Electron microscopic and optical diffraction analysis of the structure of scorpion muscle thick filaments

We rapidly and gently isolated thick filaments from scorpion tail muscle by a modification of the technique previously described for isolating Limulus thick filaments. Images of negatively stained filaments appeared to be highly periodic, with a well-preserved myosin cross-bridge array. Optical diffraction patterns of the electron micrograph images were detailed and similar to optical diffraction patterns from Limulus and tarantula thick filaments. Analysis of the optical diffraction patterns and computed Fourier transforms, together with the appearance of the filaments in the micrographs, suggested a model for the filaments in which the myosin cross-bridges were arranged on four helical strands with 12 cross-bridges per turn of each strand, thus giving the observed repeat every third cross-bridge level. Comparison of the scorpion thick filaments with those isolated from the closely related chelicerate arthropods, Limulus and tarantula, revealed that they were remarkably similar in appearance and helical symmetry but different in diameter.     

Structure and periodicities of cross-bridges in relaxation, in rigor, and during contractions initiated by photolysis of caged Ca2+.

Ultra-rapid freezing and electron microscopy were used to directly observe structural details of frog muscle fibers in rigor, in relaxation, and during force development initiated by laser photolysis of DM-nitrophen (a caged Ca2+). Longitudinal sections from relaxed fibers show helical tracks of the myosin heads on the surface of the thick filaments. Fibers frozen at approximately 13, approximately 34, and approximately 220 ms after activation from the relaxed state by photorelease of Ca2+ all show surprisingly similar cross-bridge dispositions. In sections along the 1,1 lattice plane of activated fibers, individual cross-bridge densities have a wide range of shapes and angles, perpendicular to the fiber axis or pointing toward or away from the Z line. This highly variable distribution is established very early during development of contraction. Cross-bridge density across the interfilament space is more uniform than in rigor, wherein the cross-bridges are more dense near the thin filaments. Optical diffraction (OD) patterns and computed power density spectra of the electron micrographs were used to analyze periodicities of structures within the overlap regions of the sarcomeres. Most aspects of these patterns are consistent with time resolved x-ray diffraction data from the corresponding states of intact muscle, but some features are different, presumably reflecting different origins of contrast between the two methods and possible alterations in the structure of the electron microscopy samples during processing. In relaxed fibers, OD patterns show strong meridional spots and layer lines up to the sixth order of the 43-nm myosin repeat, indicating preservation and resolution of periodic structures smaller than 10 nm. In rigor, layer lines at 18, 24, and 36 nm indicate cross-bridge attachment along the thin filament helix. After activation by photorelease of Ca2+, the 14.3-nm meridional spot is present, but the second-order meridional spot (22 nm) disappears. The myosin 43-nm layer line becomes less intense, and higher orders of 43-nm layer lines disappear. A 36-nm layer line is apparent by 13 ms and becomes progressively stronger while moving laterally away from the meridian of the pattern at later times, indicating cross-bridges labeling the actin helix at decreasing radius.     

AN ultrastructural study of cross-bridge arrangement in the frog thigh muscle thick filament.

We have developed thick filament isolation methods that preserve the relaxed cross-bridge order of frog thick filaments such that the filaments can be analyzed by the convergent techniques of electron microscopy, optical diffraction, and computer image analysis. Images of the filaments shadowed by using either unidirectional shadowing or rotary shadowing show a series of subunits arranged along a series of right-handed near-helical strands that occur every 43 nm axially along the filament arms. Optical filtrations of images of these shadowed filaments show 4-5 subunits per half-turn of the strands, consistent with a three-stranded arrangement of the cross-bridges, thus supporting our earlier results from negative staining and computer-image analysis. The optical diffraction patterns of the shadowed filaments show a departure from the pattern expected for helical symmetry consistent with the presence of cylindrical symmetry and a departure of the cross-bridges from helical symmetry. We also describe a modified negative staining procedure that gives improved delineation of the cross-bridge arrangement. From analysis of micrographs of these negatively stained filament tilted about their long axes, we have computed a preliminary three-dimensional reconstruction of the filament that clearly confirms the three-stranded arrangement of the myosin heads.     

Modeling rigor cross-bridge patterns in muscle I. Initial studies of the rigor lattice of insect flight muscle.

We have undertaken some computer modeling studies of the cross-bridge observed by Reedy in insect flight muscle so that we investigate the geometric parameters that influence the attachment patterns of cross-bridges to actin filaments. We find that the appearance of double chevrons along an actin filament indicates that the cross-bridges are able to reach 10--14 nm axially, and about 90 degrees around the actin filament. Between three and five actin monomers are therefore available along each turn of one strand of actin helix for labeling by cross-bridges from an adjacent myosin filament. Reedy's flared X of four bridges, which appears rotated 60 degrees at successive levels on the thick filament, depends on the orientation of the actin filaments in the whole lattice as well as on the range of movement in each cross-bridge. Fairly accurate chevrons and flared X groupings can be modeled with a six-stranded myosin surface lattice. The 116-nm long repeat appears in our models as "beating" of the 14.5-nm myosin repeat and the 38.5-nm actin period. Fourier transforms of the labeled actin filaments indicate that the cross-bridges attach to each actin filament on average of 14.5 nm apart. The transform is sensitive to changes in the ease with which the cross-bridge can be distorted in different directions.     

The myosin filament: V. Intermediate voltage electron microscopy and optical diffraction studies of the substructure

Using a 200 kV electron microscope (JEM 200 A), thick (up to 0.4 μm) crosssections of the myosin filaments of vertebrate striated muscle were studied. It was found that: (a) with increasing section thickness the cross-sectional profiles of the shaft of the filament were increasingly more triangular and in sections 0.4 μm thick each apex of the triangle was clearly blunted. This unique cross-sectional profile is predicted by the model proposed by Pepe (1966,1967) in which 12 parallel structural units are packed to form a triangular profile with a structural unit missing at each apex of the triangle. (b) With increasing section thickness the substructure of the myosin filament was enhanced, with the best substructure visible in sections 0.2 μm to 0.3 μm thick. This strongly supports parallel alignment of structural units in the shaft of the filament as proposed by Pepe (1966,1967). (c) The substructure spacing, determined by optical diffraction from electron micrographs of cross-sections of individual myosin filaments or groups of filaments is about 4 nm. (d) The different optical diffraction patterns observed from individual myosin filaments can be explained if the projection of each structural unit in the plane of the section has an elongated profile. With a substructure spacing of 4 nm an elongated cross-sectional profile could be produced by having two myosin molecules per structural unit. Models drawn with two myosin molecules per structural unit in the model proposed by Pepe (1966,1967) gave optical diffraction patterns similar to those observed from individual filaments. (e) The different optical diffraction patterns observed from individual myosin filaments can be explained if the elongated profiles in each structural unit are similarly oriented but with the orientation changing along the length of the filament. The change in orientation per unit length of the filament must be small enough to maintain an elongated profile for the projection of the structural unit in the plane of the sections 0.3 μm thick. All of these observations and conclusions strongly support the model for the myosin filament proposed by Pepe (1966,1967).     

The vertebrate skeletal muscle thick filaments are not three-stranded. Reinterpretation of some experimental data

Computer simulation of mass distribution within the model and Fourier transforms of images depicting mass distribution are explored for verification of two alternative modes of the myosin molecule arrangement within the vertebrate skeletal muscle thick filaments. The model well depicting the complete bipolar structure of the thick filament and revealing a true threefold-rotational symmetry is a tube covered by two helices with a pitch of 2 x 43 nm due to arrangement of the myosin tails along a helical path and grouping of all myosin heads in the crowns rotated by 240 degrees and each containing three cross-bridges separated by 0 degrees, 120 degrees, and 180 degrees. The cross-bridge crown parameters are verified by EM images as well as by optical and low-angle X-ray diffraction patterns found in the literature. The myosin tail arrangement, at which the C-terminus of about 43-nm length is near-parallel to the filament axis and the rest of the tail is quite strongly twisted around, is verified by the high-angle X-ray diffraction patterns. A consequence of the new packing is a new way of movement of the myosin cross-bridges, namely, not by bending in the hinge domains, but by unwrapping from the thick filament surface towards the thin filaments along a helical path.     

Structure of the myosin filaments of relaxed and rigor vertebrate striated muscle studied by rapid freezing electron microscopy.

Rapid freezing followed by freeze-substitution has been used to study the ultrastructure of the myosin filaments of live and demembranated frog sartorius muscle in the states of relaxation and rigor. Electron microscopy of longitudinal sections of relaxed specimens showed greatly improved preservation of thick filament ultrastructure compared with conventional fixation. This was revealed by the appearance of a clear helical arrangement of myosin crossbridges along the filament surface and by a series of layer line reflections in computed Fourier transforms of sections, corresponding to the layer lines indexing on a 43 nm repeat in X-ray diffraction patterns of whole, living muscles. Filtered images of single myosin filaments were similar to those of negatively stained, isolated vertebrate filaments and consistent with a three-start helix. M-line and other non-myosin proteins were also very well preserved. Rigor specimens showed, in the region of overlapping myosin and actin filaments, periodicities corresponding to the 36, 24, 14.4 and 5.9 nm repeats detected in X-ray patterns of whole muscle in rigor; in the H-zone they showed a disordered array of crossbridges. Transverse sections, whose Fourier transforms extend to the (3, 0) reflection, supported the view, based on X-ray diffraction and conventional electron microscopy, that in the overlap zone of relaxed muscle most of the crossbridges are detached from the thin filaments while in rigor they are attached. We conclude that the rapid freezing technique preserves the molecular structure of the myofilaments closer to the in vivo state (as monitored by X-ray diffraction) than does normal fixation.     

Recent X-ray Diffraction and Electron Microscope Studies of Striated Muscle

The sliding filament model for muscular contraction supposes that an appropriately directed force is developed between the actin and myosin filaments by some process in which the cross-bridges are involved. The cross-bridges between the filaments are believed to represent the parts of the myosin molecules which possess the active sites for ATPase activity and actin-binding ability, and project out sidewise from the backbone of the thick filaments. The arrangement of the cross-bridges is now being studied by improved low-angle X-ray diffraction techniques, which show that in a resting muscle, they are arranged approximately but not exactly in a helical pattern, and that there are other structural features of the thick filaments which give rise to additional long periodicities shown up by the X-ray diagram. The actin filaments also contain helically arranged subunits, and both the subunit repeat and the helical repeat are different from those in the myosin filaments. Diffraction diagrams can be obtained from muscles in rigor (when permanent attachment of the cross-bridges to the actin subunits takes place) and now, taking advantage of the great increase in the speed of recording, from actively contracting muscles. These show that changes in the arrangement of the cross-bridges are produced under both these conditions and are no doubt associated in contraction with the development of force. Thus configurational changes of the myosin component in muscle have been demonstrated: these take place without any significant over-all change in the length of the filaments.     

The Z lattice in canine cardiac muscle

Filtered images of mammalian cardiac Z bands were reconstructed from optical diffraction patterns from electron micrographs. Reconstructed images from longitudinal sections show connecting filaments at each 38-nm axial repeat in an array consistent with cross-sectional data. Some reconstructed images from cross sections indicate two distinctly different optical diffraction patterns, one for each of two lattice forms (basket weave and small square). Other images are more complex and exhibit composite diffraction patterns. Thus, the two lattice forms co-exist, interconvert, or represent two different aspects of the same details within the lattice. Two three-dimensional models of the Z lattice are presented. Both include the following features: a double array of axial filaments spaced at 24 nm, successive layers of tetragonally arrayed connecting filaments, projected fourfold symmetry in cross section, and layers of connecting filaments spaced at intervals of 38 nm along the myofibril axis. Projected views of the models are compared to electron micrographs and optically reconstructed images of the Z lattice in successively thicker cross sections. The entire Z band is rarely a uniform lattice regardless of plane of section or section thickness. Optical reconstructions strongly suggest two types of variation in the lattice substructure: (a) in the arrangement of connecting filaments, and (b) in the arrangement of units added side-to-side to make larger myofilament bundles and/or end-to-end to make wider Z bands. We conclude that the regular arrangement of axial and connecting filaments generates a dynamic Z lattice.     

Actin filaments in muscle: Pattern of myosin and tropomyosin/troponin attachments

X-ray patterns from lobster and crayfish muscles show very clear layer lines from the thin filaments, well separated from the myosin layer lines. The intensities in patterns from relaxed muscles include an important contribution from the regulatory proteins, and allow the arrangement of the troponin complexes to be deduced. Moreover, the troponin diffraction indirectly provides an accurate value for the pitch of the actin helix in relaxed muscle.In rigor, the attachment of cross-bridges modifies the intensities. These X-ray patterns support Reedy's (1968) concept that cross-bridges in rigor attach only to certain azimuths on the actin filaments (“target areas”); the 145 Å repeat of their origins on the thick filaments is not reflected in the pattern of attachment. Our calculations show that the observed intensities agree quantitatively with those expected for models based on such attachment, but depend significantly on the locations of the troponin complexes. The arrangement of the filament components is discussed in terms of design requirements. Our conclusions may be applicable to many other muscles, especially insect flight muscle and other invertebrate muscles.     

Cross-Bridges and Periods in Insect Flight Muscle

The periodic structure of the cross-bridge lattice of glycerinatedLethocerus flight muscle has been studied in sections by electronmicroscopy, assisted by optical diffraction, and in unfixedfiber bundles by X-ray diffraction. Diffraction patterns exhibitfirst through ninth orders of 1166 Ä, virtually all ofwhich were found to arise from the lattice of cross-bridges.Diffraction and inspection show that "horizontal" cross-bridgesof relaxation become slanted in rigor, and may push actins towardthe M line in producing the increase in tension seen with theinduction of rigor. Myosin filaments contain unexpected structural features. Cross-bridgeorigins form opposed pairs repeating every 146 Ä; and rotating67.5 degrees with each repeat, thus defining twin, left-handed,helical tracks which require 1 turns (or 8 x 146 Ä) toestablish a meridional repeat of 1166 Ä. Each origin isdual and gives rise to two bridges; thus, the unit groupingof paired origins involves four bridges. One half-turn of themyosin helix requires 388 Ä, matching the actin helix exactlyin pitch. (Actin is, however, right-handed.) The resulting matchseems awkward azimuthally (sixteenfold myosin distributes bridgesto a sixfold envelope of actin filaments), but minimizes axialmismatching between subunits of the myosin and actin and lendscredence to the theory that all bridges may swing synchronouslyduring typical, low-amplitude, oscillatory contractions.     

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.     

Frog skeletal muscle thick filaments are three-stranded

A procedure has been developed for isolating and negatively staining vertebrate skeletal muscle thick filaments that preserves the arrangement of the myosin crossbridges. Electron micrographs of these filaments showed a clear periodicity associated with crossbridges with an axial repeat of 42.9 nm. Optical diffraction patterns of these images showed clear layer lines and were qualitatively similar to published x-ray diffraction patterns, except that the 1/14.3-nm meridional reflection was somewhat weaker. Computer image analysis of negatively stained images of these filaments has enabled the number of strands to be established unequivocally. Both reconstructed images from layer line data and analysis of the phases of the inner maxima of the first layer line are consistent only with a three-stranded structure and cannot be reconciled with either two- or four-stranded models.     

Structure and paramyosin content of tarantula thick filaments

Muscle fibers of the tarantula femur exhibit structural and biochemical characteristics similar to those of other long-sarcomere invertebrate muscles, having long A-bands and long thick filaments. 9-12 thin filaments surround each thick filament. Tarantula muscle has a paramyosin:myosin heavy chain molecular ratio of 0.31 +/- 0.079 SD. We studied the myosin cross-bridge arrangement on the surface of tarantula thick filaments on isolated, negatively stained, and unidirectionally metal-shadowed specimens by electron microscopy and optical diffraction and filtering and found it to be similar to that previously described for the thick filaments of muscle of the closely related chelicerate arthropod, Limulus. Cross-bridges are disposed in a four-stranded right-handed helical arrangement, with 14.5-nm axial spacing between successive levels of four bridges, and a helical repeat period every 43.5 nm. The orientation of cross-bridges on the surface of tarantula filaments is also likely to be very similar to that on Limulus filaments as suggested by the similarity between filtered images of the two types of filaments and the radial distance of the centers of mass of the cross-bridges from the surfaces of both types of filaments. Tarantula filaments, however, have smaller diameters than Limulus filaments, contain less paramyosin, and display structure that probably reflects the organization of the filament backbone which is not as apparent in images of Limulus filaments. We suggest that the similarities between Limulus and tarantula thick filaments may be governed, in part, by the close evolutionary relationship of the two species.     

Optical diffraction of the Z lattice in canine cardiac muscle

Optical diffraction patterns from electron micrographs of both longitudinal and cross sections of normal and anomalous canine cardiac Z bands have been compared. The data indicate that anomalous cardiac Z bands resembling nemaline rods are structurally related to Z bands in showing a repeating lattice common to both. In thin sections transverse to the myofibril axis, both electron micrographs and optical diffraction patterns of the Z structure reveal a square lattice of 24 nm. This lattice is simple at the edge of each I band and centered in the interior of the Z band, where two distinct lattice forms have been observed. In longitudinal sections, oblique filaments visible in the electron micrographs correspond to a 38-nm axial periodicity in diffraction patterns of both Z band and Z rod. We conclude that the Z rods will be useful for further analysis and reconstruction of the Z lattice by optical diffraction techniques.     

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.     

Interaction of aldolase with thin filaments within I-disks, isolated from skeletal muscles

Using electron microscopy and optical diffraction, Ca2+-dependent binding of a glycolytic enzyme (aldolase) to thin filaments of isolated skeletal muscle I-disks have been revealed. On the micrographs of negatively stained I-disks the cross-striation determined by troponin-tropomyosin complex distribution has a period of about 38 nm. The width of troponin-tropomyosin stripes is 5-6 nm. On the optical diffraction patterns from isolated I-disks the meridional reflections measuring 38.5, 19.2, 12.8 nm are present. On the micrographs of isolated I-disks, treated with aldolase in the absence of Ca2+ (1 mM EGTA) the width of periodic transverse stripes (period approximately 38 nm) increases from 5-6 nm to 25-28 nm due to the interaction of aldolase with thin filaments. On the optical diffraction patterns from I-disks treated with aldolase in the absence of Ca2+ (1 mM EGTA) the strong meridional reflection equal to 38.5 nm is present, while the reflections equal to 19.2 nm are absent. The optical diffraction patterns from I-disks treated with aldolase in the presence of Ca2+ (greater than or equal to 10(-5) M) do not, as a rule, differ from those obtained from I-disks not treated with aldolase, i.e. they contain the three above reflections. The binding of aldolase to thin filaments in the absence of Ca2+ is the reason of disappearance of meridional reflections equal to 19.2 and 12.8 nm.     

The axial repeats in paracrystals of light meromyosin and its complex with C-protein

We examined the axial repeats in electron micrographs of three types of negatively stained paracrystals (two tactoid- and one sheet-like type) of rabbit light meromyosin (LMM) and its complex with C-protein characterized previously by similar axial period of about 43.0 nm. Assuming for the axial repeat in type II tactoids the value of 42.93 +/- 0.05 nm as it was determined by X-ray diffraction technique (Yagi and Offer 1981), we found average axial repeats in type I tactoid and in sheet-like paracrystal of 42.93 +/- 0.75 nm and 43.50 +/- 0.62 nm respectively. Analyzing the micrographs where the two types paracrystals are located side-by-side we determined rather accurately the average ratio of axial repeat in sheet-like paracrystal to that in type I tactoid (1.013 +/- 0.002). Taking 42.93 nm as the axial repeat in type I tactoid, the axial repeat in sheet-like paracrystal was found to be 43.50 +/- 0.08 nm. C-protein binds to LMM with the period of the underlying LMM paracrystals and independently of the value of their axial repeats. Two different axial repeats (42.9 nm and 43.5 nm) revealed for LMM paracrystals in this study precisely coincide with the average repeat periods of myosin crossbridges along the thick filaments found for different physiological states of skeletal muscles (Lednev and Kornev 1987). Molecular basis for the appearance of two structural states in LMM paracrystals and in the shafts of thick filaments are discussed.     

Resting myosin cross-bridge configuration in frog muscle thick filaments

Clear images of myosin filaments have been seen in shadowed freeze-fracture replicas of single fibers of relaxed frog semitendinosus muscles rapidly frozen using a dual propane jet freezing device. These images have been analyzed by optical diffraction and computer averaging and have been modelled to reveal details of the myosin head configuration on the right-handed, three-stranded helix of cross-bridges. Both the characteristic 430-A and 140-150-A repeats of the myosin cross-bridge array could be seen. The measured filament backbone diameter was 140-160 A, and the outer diameter of the cross-bridge array was 300 A. Evidence is presented that suggests that the observed images are consistent with a model in which both of the heads of one myosin molecule tilt in the same direction at an angle of approximately 50-70 degrees to the normal to the filament long axis and are slewed so that they lie alongside each other and their radially projected density lies along the three right-handed helical tracks. Any perturbation of the myosin heads away from their ideal lattice sites needed to account for x-ray reflections not predicted for a perfect helix must be essentially along the three helical tracks of cross-bridges. Little trace of the presence of non-myosin proteins could be seen.     

Determination of quantitative parameters of the binding of F-protein (phosphofructokinase) with myosin and localization of binding sites on the myosin molecule

To determine the localization of F-protein binding sites on myosin, the interaction of F-protein with myosin and its proteolytic fragments in 0.1 M KCl, 10 mM K-phosphate pH 6.5 was studied, using sedimentation, electron microscopic and optical diffraction methods. Sedimentation experiments showed that F-protein binds to myosin and myosin rod rather than to light meromyosin or S-1. The F-protein binding to myosin and rod is of a similar character. The calculated values of the constants of F-protein binding to myosin and rod are 2.6 X 10(5) M-1 and 2.1 X 10(5) M-1, respectively. The binding sites are probably located on the subfragment-2 portion of the myosin molecule. The number of F-protein binding sites on myosin calculated per chain weight of 80 000 is 5 +/- 1. The sedimentation results were confirmed by electron microscopic data. F-protein does not bind to light meromyosin paracrystals, but decorates myosin and rod filaments with the interval of 14.3 nm regardless of whether F-protein is added before or after filamentogenesis. A comparison of optical diffraction patterns obtained from myosin and rod filaments with those from decorated ones revealed a marked enhancement of meridional reflection at (14.3 nm)-1 in the latter case.