John M. Squire

Structural role of tropomyosin in muscle regulation: Analysis of the X-ray diffraction patterns from relaxed and contracting muscles

Abstract Previous work has shown that there are significant differences in the X-ray diffraction patterns obtained from relaxed and contracting muscles. We show that some of these changes can be explained in terms of a small movement (~ 5 to 15 A) of the tropomyosin molecules in the groove of the actin helix. The position of the tropomyosin in relaxed skeletal muscle is such that it might physically block or at least structurally alter the cross-bridge attachment site on actin, whereas in contracting skeletal muscle the tropomyosin moves to a position well clear of the attachment site. The movement of the tropomyosin molecules is apparently smaller in molluscan muscles during tonic contraction than in vertebrate skeletal muscle. We suggest a possible relationship between the smaller movement of the tropomyosin and the “catch” response of molluscan muscles. We also show that any increase of intensity on the 59 A and 51 A layer-lines is most likely to be associated with some extra mass (HMM S-1) attaching to the actin molecules. Such a change cannot be explained in terms of a change in tropomyosin structure or in the order within the thin filaments. Since changes on these two layer-lines have been observed during contraction, this provides good evidence for cross-bridge attachment to actin in contracting muscles.

Fifty years of coiled-coils and α-helical bundles : A close relationship between sequence and structure

alpha-Helical coiled coils are remarkable for the diversity of related conformations that they adopt in both fibrous and globular proteins, and for the range of functions that they exhibit. The coiled coils are based on a heptad (7-residue), hendecad (11-residue) or a related quasi-repeat of apolar residues in the sequences of the alpha-helical regions involved. Most of these, however, display one or more sequence discontinuities known as stutters or stammers. The resulting coiled coils vary in length, in the number of chains participating, in the relative polarity of the contributing alpha-helical regions (parallel or antiparallel), and in the pitch length and handedness of the supercoil (left- or right-handed). Functionally, the concept that a coiled coil can act only as a static rod is no longer valid, and the range of roles that these structures have now been shown to exhibit has expanded rapidly in recent years. An important development has been the recognition that the delightful simplicity that exists between sequence and structure, and between structure and function, allows coiled coils with specialized features to be designed de novo.

A new look at thin filament regulation in vertebrate skeletal muscle

It is 30 years since Ebashi and colleagues showed that Ca2+ ions directly affect regulation of the myosin‐actin interaction in muscle through the action of tropomyosin and troponin on muscle thin filaments. It is more than 20 years since the idea was put forward that tropomyosin might act, at least in part, by changing its position on actin, thus uncovering or modifying the myosin binding site on actin when troponin molecules take up Ca2+. Since that time, a great deal of evidence for and against this steric blocking mechanism has been published: a structure for actin filaments at close to atomic resolution has been proposed, and the whole regulation story has become both more complicated and more subtle. Here we review structural and biochemical aspects of regulation in vertebrate skeletal muscle. We show that some basic ideas of the steric blocking mechanism remain valid. We also show that additional factors, such as troponin movements and structural changes within the actin monomers themselves, may be crucial. A number of the resulting regulation scenarios need to be distinguished.—Squire, J. M., Morris, E. P. A new look at thin filament regulation in vertebrate skeletal muscle. FASEB J. 12, 761–771 (1998)

Architecture and function in the muscle sarcomere

Striated muscle sarcomeres in vertebrates comprise ordered arrays of actin and myosin filaments, organized by an elaborate protein scaffold. Recent innovative work in a number of laboratories has greatly improved our knowledge of these structures, their organization and their interactions. Structural details have been reported on myosin filaments, actin filaments, Z-bands, M-bands, titin, and nebulin. Time-resolved X-ray diffraction and electron microscopy are revealing the molecular movements involved in force production and regulation.

Fine structure of the A-band in cryo-sections: The structure of the A-band of human skeletal muscle fibres from ultra-thin cryo-sections negatively stained

The technique of cryo-ultramicrotomy has been applied to the study of the ultrastructure of the A-band in vertebrate skeletal muscle. The tissue (open biopsies from human musculus tibialis anterior ) was fixed briefly in glutaraldehyde, then treated with glycerol and rapidly frozen. Thin sections of frozen tissue were then negatively stained and viewed in the electron microscope. Preparations obtained in this way showed a marked improvement in preservation of certain types of order in the A-band. Most A-bands showed the same characteristic, but complex, pattern of transverse striations from the M-band to the outer edges of the A-band. Our purpose here is to give a general description of the cryo-sectioned A-band in terms of the relative spacings and densities of the various striations. A new nomenclature for different parts of the A-band is also suggested to facilitate future reference to the striations. The outstanding features of the striation pattern were: (1) A pattern of three or five strong lines (M-bridges) in the M-band depending on fibre type. (2) A pseudo-H-zone (here called the M-region) with a measured length of 1620 A (±36 A) and containing many closely spaced weak lines in addition to the M-bridge lines. Most of these were found to lie on one of two over-lapping periodicities of about 145 A. (3) Striations outside the M-region spaced about 140 A to 150 A and showing up clearly the myosin crossbridge repeat already known to be 143 A. Different striations had different characteristic densities and their positions often appeared to be perturbed in a characteristic way from positions on a regular 143 A repeat. The overall pattern therefore showed considerable complexity. (4) A rapidly chaing pattern of striations everywhere except in the region of the A-band (here called the C-zone), where in other muscles C-protein is known to be located. But even in this region subtle changes in the pattern consistently appeared. Since the myosin repeat and the C-protein repeat have previously been reported to be identical (430 A), no changes in pattern every 430 A would be expected to occur. That changes do occur in cryo-sections must mean one of three things. Either the underlying packing arrangement of the myosin molecules must change throughout the C-zone, or actin filaments in the muscle must in some way cause the perturbation of the regular crossbridge arrangement, or the myosin packing is constant but the periodicities of myosin and C-protein are different. Because of the importance of the latter in the context of a possible length-determining mechanism in myosin filaments, we confine ourselves here to a preliminary description of the C-zone. An account of a continuing detailed study and analysis of the C-zone will be given elsewhere. That the complex pattern of transverse striations is representative of the axial structure in living muscle is indicated by the fact that optical diffraction patterns from whole cryo-sectioned A-bands are comparable to the meridional X-ray diffraction patterns from living vertebrate skeletal muscle ( Huxley & Brown, 1967 ). This suggests that the cryo-sectioning technique has preserved for the first time the axial periodicities in the A-band and that this technique may help to bridge the gap between electron microscopy and X-ray diffraction.0115

Structural Evidence for the Interaction of C-protein (MyBP-C) with Actin and Sequence Identification of a Possible Actin-binding Domain

C-protein (MyBP-C) is a myosin-binding protein that is usually seen in two sets of seven to nine positions in the C-zones in each half of the vertebrate striated muscle A-band. Skeletal muscle C-protein is a modular structure containing ten sub-domains (C1 to C10) of which seven are immunoglobulin-type domains and three (C6, C7 and C9) are fibronectin-like domains. Cardiac muscle C-protein has an extra N-terminal domain (C0) and also some sequence insertions, one of which provides phosphorylation sites. It is conceivable that C-protein has both a structural and regulatory role within the sarcomere. The precise mode of binding of C-protein to the myosin filament has not been determined. However, detailed ultrastructural studies have suggested that C-protein, which binds to myosin, can give rise to a longer periodicity (about 435A) than the intrinsic myosin filament repeat of 429A. The reason for this has remained a puzzle for over 25 years. Here we show by modelling and computation that the presence of this longer periodicity could be explained if the myosin-binding part of C-protein binds to myosin with the expected 429A repeat, but if there are systematic interactions of the N-terminal end of C-protein with the neighbouring actin filaments in the hexagonal lattice of filaments in the A-band. We also show that if they occur these interactions would probably only arise in defined muscle states. Further analysis of the MyBP-C sequence identifies a possible actin-binding domain in the Pro-Ala-rich sequence found at the N terminus of skeletal MyBP-C and between domains C0 and C1 in the cardiac sequence.

General model of myosin filament structure: III. Molecular packing arrangements in myosin filaments

Abstract Following the original proposals about myosin filament structure put forward as part of a general myosin filament model (Squire, 1971, 1972) it is here shown what the most likely molecular packing arrangements within the backbones of certain myosin filaments would be assuming that the model is correct. That this is so is already indicated by recently published experimental results which have confirmed several predictions of the model (Bullard and Reedy, 1972; Reedy et al. , 1972; Tregear and Squire, 1973). The starting point in the analysis of the myosin packing arrangements is the model for the myosin ribbons in vertebrate smooth muscle proposed by Small & Squire (1972). It is shown that there is only one reasonable type of packing arrangement for the rod portions of the myosin molecules which will account for the known structure of the ribbons and which is consistent with the known properties of myosin molecules. The dominant interactions in this packing scheme are between parallel myosin molecules which are related by axial shifts of 430 A and 720 A. In this analysis the myosin rods are treated as uniform rods of electron density and only the general features of two-strand coiled-coil molecules are considered. Since the general myosin filament model is based on the assumption that the structures of different types of myosin filament must be closely related, the packing scheme derived for the myosin ribbons is used to deduce the structures of the main parts (excluding the bare zones) of the myosin filaments in a variety of muscles. It is shown in each case that there is only one packing scheme consistent with all the available data on these filaments and that in each filament type exactly the same interactions between myosin rods are involved. In other words the myosin-myosin interactions involved in filament formation are specific, they involve molecular shifts of either 430 A or 720 A, and are virtually identical in all the different myosin filaments which have been considered. Apart from the myosin ribbons, these are the filaments in vertebrate skeletal muscle, insect flight muscle and certain molluscan muscles. In the case of the thick filaments in vertebrate skeletal muscle the form of the myosin packing arrangement in the bare zone is considered and a packing scheme proposed which involves antiparallel overlaps between myosin rods of 1300 A and 430 A. It is shown that this scheme readily explains the triangular profiles of the myosin filaments in the bare zone (Pepe, 1967, 1971) and many other observations on the form of these myosin filaments. Finally it is shown that the cores of several different myosin filaments, assuming they contain protein, may consist of different arrangements of one or other of two types of core subfilament.

Myosin content and filament structure in smooth and striated muscle

Abstract Fibres from four different muscles (rabbit psoas, guinea pig taenia coli, Lethocerus flight and leg) were glycerol-extracted, homogenized and dissolved in a sodium dodecyl sulphate solution. The relative mass of the myosin heavy chain and actin polypeptides present in these extracts was measured by polyacrylamide gel electrophoresis. The ratio was found to be consistent for each muscle and to differ widely between muscles. The results were used to calculate the number of myosin molecules per subunit repeat along the thick filaments of the striated muscles and ribbon-like filaments, and so to test a theory of filament structure.

Three-dimensional structure of the vertebrate muscle A-band: III. M-region structure and myosin filament symmetry☆

Ultrathin transverse sections of the body muscle of bony fish and the sartorius muscle of frog have been analysed in detail by optical diffraction and image averaging to reveal the ultrastructures of the myosin filaments both in the M-region and in the outer ends of the filament (the tip region). The evidence is unequivocal that the myosin filaments in both muscle types have 3-fold rotational symmetry in all of the regions where symmetry can be seen. Using the nomenclature of Sjostrom & Squire (1977a), the appearances of cross-sections in fish at different axial locations are as follows. (Note that fish myosin filaments are arranged in a simple hexagonal lattice.) 1. (1)At M1 (central M-bridge line) the myosin filament profile is almost circular, but does sometimes show a three-component structure. There are six M-bridges from each filament. 2. (2) At M4 the filament backbone comprises three kidney-shaped subunits related by a triad axis, together with six M-bridges. There are two types of M-bridge interaction site. 3. (3) At M6 there are three kidney-shaped subunits related by a triad. There are no M-bridges but there does seem to be extra protein here. 4. (4) In the bare region there are three nearly circular subunits related by a triad. 5. (5) At M9 or the start of the bridge region there is a Y-shaped filament profile from which three projections radiate towards three of the six actin filament positions (trigonal points). 6. (6) There is an apparent change of hand of the filament profiles across the M-band when viewed in the same section (i.e. from M4 to M4′ or M6 to M6′). This implies that the bipolar myosin filament has the symmetry of the dihedral point group 32. 7. (7) There is a change in orientation of the 3-fold myosin filament profile across the M-band so that the bare region profiles are about 40 ° apart. Less detail has been seen in frog sartorius muscle sections because of the presence of the superlattice (see paper II in this series, Luther & Squire, 1980). However, the observations are entirely compatible with a myosin filament with the same structure as in fish but arranged in a superlattice. The superlattice is apparent not only in the bare region in frog, but also at the M-bridge level M4. There are two classes of M-bridge interaction at M4 just as in fish. At the A-band edges of both muscles (outer D-zone) the filament profiles appear either triangular, Y-shaped or composed of three subunits. The lattices are insufficiently ordered for satisfactory image averaging to be carried out. As yet the main part of the bridge region shows little ultrastructural detail. However, the present results show clearly that the vertebrate myosin filament has 3-fold rotational symmetry; there can be little doubt that the symmetry of the crossbridge array approximates to that of a three-stranded helix (Squire, 1972). A model is proposed for the structure of the myosin filament in the vertebrate M-region. It explains the observed appearances without going into molecular detail. It is also suggested that it is the M-bridges at M4 that are primarily responsible for defining the two types of A-band structure (simple lattice and superlattice) and that the M1 bridges may have a secondary role.

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 101 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.