filaments where 50% of the caldesmon was dissociated (CD50) ranged from 0.2 microM (actin monomer) at 10.03 to 8 microM at 10.16 in a 5 mM-MgCl2, pH 7.1, buffer. Mg2+, 25 mM at constant I, increased CD50 4-fold. CD50 was 4-fold greater at 10(-4) M-Ca2+ than at 10(-9) M-Ca2+. Aorta heavy meromyosin (HMM).ADP.Pi complex (2.5 microM excess over thin filaments) strongly antagonized caldesmon dissociation, but skeletal-muscle HMM.ADP.Pi did not. The behaviour of caldesmon in native thin filaments was indistinguishable from caldesmon in reconstituted synthetic thin filaments. The variability of Ca2(+)-sensitivity with conditions observed in thin filament preparations was shown to be related to dissociation of regulatory caldesmon from the thin filament.