The effect of calcium activation of skinned fiber bundles on the structure of Limulus thick filaments

Here we present evidence that strongly suggests that the well-documented phenomenon of A-band shortening in Limulus telson muscle is activation dependent and reflects fragmentation of thick filaments at their ends. Calcium activation of detergent-skinned fiber bundles of Limulus telson muscle results in large decreases in A-band (from 5.1 to 3.3 microns) and thick filament (from 4.1 to 3.3 microns) lengths and the release of filament end fragments. In activated fibers, maintained stretched beyond overlap of thick and thin filaments, these end fragments are translocated to varying depths within the I-bands. Here they are closely associated with fine filamentous structures that also span the gap between A- and I-bands and attach to the distal one-third of the thick filaments. End-fragments are rarely, if ever, present in similarly stretched and skinned, but unstimulated fibers, although fine "gap filaments" persist. Negatively stained thick filaments, separated from skinned, calcium-activated, fiber bundles, allowed to shorten freely, are significantly shorter than those obtained from unstimulated fibers, but are identical to the latter with respect to both the surface helical array of myosin heads and diameters. Many end-fragments are present on grids containing thick filaments from activated fibers; few, if any, on those from unstimulated fibers. SDS-PAGE shows no evidence of proteolysis due to activation and demonstrates the presence of polypeptides with very high molecular weights in the preparations. We suggest that thick filament shortening is a direct result of activation in Limulus telson muscle and that it occurs largely by breakage within a defined distal region of each polar half of the filament. It is possible that at least some of the fine "gap filaments" are composed of a titin-like protein. They may move the activation-produced, fragmented ends of thick filaments to which they attach, into the I-bands by elastic recoil, in highly stretched fibers.     

Localized deposition of lead phosphate precipitate in glycerinated rabbit psoas muscle during ATP-induced contraction

The location of ATP-ase sites within sarcomeres was studied by allowing glycerinated rabbit psoas muscle to shorten under load in a solution containing ATP and lead nitrate. The lead phosphate precipitate formed during the contraction is located mainly within the A-band of shortened sarcomeres. In fibers glycerinated at rest length, the heaviest precipitate deposits are located at the center of the A-band, roughly corresponding to the double overlap zone, with lesser precipitate concentrations at the A-l boundary contraction bands. In fibers glycerinated at stretched length, shortened sarcomeres with heavy contraction bands at the A-l boundary and patent H-bands have heavy precipitate deposits over the contraction bands and no localization to the center of the A-band. The precipitate is thus seen to be most concentrated at those regions where one would expect to find the best contact between thick and thin filaments. Physiological experiments were performed. Evidence is presented that lead ions can substitute for calcium in the contraction process, altering the time course of contraction.     

Ultrastructure of muscle fibers of an extraocular muscle of the pigeon

An electron microscopic analysis of the internal rectus muscle of the eye of the pigeon permitted identification of three types of muscle fibers: the first type shows the features previously described in vertebrate twitch fibers. The second type has very scarce sarcoplasmic reticulum at the A-band, their myofibrils fuse together at this level; the Z-line is large and the M-line is not present; the thick filaments are more abundant per unit area than in the first type of fibers, their hexagonal array is slightly disrupted and the fibers appear more opaque than the other two fiber types. The third type of fibers has bundles of myofibrils incompletely surrounded by sarcoplasmic reticulum at the A-band; the Z-line is large; the M-line is present and the hexagonal array of the thick filaments is maintained.     

Donnan potentials from striated muscle liquid crystals. Sarcomere length dependence.

Donnan potentials from A-bands and I-bands were measured as a function of sarcomere length in skinned long-tonic muscle fibers of the crayfish. These measurements were made using standard electrophysiological technique. Simultaneously, the relative cross-sectional area of the fibers was determined. Lattice plane spacings and hence unit-cell volumes were determined by low-angle x-ray diffraction. At a sarcomere length at which the myosin filaments and actin filaments nominally do not overlap, measurements of potential, relative cross-sectional area, and unit-cell volume were used in conjunction with Donnan equilibrium theory to calculate the effective linear charge densities along the myosin filament (6.6 X 10(4) e-/mu) and actin filament (6.8 X 10(3) e-/mu). Using these linear charge densities, unit-cell volumes and Donnan equilibrium theory, an algorithm was developed to predict A-band and I-band potentials at any sarcomere length. Over the range of sarcomere lengths investigated, the predicted values coincide with the experimental data. The ability of the model to predict the data demonstrates the applicability of Donnan equilibrium theory to measurements of electrochemical potential from liquid-crystalline systems.     

Donnan potentials from striated muscle liquid crystals. A-band and I-band measurements.

A-band and I-band potentials are measured selectively in crayfish skinned long-tonic muscle fibers. The microelectrode tip diameters used in this study are shown to be sufficiently small to permit the discrete placement of an electrode into either an A-band or I-band. Random and directed impalements into mechanically skinned fibers with microelectrodes yields reproducible trimodal distributions of potentials where the modalities represent the A-band (-1.80 mV), the I-band (-0.76 mV), and the Z-line vicinity (-3.63 mV). In conjunction with Donnan equilibrium theory, fixed charge concentrations are calculated from the measured potentials for the A-band (25.9 mmol e-/l), I-band (10.9 mmol e-/l), and Z-line vicinity (52.3 mmol e-/l). When skinned fibers are treated with Triton X-100, the mean potentials (and charge concentrations) decrease: A-band to -1.71 mV (24.6 mmol e-/l), I-band to -0.71 mV (10.2 mmol e-/l), and the Z-line vicinity to -3.40 mV (49.0 mmol e/l). In the A-band this represents a loss of 1.3 mmol e-/l while in the I-band 0.7 mmol e-/l are lost; both decreases are attributed to the removal of internal membranous structures. In the rigor condition the A-band increases to -2.18 mV (33.1 mmol e-/l) and the I-band increases to -0.88 mV (13.3 mmol e-/l). Relative to the relaxed condition, this represents an increase of 8.5 mmol e-/l and 3.1 mmol e-/l in the A-band and I-band, respectively. The evidence shows that it is practical to measure A-band and I-band potentials selectively. Further, it is demonstrated that similar measurements can be obtained from agar, another polyelectrolyte gel system (see Appendix).     

Degree of polarization of light diffracted from resting striated muscle

A laser light diffractometer has been developed to measure directly the total degree of polarization of (alpha t) of light diffracted and randomly scattered from striated muscle fibers. From alpha t the degree of polarization (alpha d) of light diffracted from the periodically arranged contractile filaments is determined. Measurements on single muscle fibers and small fiber bundles indicate that both alpha t and alpha d of the first-order diffraction decrease monotonically with sarcomere length. For the second-order diffraction, alpha t and alpha d exhibit a peak at sarcomere length of about 3.0 micron. A proposed theory based on the anisotropic light scattering efficiencies of the thick and thin filaments can account for the measurements. The comparison between the theory and measurements indicates that the A-band, as well as the I-band, are optically anisotropic.     

Oblique section 3-D reconstruction of relaxed insect flight muscle reveals the cross-bridge lattice in helical registration.

In this work we examined the arrangement of cross-bridges on the surface of myosin filaments in the A-band of Lethocerus flight muscle. Muscle fibers were fixed using the tannic-acid-uranyl-acetate, ("TAURAC") procedure. This new procedure provides remarkably good preservation of native features in relaxed insect flight muscle. We computed 3-D reconstructions from single images of oblique transverse sections. The reconstructions reveal a square profile of the averaged myosin filaments in cross section view, resulting from the symmetrical arrangement of four pairs of myosin heads in each 14.5-nm repeat along the filament. The square profiles form a very regular right-handed helical arrangement along the surface of the myosin filament. Furthermore, TAURAC fixation traps a near complete 38.7 nm labeling of the thin filaments in relaxed muscle marking the left-handed helix of actin targets surrounding the thick filaments. These features observed in an averaged reconstruction encompassing nearly an entire myofibril indicate that the myosin heads, even in relaxed muscle, are in excellent helical register in the A-band.     

A new muscle contractile system composed of a thick filament lattice and a single actin filament

To bridge the gap between the contractile system in muscle and in vitro motility assay, we have devised an A-band motility assay system. A glycerinated skeletal myofibril was treated with gelsolin to selectively remove the thin filaments and expose a single A-band. A single bead-tailed actin filament trapped by optical tweezers was made to interact with the inside or the outer surface of the A-band, and the displacement of the bead-tailed filament was measured in a physiological ionic condition by phase-contrast and fluorescence microscopy. We observed large back-and-forth displacement of the filament accompanied by a large change in developed force. Despite this large tension fluctuation, we found that the average force was proportional to the overlap inside and outside the A-band up to approximately 150 nm and 300 nm from the end of the A-band, respectively. Consistent with the difference in the density of myosin molecules, the average force per unit length of the overlap inside the A-band (the time-averaged force/myosin head was approximately 1 pN) was approximately twice as large as that outside. Thus, we conclude that the A-band motility assay system described here is suitable for studying force generation on a single actin filament, and its sliding movement within a regular three-dimensional thick filament lattice.     

Titin organisation and the 3D architecture of the vertebrate-striated muscle I-band

The giant muscle protein titin (connectin) is known to serve as a cytoskeletal element in muscle sarcomeres. It elastically restrains lengthening sarcomeres, it aids the integrity and central positioning of the A-band in the sarcomere and it may act as a template upon which some sarcomeric components are laid down during myogenesis. A puzzle has been how titin molecules, arranged systematically within the hexagonal A-band lattice of myosin filaments, can redistribute through the I-band to their anchoring sites in the tetragonal Z-band lattice. Recent work by Liversage and colleagues has suggested that there are six titin molecules per half myosin filament. Since there are two actin filaments per half myosin filament in a half sarcomere, this means that there are three titin molecules interacting with each Z-band unit cell containing one actin filament in the same sarcomere and one of opposite polarity from the next sarcomere. Liversage et al. suggested that the three titins might be distributed with two on an actin filament of one polarity and one on the filament of opposite polarity. Here, we build on this suggestion and discuss the transition of titin from the A-band to the Z-band. We show that there are good structural and mechanical reasons why titin might be organised as Liversage et al., suggested and we discuss the possible relationships between A-band arrangements in successive sarcomeres along a myofibril.     

Differential localization of two myosins within nematode thick filaments

The body wall muscle cells of the nematode, Caenorhabditis elegans, contain two unique types of myosin heavy chain, A and B. We have utilized an immunochemical approach to define the structural location of these two myosins within body wall muscle thick filaments. By immunofluorescence microscopy, myosin B antibodies label the thick filament-containing A-bands of body wall muscle with the exception of a thin gap at the center of each A-band, and myosin A antibodies react to form a medial fluorescent stripe within each A-band. The complexes of these monoclonal antibodies with isolated thick filaments were negatively stained and studied by electron microscopy. The myosin B antibody reacts with the polar regions of all filaments but does not react with a central 0.9 μm zone. The myosin A antibody reacts with a central 1.8 μm zone in all filaments but does not react with the polar regions.     

Degree of polarization of light diffracted from resting striated muscle

A laser light diffractometer has been developed to measure directly the total degree of polarization of ({ie145-1}) of light diffracted and randomly scattered from striated muscle fibers. From {ie145-2} the degree of polarization ({ie145-3}) of light diffracted from the periodically arranged contractile filaments is determined. Measurements on single muscle fibers and small fiber bundles indicate that both {ie145-4} and {ie145-5} of the firstorder diffraction decrease monotonically with sarcomere length. For the second-order diffraction, {ie145-6} and {ie145-7} exhibit a peak at sarcomere length of about 3.0 μm. A proposed theory based on the anisotropic light scattering efficiencies of the thick and thin filaments can account for the measurements. The comparison between the theory and measurements indicates that the A-band, as well as the I-band, are optically anisotropic.     

The molecular composition of the sarcomeric M-band correlates with muscle fiber type

The M-band is the transverse structure that cross-links the thick filaments in the center and provides a perfect alignment of the A-band in the activated sarcomere. The molecular composition of the M-bands in adult mouse skeletal muscle is fiber-type dependent. All M-bands in fast fibers contain M-protein while M-bands in slow fibers contain a significant proportion of the EH-myomesin isoform, previously detected only in embryonic heart muscle. This fiber-type specificity develops during the first postnatal weeks. However, the ratio between the amounts of myosin and of myomesin, taken as sum of both isoforms, remains nearly constant in all studied muscles. Ultrastructural analysis demonstrates that some of the soleus fibers show a diffuse appearance of the M-band, resembling the situation in the embryonic heart. A model is proposed to explain the functional consequence of differential M-band composition for the physiological and morphological properties of sarcomeres in different muscle types.     

Donnan potentials from striated muscle liquid crystals. Lattice spacing dependence.

Electrochemical potentials were measured as a function of myofilament packing density in crayfish striated muscle. The A-band striations are supramolecular smectic B1 lattice assemblies of myosin filaments and the I-band striations are nematic liquid crystals of actin filaments. Both A- and I-bands generate potentials derived from the fixed charge that is associated with structural proteins. In the reported experiments, filament packing density was varied by osmotically reducing lattice volume. The electrochemical potentials were measured from the A- and I-bands in the relaxed condition over a range of lattice volumes. From the measurements of relative cross-sectional area, unit-cell volume (obtained by low-angle x-ray diffraction) and previously determined effective linear charge densities (Aldoroty, R.A., N.B. Garty, and E.W. April, 1985, Biophys. J., 47:89-96), Donnan potentials can be predicted for any amount of compression. In the relaxed condition, the predicted Donnan potentials correspond to the measured electrochemical potentials. In the rigor condition, however, a net increase in negative charge associated with the myosin filament is observed. The predictability of the data demonstrates the applicability of Donnan equilibrium theory to the measurement of electrochemical potentials from liquid-crystalline systems. Moreover, the relationship between filament spacing and the Donnan potential is consistent with the concept that surface charge provides the necessary electrostatic force to stabilize the myofilament lattice.     

Three-dimensional structure of the vertebrate muscle A-band: II. The myosin filament superlattice

The three-dimensional arrangement of the myosin filaments in the A-band of frog sartorius muscle was studied using electron micrographs of very thin and accurately cut transverse sections through the bare region (on each side of the M-band) where the thick filament shafts are roughly triangular in shape. It was found that the orientations of these triangular profiles are arranged to give a superlattice of the same size and shape as that proposed by Huxley & Brown (1967) on the basis of X-ray diffraction evidence, but the contents of the superlattice may not be as they suggested. The results from detailed image analysis strongly suggest that myosin filaments (which have been shown to have 3-fold rotational symmetry, Luther, 1978; Luther, Munro and Squire, unpublished results) are arranged with one of two orientations which are 60 ° (or 180 °) apart. This arrangement of filaments with 3-fold symmetry is not that predicted for a superlattice with the symmetry suggested by Huxley & Brown.Two rules define the way in which the orientations of neighbouring filaments are defined. Rule (1): no three mutually adjacent filaments in the hexagonal array of filaments in the A-band can all have identical orientations; and rule (2): no three successive filaments along a 10$\text{1}\text{?}$ row in the filament array can have identical orientations. These two no-three-alike rules are sufficient to describe the observed arrangement of filament profiles in the frog bare region (except for some minor violations discussed in the text), and they lead automatically to the generation of the required superlattice. The A-band structure in fish muscle is different; there is no superlattice and the triangular bare region profiles have only one orientation. The frog superlattice and fish simple lattice are explained directly in terms of different interactions between the M-bridges in the M-bands of these muscles. The observed structures require that the myosin filament symmetry at the centre of the M-band is that of the dihedral point group 32. The two possible forms of interaction between filaments with this symmetry (apart from a completely random structure) give rise to the observed A-band lattices in frog and fish muscles. The 3-fold rotational symmetry of the myosin filaments required to explain the observed micrographs also requires that the myosin crossbridge arrangements around the actin filaments in frog and fish muscles will be different. It is suggested that the structure in the frog A-band (and in the A-bands of other higher vertebrates) has evolved from that in fish to improve the distribution of crossbridges around the aotin filaments. The X-ray diffraction evidence of Huxley & Brown (1967) will be accounted for in terms of the proposed A-band structure in a further paper in this series.     

Ultrastructure of the myocardial cell and its membrane systems in the adult fly Calliphora erythrocephala (Insecta: Diptera)

Summary Calliphora erythrocephala has cross-striated cardiac muscle cells with A, I and Z-bands. The diameters of the myosin and actin filaments are 200–250 Å and 85 Å respectively and the length of the myosin filaments (A-band) is approximately 1.5 . Usually 8–10 actin filaments surround each myosin filament.The myocardial cells show a well-developed membrane system and interior couplings. A perforated sheet of SR envelopes the myofibrils at the A-band, dilates into flattened cisternae at both A-I band levels before it merges into a three-dimensional net-work between the actin filaments of the I-bands and between the dense bodies of the discontinuous Z-discs. The T-system consists of broad flattened tubules running between the myofibrils at the A-I band levels forming dyads with the SR-cisternae. Longitudinal connections between the transverse (T-) tubules often occur.It is suggested that this well-developed SR may be an adaptation to facilitate a rapid contraction/relaxation frequency by an effective Ca²⁺ uptake.     

Lesions in the rat soleus muscle following eccentrically biased exercise

Morphological changes in skeletal muscle related to lengthening (eccentric) contractions have been noted by several laboratories. However, a systematic examination of skeletal muscle following repetitive eccentric contractions is lacking. This study was undertaken to study lesions and determine their relative densities in rat soleus muscle 30 min following level or downhill treadmill exercise. Following fixation in situ by vascular perfusion, toluidine-blue-stained 1-micron sections of the muscle samples selected at 73-micron intervals were scanned with a light microscope. Three types of lesions were noted: focal disruptions in the A-band, localized dissolution of Z-lines, and clotting of muscle fibers. Soleus muscle from the caged controls and the tibialis anterior muscle from downhill-exercised rats were essentially free of lesions. Eighty-nine percent of the soleus m. lesions in the downhill runner group and 97% of those in the level runner group were A-band disruptions. A-band lesion density was significantly higher in the soleus muscle of the downhill runners compared to level runners with the highest incidence in the distal half. A-band lesion density was lower in soleus muscles from level runners; however, the highest intramuscular incidence was in the proximal rather than the distal end. The results indicate that a disruption of the A-band is a principal change within some skeletal muscle fibers 30 min following repetitive eccentric contractions.     

The positional stability of thick filaments in activated skeletal muscle depends on sarcomere length: evidence for the role of titin filaments

Electron microscopy was used to study the positional stability of thick filaments in isometrically contracting skinned rabbit psoas muscle as a function of sarcomere length at 7 degrees C. After calcium activation at a sarcomere length of 2.6 micron, where resting stiffness is low, sarcomeres become nonuniform in length. The dispersion in sarcomere length is complete by the time maximum tension is reached. A-bands generally move from their central position and continue moving toward one of the Z-discs after tension has reached a plateau at its maximum level. The lengths of the thick and thin filaments remain constant during this movement. The extent of A-band movement during contraction depends on the final length of the individual sarcomere. After prolonged activation, all sarcomeres between 1.9 and 2.5 micron long exhibit A-bands that are adjacent to a Z-disc, with no intervening I-band. Sarcomeres 2.6 or 2.7 micron long exhibit a partial movement of A-bands. At longer sarcomere lengths, where the resting stiffness exceeds the slope of the active tension-length relation, the A-bands remain perfectly centered during contraction. Sarcomere symmetry and length uniformity are restored upon relaxation. These results indicate that the central position of the thick filaments in the resting sarcomere becomes unstable upon activation. In addition, they provide evidence that the elastic titin filaments, which join thick filaments to Z-discs, produce almost all of the resting tension in skinned rabbit psoas fibers and act to resist the movement of thick filaments away from the center of the sarcomere during contraction.     

ULTRASTRUCTURE OF BARNACLE GIANT MUSCLE FIBERS

Increasing use of barnacle giant muscle fibers for physiological research has prompted this investigation of their fine structure. The fibers are invaginated by a multibranched system of clefts connecting to the exterior and filled with material similar to that of the basement material of the sarcolemmal complex. Tubules originate from the surface plasma membrane at irregular sites, and also from the clefts They run transversely, spirally, and longitudinally, making many diadic and some triadic contacts with cisternal sacs of the longitudinal sarcoplasmic reticulum. The contacts are not confined to any particular region of the sarcomere. The tubules are wider and their walls are thicker at points of contact with Z material. Some linking of the Z regions occurs across spaces within the fiber which contain large numbers of glycogen particles. A-band lengths are extremely variable, in the range 2.2 µm–20.3 µm (average 5.2 µm) Individual thick filaments have thin (110 Å) hollow regions alternating with thick (340 Å) solid ones. Bridges between thick filaments occur at random points and are not concentrated into an M band The thin:thick filament ratio is variable in different parts of a fiber, from 3:1 to 6:1. Z bands are basically perforated, but the number of perforations may increase during contraction.     

The myosin filament superlattice in the flight muscles of flies: A-band lattice optimisation for stretch-activation?

Low-angle X-ray diffraction patterns from relaxed fruitfly (Drosophila) flight muscle recorded on the BioCat beamline at the Argonne Advanced Photon Source (APS) show many features similar to such patterns from the "classic" insect flight muscle in Lethocerus, the giant water bug, but there is a characteristically different pattern of sampling of the myosin filament layer-lines, which indicates the presence of a superlattice of myosin filaments in the Drosophila A-band. We show from analysis of the structure factor for this lattice that the sampling pattern is exactly as expected if adjacent four-stranded myosin filaments, of repeat 116 nm, are axially shifted in the hexagonal A-band lattice by one-third of the 14.5 nm axial spacing between crowns of myosin heads. In addition, electron micrographs of Drosophila and other flies (e.g. the house fly (Musca) and the flesh fly (Sarcophaga)) combined with image processing confirm that the same A-band superlattice occurs in all of these flies; it may be a general property of the Diptera. The different A-band organisation in flies compared with Lethocerus, which operates at a much lower wing beat frequency (approximately 30 Hz) and requires a warm-up period, may be a way of optimising the myosin and actin filament geometry needed both for stretch activation at the higher wing beat frequencies (50 Hz to 1000 Hz) of flies and their need for a rapid escape response.     

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.