Peter Vibert

Electron microscopy of thin filaments decorated with a Ca2+-regulated myosin

Abstract Scallop thin filaments decorated with proteolytic fragments of scallop myosin display two forms of “arrowhead” complex by electron microscopy, depending on the presence or absence of the regulatory light chain. The arrowhead pattern obtained with heavy meromyosin or myosin subfragment-1 from which this light chain has been removed resembles that previously obtained by Moore et al. (1970) with rabbit myosin subfragment-1. We term this form “blunted”. When the regulatory light chain is present, the arrowhead profiles look distinctly different and appear more “barbed”. The attachment of individual heads or pairs of heads can also be observed when lower concentrations of myosin fragments are used. The angles of attachment and the shape of the heads can be seen in this case without the superposition that occurs in fully decorated filaments. Both heads of heavy mero-myosin appear to attach to the same actin strand of a single thin filament, usually to adjacent actin monomers. The heads are long (about 20 nm), narrow (about 4 nm wide), and distinctly curved. Both attach at approximately the same angle to actin and join to the rod by apparent elongation and bending of the leading head of the pair. There is some evidence that the binding of myosin heads to thin filaments may be co-operative. In preliminary experiments, we have duplicated most of the above observations using rabbit skeletal muscle proteins.

Three-dimensional reconstruction of thin filaments decorated with a Ca2+-regulated myosin☆

Abstract Three-dimensional reconstructions of “barbed” and “blunted” arrowheads (Craig et al. , 1980) show that these two forms arise from arrangement of scallop myosin subfragments (S1) that appear about 40 A longer in the presence of the regulatory light chain than in its absence. A similar difference in apparent length is indicated by images of single myosin subfragments in partially decorated filaments. The extra mass is located at the end of the subfragment furthest from actin, and probably comprises part of the regulatory light chain as well as a segment of the myosin heavy chain. The fact that barbed arrowheads are also formed by myosin subfragments from vertebrate striated and smooth muscles implies that the homologous light chains in these myosins have locations similar to that of the scallop light chain. The scallop light chain probably does not extend into the actin-binding site on the myosin head, and is therefore unlikely to interfere physically with binding. Rather, regulation of actin-myosin interaction by light chains may involve Ca 2+ -dependent changes in the structure of a region near the head-tail junction of myosin. The reconstructions suggest locations for actin and tropomyosin relative to myosin that are similar to those proposed by Taylor & Amos (1981) and are consistent with a revised steric blocking model for regulation by tropomyosin. The identification of actin from these reconstructions is supported by images of partially decorated filaments that display the polarity of the actin helix relative to that of bound myosin subfragments.

Mini-titins in striated and smooth molluscan muscles: structure, location and immunological crossreactivity

SummaryInvertebrate mini-titins are members of a class of myosin-binding proteins belonging to the immunoglobulin superfamily that may have structural and/or regulatory properties. We have isolated mini-titins from three molluscan sources: the striated and smooth adductor muscles of the scallop, and the smooth catch muscles of the mussel. Electron microscopy reveals flexible rod-like molecules about 0.2 μm long and 30 Å wide with a distinctive polarity. Antibodies to scallop mini-titin label the A-band and especially the A/I junction of scallop striated muscle myofibrils by indirect immunofluorescence and immuno-electron microscopy. This antibody crossreacts with mini-titins in scallop smooth and Mytilus catch muscles, as well as with proteins in striated muscles from Limulus, Lethocerus (asynchronous flight muscle), and crayfish. It labels the A/I junction (I-region in Lethocerus) in these striated muscles as well as in chicken skeletal muscle. Antibodies to the repetitive immunoglobulin-like regions and also to the kinase domain of nematode twitchin crossreact with scallop mini-titin and label the A-band of scallop myofibrils. Electron microscopy of single molecules shows that antibodies to twitchin kinase bind to scallop mini-titin near one end of the molecule, suggesting how the scallop structure might be aligned with the sequence of nematode twitchin.

Dimer ribbons in the three-dimensional structure of sarcoplasmic reticulum☆

The three-dimensional structure of scallop sarcoplasmic reticulum membranes has been determined from electron micrographs of two classes of stain-filled tubules by helical reconstruction methods. These structures are characterized by dimer ribbons of Ca2+-ATPase molecules running diagonally around the tube wall. Deep right-handed grooves separate the ribbons. The elongated, curved units of the dimer (approximately 95 A long in the radial direction; 60 to 70 A axially, and about 30 A wide) are displaced axially by approximately 34 A and are connected at their outer ends by a bridge running nearly parallel to the tube axis. The monomers make a second contact at their inner ends. Adjacent units with the same orientation form a strong contact that is responsible for the ribbon appearance. Comparison of tubules of different diameter shows that one set of connections between the dimer ribbons is conserved: the inner ends of axially displaced dimers appear to make contact along a left-handed path almost perpendicular to the major grooves. The lipid bilayer cannot be clearly identified. The two-dimensional map obtained from flattened tubules is consistent with the three-dimensional reconstruction in showing dimer ribbons connected by a weak contact across the grooves, strongly resembling the inter-dimer bond observed in three dimensions. The two-dimensional map shows a 2-fold axis relating units of the dimer, but the three-dimensional tubes show a slight axial polarity that may arise from the presence of proteins other than the Ca2+-ATPase.

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

Abstract 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 Reedys (1968) concept that cross-bridges in rigor attach only to certain azimuths on the actin filaments (“target areas”); the 145 A 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-bridge arrangements in Limulus muscle☆

Abstract X-ray diffraction patterns show Limulus muscle to have a structure in rigor similar to that of insect flight muscle, except that the thick filaments are staggered. Myosin filaments in relaxed muscle bear a highly ordered helical array of cross-bridges which, however, is very labile. The array undergoes a reversible transition between order and disorder in response to changes in ionic strength.

Structure of myosin/paramyosin filaments from a molluscan smooth muscle

Small-angle X-ray diffraction patterns of chemically skinned pedal retractor muscles from Mytilus (PRM) in the relaxed state show a set of diffuse off-meridional reflections that arise from a helical array of myosin crossbridges with 8/3 screw symmetry. Experiments involving extraction of myosin as well as analysis of the rigor pattern have been used to confirm the origin of these reflections. The relatively high myosin/paramyosin molar ratio (1.3 to 1.6) in PRM compared to other molluscan smooth muscles may account for the observation of the relatively stronger diffraction from the myosin array. Thick filaments isolated from PRM and contrasted by negative staining for electron microscopy appear to be very long (up to 17 micron), and to have a rather small diameter (about 40 nm at the center); they taper gradually toward the ends. These filaments show a clear transverse band pattern repeating at 14.4 nm and elongated projections (crossbridges) at the surface except in the central bare zone. Optical diffraction patterns show reflections from crossbridges consistent with the X-ray patterns of the relaxed whole muscle. Filaments unidirectionally shadowed with platinum show diagonal striations running at an angle of about 17 degrees to the filament axis, revealing that the crossbridges are arrayed in a right-handed helix. The paramyosin core is clearly seen upon extraction of myosin. Observations on both negatively stained and sectioned material are consistent with the results of Elliott (1979) and Bennett & Elliott (1981) suggesting a layered structure of the core. Cores stripped of myosin, however, appear to undergo some distortion indicating that the three-dimensional structure is not yet completely solved. The assembly of these thick filaments presents intriguing structural problems since the myosin surface lattice does not appear to have the same symmetry as the underlying paramyosin core.

Helical reconstruction of frozen-hydrated scallop myosin filaments

Native myosin filaments from scallop striated muscle that have been rapidly frozen in relaxing solutions appear to be well preserved in vitreous ice. Electron micrographs of samples at -177 degrees C were recorded with an electron dose of 10 e/A2 at 1.5 microns defocus. After filament images were straightened by spline-fitting, several transforms showed well-defined layer-lines arising from the helical structure of the filament. A set of 17 near-meridional layer-lines has been collected and corrected for background and for phase and amplitude contrast functions. Preliminary helical reconstructions from this still incomplete data set reveal aspects of structure that were not apparent from earlier analysis of negatively stained filaments from scallop muscle. Individual pear-shaped myosin heads now appear to be well resolved from each other and from the filament backbone. The two heads of each myosin molecule appear to be splayed apart axially. The reconstructions also reveal that the filament backbone has a polygonal shape in cross-section, and that it appears to contain seven peripherally located subfilaments.

Electron microscopy of cross-linked scallop myosin☆

The N-terminal regions of the regulatory light chains on the two heads of scallop myosin can be cross-linked to one another. Electron microscopy of cross-linked myosin molecules, and of dimers of myosin subfragment-1 produced by digesting them with papain, shows that the site of cross-linking is very close to the head-rod junction.

Location of paramyosin in relation to the subfilaments within the thick filaments of scallop striated muscle

SummaryMyosin co-assembles with paramyosin in the thick filaments of invertebrate muscles. The molar ratio of the two proteins varies greatly but where sufficient paramyosin is present it forms the filament core with myosin arranged on its surface. In the fastest acting striated muscles, paramyosin is present in small amounts, and neither its location nor the nature of its interactions with myosin has previously been established. Antibodies to paramyosin have now been used in an attempt to locate the protein in thick filaments that have been isolated from the striated adductor muscle of the scallop and then frayed apart into their constituent subfilaments. Using a gold-conjugated secondary antibody, the location of paramyosin in relation to the subfilaments has been determined by electron microscopy of negatively stained samples. The labelling indicates that paramyosin extends throughout the length of the scallop filaments and appears to be associated with each subfilament, raising the possibility that in these filaments paramyosin may not be confined to a central core domain.