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 electron microscopic and optical diffraction analysis of the structure of Limulus telson muscle thick filaments

Long, thick filaments (greater than 4.0 micrometer) rapidly and gently isolated from fresh, unstimulated Limulus muscle by an improved procedure have been examined by electron microscopy and optical diffraction. Images of negatively stained filaments appear highly periodic with a well-preserved myosin cross-bridge array. Optical diffraction patterns of the electron micrographs show a wealth of detail and are consistent with a myosin helical repeat of 43.8 nm, similar to that observed by x-ray diffraction. Analysis of the optical diffraction patterns, in conjunction with the appearance in electron micrographs of the filaments, supports a model for the filament in which the myosin cross-bridges are arranged on a four-stranded helix, with 12 cross-bridges per turn or each helix, thus giving an axial repeat every third level of cross-bridges (43.8 nm).     

The mammalian cardiac muscle thick filament: crossbridge arrangement

Although skeletal muscle thick filaments have been extensively studied, information on the structure of cardiac thick filaments is limited. Since cardiac muscle differs in many physiological properties from skeletal muscle it is important to elucidate the structure of the cardiac thick filament. The structure of isolated and negatively stained rabbit cardiac thick filaments has been analyzed from computed Fourier transforms and image analysis. The transforms are detailed, showing a strong set of layer lines corresponding to a 42.9 nm quasi-helical repeat. The presence of relatively strong "forbidden" meridional reflections not expected from ideal helical symmetry on the second, fourth, fifth, seventh, eighth, and tenth layer lines suggest that the crossbridge array is perturbed from ideal helical symmetry. Analysis of the phase differences for the primary reflections on the first layer line of transforms from 15 filaments showed an average difference of 170 degrees, close to the value of 180 degrees expected for an odd-stranded structure. Computer-filtered images of the isolated thick filaments unequivocally demonstrate a three-stranded arrangement of the crossbridges on the filaments and provide evidence that the crossbridge arrangement is axially perturbed from ideal helical symmetry.     

The structure of isolated cardiac Myosin thick filaments from cardiac Myosin binding protein-C knockout mice

Mutations in the thick filament associated protein cardiac myosin binding protein-C (cMyBP-C) are a major cause of familial hypertrophic cardiomyopathy. Although cMyBP-C is thought to play both a structural and a regulatory role in the contraction of cardiac muscle, detailed information about the role of this protein in stability of the thick filament and maintenance of the ordered helical arrangement of the myosin cross-bridges is limited. To address these questions, the structure of myosin thick filaments isolated from the hearts of wild-type mice containing cMyBP-C (cMyBP-C^+/+) were compared to those of cMyBP-C knockout mice lacking this protein (cMyBp-C^−/−). The filaments from the knockout mice hearts lacking cMyBP-C are stable and similar in length and appearance to filaments from the wild-type mice hearts containing cMyBP-C. Both wild-type and many of the cMyBP-C^−/− filaments display a distinct 43 nm periodicity. Fourier transforms of electron microscope images typically show helical layer lines to the sixth layer line, confirming the well-ordered arrangement of the cross-bridges in both sets of filaments. However, the “forbidden” meridional reflections, thought to derive from a perturbation from helical symmetry in the wild-type filament, are weaker or absent in the transforms of the cMyBP-C^−/− myocardial thick filaments. In addition, the cross-bridge array in the absence of cMyBP-C appears more easily disordered.     

Flash and smash: rapid freezing of muscle fibers activated by photolysis of caged ATP.

A new approach was used to study transient structural states of cross-bridges during activation of muscle fibers. Rabbit skinned muscle fibers were rapidly and synchronously activated from the rigor state by photolysis of caged ATP in the presence of Ca2+. At several different times during the switch from rigor to fully active tension development, the fibers were rapidly frozen on a liquid helium-cooled metal block, freeze-substituted, and examined in an electron microscope. The limits of structural preservation and resolution with this technique were analyzed. We demonstrate that the resolution of our images is sufficient to draw the following conclusions about cross-bridge structure. Rigor cross-bridges point away from the Z-line and most of them are wider near the thin filaments than near the backbone of the thick filaments. In contrast, cross-bridges in actively contracting fibers stretch between the thick and thin filaments at a variable angle, and are uniformly thin. Diffraction patterns computed from contracting muscle show layer lines both at 38 and 43 nm indicating that active cross-bridges contribute mass to both the actin- and myosin-based helical periodicities. The images obtained from fibers frozen 20 ms after release of ATP show a mixture of rigor and active type cross-bridge configurations. There is little evidence of cross-bridges with the rigor shape by 50 ms, and the difference in configurations between 50 and 300 ms after photolysis is surprisingly subtle.     

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.     

Unfixed cryosections of striated muscle to study dynamic molecular events.

The structures of the actin and myosin filaments of striated muscle have been studied extensively in the past by sectioning of fixed specimens. However, chemical fixation alters molecular details and prevents biochemically induced structural changes. To overcome these problems, we investigate here the potential of cryosectioning unfixed muscle. In cryosections of relaxed, unfixed specimens, individual myosin filaments displayed the characteristic helical organization of detached cross-bridges, but the filament lattice had disintegrated. To preserve both the filament lattice and the molecular structure of the filaments, we decided to section unfixed rigor muscle, stabilized by actomyosin cross-bridges. The best sections showed periodic, angled cross-bridges attached to actin and their Fourier transforms displayed layer lines similar to those in x-ray diffraction patterns of rigor muscle. To preserve relaxed filaments in their original lattice, unfixed sections of rigor muscle were picked up on a grid and relaxed before negative staining. The myosin and actin filaments showed the characteristic helical arrangements of detached cross-bridges and actin subunits, and Fourier transforms were similar to x-ray patterns of relaxed muscle. We conclude that the rigor structure of muscle and the ability of the filament lattice to undergo the rigor-relaxed transformation can be preserved in unfixed cryosections. In the future, it should be possible to carry out dynamic studies of active sacromeres by cryo-electron microscopy.     

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 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.     

Three-dimensional image reconstruction of insect flight muscle. I. The rigor myac layer

We have obtained detailed three-dimensional images of in situ cross-bridge structure in insect flight muscle by electron microscopy of multiple tilt views of single filament layers in ultrathin sections, supplemented with data from thick sections. In this report, we describe the images obtained of the myac layer, a 25-nm longitudinal section containing a single layer of alternating myosin and actin filaments. The reconstruction reveals averaged rigor cross-bridges that clearly separate into two classes constituting lead and rear chevrons within each 38.7-nm axial repeat. These two classes differ in tilt angle, size and shape, density, and slew. This new reconstruction confirms our earlier interpretation of the lead bridge as a two-headed cross-bridge and the rear bridge as a single-headed cross-bridge. The importance of complementing tilt series with additional projections outside the goniometer tilt range is demonstrated by comparison with our earlier myac layer reconstruction. Incorporation of this additional data reveals new details of rigor cross-bridge structure in situ which include clear delineation of (a) a triangular shape for the lead bridge, (b) a smaller size for the rear bridge, and (c) density continuity across the thin filament in the lead bridge. Within actin's regular 38.7-nm helical repeat, local twist variations in the thin filament that correlate with the two cross-bridge classes persist in this new reconstruction. These observations show that in situ rigor cross-bridges are not uniform, and suggest three different myosin head conformations in rigor.     

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.     

Changes of thick filament structure during contraction of frog striated muscle.

The strongest myosin-related features in the low-angle axial x-ray diffraction pattern of resting frog sartorius muscle are the meridional reflections corresponding to axial spacings of 21.4 and 14.3 nm, and the first layer line, at a spacing 42.9 nm. During tetanus the intensities of the first layer line and the 21.4-nm meridional decrease by 62 and 80% respectively, but, when the muscle is fresh, the 14.3-nm meridional intensity rises by 13%, although it shows a decrease when the muscle is fatigued. The large change in the intensity of the 21.4-nm meridional reflection suggests that the projected myosin cross-bridge density onto the thick filament axis changes during contraction. The model proposed by Bennett (Ph.D. Thesis, University of London, 1977) in which successive cross-bridge levels are at 0,3/8, and 5/8 of the 42.9-nm axial repeat in the resting muscle, passing to 0, 1/3, and 2/3 in the contracting state, can explain why the 21.4-nm reflection decreases in intensity while the 14.3-nm increases when the muscle is activated. The model predicts a rather larger increase of the 14.3-nm reflection intensity during contraction than that observed, but the discrepancy may be removed if a small change of shape or tilt of the cross-bridges relative to the thick filament axis is introduced. The decrease of the intensity of the first layer line indicates that the cross-bridges become disordered in the plane perpendicular to the filament axis.     

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.     

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.     

The mammalian cardiac muscle thick filament: backbone contributions to meridional reflections

Information about the structure of the vertebrate striated muscle thick filament backbone is important for understanding the arrangement of both the rod portion of the myosin molecule and the accessory proteins associated with the backbone region of the filament. Although models of the backbone have been proposed, direct data on the structure of the backbone is limited. In this study, we provide evidence that electron micrographs of isolated negatively stained cardiac thick filaments contain significant information about the filament backbone. Computed Fourier transforms from isolated cardiac thick filaments show meridional (or near meridional) reflections on the 10th and 11th layer lines that are particularly strong. Comparison of Fourier filtrations of the filaments that exclude, or include, these reflections, provide evidence that these reflections originate at least in part from a series of striations on the backbone at a approximately 4 nm spacing. The striations are likely to result either from the packing of the myosin rods, or from proteins such as titin associated with the filament backbone.     

Dynamic analysis of the structure and function of sarcomeres

We attempted to analyze the relationships between the steric structure of the sarcomere and its physiological functions by the use of a sarcomere model of muscle contraction, which includes the geometric arrangement of the thick and thin filaments of the sarcomere, as well as of the cross-bridges and actin sites. Motions of both cross-bridges and myofilaments were considered in terms of our three-state model of the elementary cycle under constraints caused by the steric structure of the sarcomere proposed by Huxley and Brown. Each cross-bridge moves in a molecular potential of our three-state model under the influence of the sliding motions of myofilaments. The sarcomere model described well the tension-velocity relation and isotonic transient processes quantitatively and consistently. In addition, it allowed independence of the no-load shortening velocity upon the overlap of the thick and thin filaments, although the motions of cross-bridges were not independent. Effects of the helical periodicities of the thick and thin filaments and of the number of cross-bridges upon muscle contraction were studied, and the conditions for smooth and efficient contraction of muscle were obtained.     

The effects of pH on force and stiffness development in mouse muscles

Changes in force and stiffness during contractions of mouse extensor digitorum longus and soleus muscles were measured over a range of extracellular pH from 6.4 to 7.4. Muscle stiffness was measured using small amplitude (less than 0.1% of muscle length), high frequency (1.5 kHz) oscillations in length. Twitch force was not significantly affected by changes in pH, but the peak force during repetitive stimulation (2, 3, and 20 pulses) was decreased significantly as the pH was reduced. Changes in muscle stiffness with pH were in the same direction, but smaller in extent. If the number of attached cross-bridges in the muscle can be determined from the measurement of small amplitude, high frequency muscle stiffness, then these findings suggest that (a) the number of cross-bridges between thick and thin filaments declines in low pH and (b) the average force per cross-bridge also declines in low pH. The decline in force per cross-bridge could arise from a reduction in the ability of cross-bridges to generate force during their state of active force production and (or) in an increased percentage of bonds in a low force, "rigor" state.     

X-ray diffraction evidence for cross-bridge formation in relaxed muscle fibers at various ionic strengths

Equatorial x-ray diffraction patterns from single skinned rabbit psoas fibers were studied at various ionic strengths to obtain structural information regarding cross-bridge formation in relaxed muscle fibers. At ionic strengths between 20 and 50 mM, the intensity of the 11 reflection, I11, of the relaxed state was close to that of the rigor state, whereas the intensity of the 10 reflection, I10, was approximately twice that of rigor reflection. Calculations by two-dimensional Fourier synthesis indicated that substantial extra mass was associated with the thin filaments under these conditions. With increasing ionic strength between 20 and 100 mM, I10 increased and I11 decreased in an approximately linear way, indicating net transfer of mass away from the thin filaments towards the thick filaments. These results provided evidence that cross-bridges were formed in a relaxed fiber at low ionic strengths, and that the number of cross-bridges decreased as ionic strength was raised. Above mu = 100 mM, I10 and I11 both decreased, indicating the onset of increasing disorder within the filament lattice.     

Analysis of equatorial x-ray diffraction patterns from muscle fibers: factors that affect the intensities.

Previously we have shown that cross-bridge attachment to actin and the radial position of the myosin heads surrounding the thick filament backbone affect the equatorial x-ray diffraction intensities in different ways (Yu, 1989). In the present study, other factors frequently encountered experimentally are analyzed by a simple model of the filament lattice. It is shown that the ordering/disordering of filaments, lattice spacing changes, the azimuthal redistributions of cross-bridges, and variations in the ordered/disordered population of cross-bridges surrounding the thick filaments can distinctly affect the equatorial intensities. Consideration of Fourier transforms of individual components of the unit cell can provide qualitative explanations for the equatorial intensity changes. Criteria are suggested that can be used to distinguish the influence of some factors from others.