Invertebrate myosin filament: subfilament arrangement in the wall of tubular filaments of insect flight muscles

Transverse sections (100 to 140 nm thick) of the flight muscles of the fleshfly Phormia terrae-novae and the housefly Musca domestica were studied. The images of 56 tubular myosin filaments of the fleshfly and 62 filaments of the housefly were digitized and computer processed by rotational averaging. The rotational power spectra of more than 80% of the filaments showed peaks for 6-fold rotational frequency. The average of these images for each species showed a characteristic pattern consisting of 12 subunits arranged in six pairs around the wall of the filament. This pattern was enhanced by rotationally filtering the average images using the 6-fold components of the rotational power spectrum. On tilting individual images, the subunits behaved like rods perpendicular to the plane of the transverse section and they were therefore considered to be subfilaments essentially parallel to the long axis of the filament. The center-to-center spacing between the subfilaments of a pair is 2.8 nm, and the center-to-center spacing between the adjacent subfilaments of neighboring pairs is 4.0 nm. The observation of 12 subfilaments is consistent with a four-stranded helical arrangement of myosin cross-bridges on the surface of the filaments.     

The myosin filament. X. Observation of nine subfilaments in transverse sections

The molecular packing of the subfilaments in muscle thick filaments has been investigated by electron microscopy. Thin (80-100 nm) transverse sections of vertebrate skeletal muscle were cut, and 129 electron microscope images of thick filaments from 15 different areas including seven to ten images in each area were analyzed by computer image processing. The transverse sections were limited to the portion of the filaments between the bare zone and the C-protein bearing region. Of the 129 images, six were discarded because they were structurally disrupted, 17 did not show evidence for the presence of subfilaments from the autocorrelation function, and four did not show evidence for three-fold rotational symmetry from the power spectrum. The remaining 102 filaments all showed evidence for three-fold rotational symmetry, consistent with other available evidence (Pepe, 1982). From the analysis of these images by rotational filtering, we have found that the vertebrate skeletal myosin filament is made up of nine subfilaments and that the image appears to have trigonal symmetry. Of these subfilaments, six are arranged with a center-to-center spacing of about 4 nm and the other three on the surface of the filament are distorted from this arrangement. Three additional densities, which together with the other nine, correspond to the pattern of 12 densities previously observed in more highly selected images (Stewart et al., 1981; Pepe and Drucker, 1972) were observed in 5% of the images. Another pattern of nine subfilaments peripherally arranged around the circumference of the filament was observed occasionally. This latter image may represent the organization of the subfilaments in the bare zone region of the filament, resulting from sampling of individual filaments displaced longitudinally relative to the other filaments in the A-band.     

The myosin filament XIV backbone structure.

The substructure of the thick filaments of chemically skinned chicken pectoralis muscle was investigated by electron microscopy. Images of transverse sections of the myosin filaments were determined to have threefold symmetry by cross-correlation analysis, which gives an unbiased determination of the rotational symmetry of the images. Resolution, using the phase residual test (Frank et al. 1981. Science [Wash. DC]. 214:1353-1355), was found to be between 3.2 and 3.6 nm. Three arrangements of nine subfilaments in the backbone were found in all regions of the filament at ionic strengths of 20 and 200 mM. In the average images of two of these, there were three dense central subfilaments and three pairs of subfilaments on the surface of the thick filament. In the average image of the third arrangement, all of the protein mass of the nine subfilaments was on the surface of the filament with three of them showing less variation in position than the others. A fourth arrangement appearing to be transitional between two of these was seen often at 200 mM ionic strength and only rarely at 20 mM. On average, the myosin subfilaments were parallel to the long axis of the filament. The different arrangements of subfilaments appear to be randomly distributed among the filaments in a transverse section of the A-band. Relative rotational orientations with respect to the hexagonal filament lattice, using the three densest subfilaments as reference showed a major clustering (32%) of filaments within one 10 degrees spread, a lesser clustering (15%) at 90 degrees to the first, and the remainder scattered thinly over the rest of the 120 degrees range. There was no obvious pattern of distribution of the two predominant orientations that could define a superlattice in the filament lattice.     

Substructures in the core of thick filaments: arrangement and number in relation to the paramyosin content of insect flight muscles

Transverse sections (100-140 nm thick) of solid myosin filaments of the flight muscles of the honeybee, Apis mellifica, the fleshfly, Phormia terrae-novae and the waterbug, Lethocerus uhleri, were photographed in a JEM-200 electron microscope at 200 kV. The images were digitized and computer processed by rotational filtering. The power spectra of the images of each of these filaments showed six-fold symmetry for the outer wall region and three-fold symmetry for the inner wall region. Images of the honeybee additionally showed three-fold symmetry for the center of the filament. Considering both paramyosin content of the myosin filaments and the results of the rotational filtering, we suggest the existence of 3 paramyosin strands in the myosin filaments of the fleshfly, 6 paramyosin strands in the honeybee filaments and 5 strands in the myosin filaments of the waterbug. In the case of the honeybee, the 3 paramyosin strands of the inner wall are positioned directly opposite the myosin subfilaments, while the 3 strands of the center seem to be arranged opposite the gaps between the myosin subfilaments. The paramyosin filaments of the fleshfly wobble between 2 myosin subfilaments, without loosing their three-fold symmetry arrangement in the inner wall. The 3 paramyosin strands in the inner wall of the waterbug myosin filaments are either arranged opposite the myosin subfilaments or opposite the gaps between the subfilaments. Finally, we were able to generate a 3-dimensional reconstruction of the myosin filament of the honeybee, showing the parallel arrangement of both, myosin subfilaments and paramyosin strands, relative to the long filament axis.     

Invertebrate myosin filament: Parallel subfilament arrangement in the wall of solid filaments from the honeybee, Apis mellifica

Transverse serial sections (100-140 nm thick) of solid myosin filaments of the honeybee, Apis mellifica, were photographed in a JEM-200 electron microscope at 200 kV. The images were digitized and computer processed by rotational filtering. 87% of the myosin filaments showed 6-fold symmetry in their power spectra, confirming the results of earlier works (Beinbrech et al., 1988, 1991). To determine if the subfilaments were arranged parallel to the filament backbone, two methods were used. First, the three images of each myosin filament in the three serial sections were superimposed. 85% of the resulting images showed a strong peak for 6-fold symmetry and the averaged images showed 6 pairs of subfilaments, which gives evidence for parallel arrangement of the subfilaments relative to the filament axis. This result was confirmed by the second method in which a 3-dimensional reconstruction was made. An average image was made from the images of the same 17 myosin filaments from each of the three sections. The data for the 3-dimensional reconstruction were collected by tracing the outlines of the structures in the three successive sections. The resulting stereo image shows a parallel arrangement of the subfilaments.     

The invertebrate myosin filament: subfilament arrangement of the solid filaments of insect flight muscles.

Transverse sections (approximately 140 nm thick) of solid myosin filaments of the flight muscles of the fleshfly, Phormia terrae-novae, the honey bee, Apis mellifica, and the waterbug, Lethocerus uhleri, were photographed in a JEM model 200A electron microscope at 200 kV. The images were digitized and computer processed by rotational filtering. In each of these filaments it was found that the symmetry of the core and the wall was not the same. The power spectra of the images showed sixfold symmetry for the wall and threefold symmetry for the core of the filaments. The images of the filaments in each muscle were superimposed according to the sixfold center of the wall. These averaged images for all three muscles showed six pairs of subunits in the wall similar to those found in the wall of tubular filaments. From serial sections of the fleshfly filaments, we conclude that the subunits in the wall of the filaments represent subfilaments essentially parallel to the long axis of the filament. In each muscle there are additional subunits in the core, closely related to the subunits in the wall. Evaluation of serial sections through fleshfly filaments suggests that the relationship of the three subunits observed in the core to those in the wall varies along the length of the filaments. In waterbug filaments there are three dense and three less dense subunits for a total of six all closely related to the wall. Bee filaments have three subunits related to the wall and three subunits located eccentrically in the core of the filaments. The presence of core subunits can be related to the paramyosin content of the filaments.     

THE MYOFILAMENT ARRANGEMENT IN THE FEMORAL MUSCLE OF THE COCKROACH, LEUCOPHAEA MADERAE FABRICIUS

The structure of the femoral muscle of the cockroach, Leucophaea maderae, was investigated by light and electron microscopy. The several hundred fibers of either the extensor or flexor muscle are 20 to 40 µ in diameter in transverse sections and are subdivided into closely packed myofibrils. In glutaraldehyde-fixed and epoxy resin-embedded material of stretched fibers, the A band is about 4.5 µ long, the thin filaments are about 2.3 µ in length, the H zone and I band vary with the amount of stretch, and the M band is absent. The transverse sections of the filaments reveal in the area of a single overlap of thick and thin filaments an array of 10 to 12 thin filaments encircling each thick filament; whereas, in the area of double overlap in which the thin filaments interdigitate from opposite ends of the A band, the thin filaments show a twofold increase in number. The thick filament is approximately 205 to 185 A in diameter along most of its length, but at about 0.2 µ from the end it tapers to a point. Furthermore, some well oriented, very thin transverse sections show these filaments to have electron-transparent cores. The diameter of the thin filament is about 70 A. Transverse sections exhibit the sarcolemma invaginating clearly at regular intervals into the lateral regions of the A band. Three distinct types of mitochondria are associated with the muscle: an oval, an elongate, and a type with three processes. It is evident, in this muscle, that the sliding filament hypothesis is valid, and that perhaps the function of the extra thin filaments is to increase the tensile strength of the fiber and to create additional reactive sites between the thick and thin filaments. These sites are probably required for the functioning of the long sarcomeres.     

The myosin filament: VII. Changes in internal structure along the length of the filament

Improved fixation procedures have enabled substructure to be observed by electron microscopy in transverse sections of vertebrate skeletal muscle thick filaments as thin as 140 nm. Optical diffraction combined with digital autocorrelation analysis, focal series and tilting experiments have confirmed the presence of a regular substructure having a repeat near 4 nm and shown that it is highly unlikely to be an artifact associated with the electron microscope imaging system. The results obtained strongly suggest that the thick filament is constructed from a bundle of rod-like subfilaments arranged parallel to the thick filament axis to within less than a degree. This cannot easily be reconciled with the general theory of thick filament structure proposed by Squire (1973), but it is consistent with the model proposed by Pepe, 1966, Pepe, 1967. Optical diffraction of 140 nm thick serial transverse sections has also suggested a structural change along the length of the filament that is manifest by a variation in the proportion of filaments showing strong diffraction maxima in one, two or three directions.     

Two kinds of thick filament in smooth muscle cells in the adductor of a clam, Chlamys nobilis

The ultrastructure of the opaque portion of the adductor muscle in the pecten Chlamys nobilis was investigated. The opaque portion was composed of smooth muscle cells that contained thin and thick filaments. The thick filaments were classified into two kinds, thinner and thicker, according to the statistical analysis of diameters. They were also classified as being shorter and longer, when isolated native filaments were examined. The thick filaments may consequently be classified into two kinds: thinner and shorter filaments, and thicker and longer ones. The thinner and shorter filaments were about 26.5 nm in diameter and 7.5 mum in length, and the thicker and longer ones were about 42.0 nm in diameter and 13.0 mum in length, respectively. A regular periodicity was apparent on the surface of the core after removal of myosin molecules from its surface. The periodicity seemed similar for the two kinds of thick filament.     

A model for length-regulation in thick filaments of vertebrate skeletal myosin.

A mechanism for length regulation in the parallel-packed section of the thick filament is proposed. It is based on experiments done on synthetic, mini- and native filaments, and its primary purpose is to explain the physical basis of the kinetic mechanism for the assembly of synthetic thick filaments from myosin alone. Kinetically, length is regulated by a dissociation rate constant that increases exponentially as the filament grows bi-directionally from its center. Growth ceases at the point of equilibrium between invariant on and length-dependent off rates. The three subfilaments structure of the parallel-packed region of the thick filament is fundamental to the proposed scheme. The intra-subfilament bonding is strong and predominantly ionic in character, whereas the inter-subfilament bonding is relatively weak. These strong and weak interactions participate directly in the strictly sequential mechanism of assembly of dimer subunit observed in the kinetics. A third domain, independent of the sequential mechanism, consists of opposing negative charges on the subfilament surface, juxtaposed at or close to the thick filament axis. The weak and repulsive domains are additively coupled to each other through the rigidity in the subfilaments. Length regulation occurs through the repulsive component rising in intensity more rapidly with length than the initially stronger positive interactions. Growth ceases at the point where the repulsive interactions weaken the attractive interactions to the extent that equilibrium is established between head-to-tail dimer subunit and its binding sites at the tips of the arms of thick filament.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Substructure and accessory proteins in scallop myosin filaments

Native myosin filaments from scallop striated muscle fray into subfilaments of approximately 100-A diameter when exposed to solutions of low ionic strength. The number of subfilaments appears to be five to seven (close to the sevenfold rotational symmetry of the native filament), and the subfilaments probably coil around one another. Synthetic filaments assembled from purified scallop myosin at roughly physiological ionic strength have diameters similar to those of native filaments, but are much longer. They too can be frayed into subfilaments at low ionic strength. Synthetic filaments share what may be an important regulatory property with native filaments: an order-disorder transition in the helical arrangement of myosin cross-bridges that is induced on activation by calcium, removal of nucleotide, or modification of a myosin head sulfhydryl. Some native filaments from scallop striated muscle carry short "end filaments" protruding from their tips, comparable to the structures associated with vertebrate striated muscle myosin filaments. Gell electrophoresis of scallop muscle homogenates reveals the presence of high molecular weight proteins that may include the invertebrate counterpart of titin, a component of the vertebrate end filament. Although the myosin molecule itself may contain much of the information required to direct its assembly, other factors acting in vivo, including interactions with accessory proteins, probably contribute to the assembly of a precisely defined thick filament during myofibrillogenesis.     

Ultrastructural study of crystalloids in Sertoli cells of the three-toed sloth (Bradypus tridactylus)

Summary Crystalloids were found in Sertoli cells of the testis of the three-toed sloth by examination at the lightand electron-microscopic levels. Needle-, or spindle-shaped crystalloids, varying in length, were located in the basal part of the Sertoli cells. They consisted of bundles of filaments each measuring ~ 11 nm in diameter. Several filaments were packed hexagonally to form a bundle. The center-to-center distance between individual filaments of a bundle was ~ 17 nm. Periodical lateral projections emanated from the filaments. Cross sections of crystalloids showed that the projections radiated from each filament in three directions, forming an equilateral triangle with a side length of ~ 15 nm. Scattered polyribosomes were found between and around the bundles.     

Three-dimensional reconstruction of a simple Z-band in fish muscle

The three-dimensional structure of the Z-band in fish white muscle has been investigated by electron microscopy. This Z-band is described as simple, since in longitudinal sections it has the appearance of a single zigzag pattern connecting the ends of actin filaments of opposite polarity from adjacent sarcomeres. The reconstruction shows two pairs of links, the Z-links, between one actin filament and the facing four actin filaments in the adjacent sarcomere. The members of each pair have nearly diametrically opposed origins. In relation to one actin filament, one pair of links appears to bind along the final 10 nm of the actin filament (proximal site) and the other pair binds along a region extending from 5 to 20 nm from the filament end (distal site). Between one pair and the other, there is a rotation of approximately 80 degrees round the filament axis. A Z-link with a proximal site at the end of one actin filament attaches at a distal site on the oppositely oriented actin filaments of the facing sarcomere and vice versa. The length of each Z-link is consistent with the length of an alpha-actinin molecule. An additional set of links located 10-15 nm from the center of the Z-band occurs between actin filaments of the same polarity. These polar links connect the actin filaments along the same direction on each side of the Z-band. The three-dimensional structure appears to have twofold screw symmetry about the central plane of the Z-band. Only approximate twofold rotational symmetry is observed in directions parallel to the actin filaments. Previous models of the Z-band in which four identical and rotationally symmetrical links emanate from the end of one actin filament and span across to the ends of four actin filaments in the adjacent sarcomere are therefore incorrect.     

Microvoids in Bombyx mori silk. An electron microscope study.

The size and distribution of microvoids in Bombyx mori silk were examined by transmission electron microscopy of silver sulphide 'stained' filaments. Silver sulphide deposited in voids and accessible regions of molecular structure appears as dense particles in thin transverse and longitudinal sections of silk filaments. Small particles (about 8 nm or less in diameter) occur around or adjacent to the periphery of the filaments. Larger particles (around 10-15 nm in diameter) occur in the form of dendritic arrays in the core region of the filaments. The leading edges of the dendritic arrays are oriented towards the fibre periphery. The particles (microvoids) appear to be either spherical or rod-like in shape and are aligned parallel to the long axis of the filament. A skin/core structure is proposed.     

Dynamic light scattering study of the effect of Mg2+ and ATP on synthetic myosin filaments.

The dynamic light scattering (DLS) method provides us with information about the apparent diffusion coefficient, Dapp, as well as the static scattering intensity, Is, of particles in solution. For long but thin rods with length L and diameter d, the dependence on L and d of Dapp is quite different from that of Is. By means of DLS we studied synthetic myosin filaments of rabbit skeletal muscle in solution at pH 8.3 and 10 degrees C. It appeared that Mg2+ ions induced thickening and lengthening of the filaments, whereas ATP (and ADP) induced thinning and shortening (depolymerization) of the filaments. When ATP was added to the filament preparation in the presence of Mg2+ ions, it was clearly observed that thinning of the filament (or splitting into subfilaments) occurred before shortening (or depolymerization).     

Myosin and paramyosin are organized about a newly identified core structure

Myosin isoforms A and B are differentially localized to the central and polar regions, respectively, of thick filaments in body wall muscle cells of Caenorhabditis elegans (Miller, D. M. III, I. Ortiz, G. C. Berliner, and H. F. Epstein, 1983, Cell, 34:477-490). Biochemical and electron microscope studies of KCl-dissociated filaments show that the myosin isoforms occupy a surface domain, paramyosin constitutes an intermediate domain, and a newly identified core structure exists. The diameters of the thick filaments vary significantly from 33.4 nm centrally to 14.0 nm near the ends. The latter value is comparable to the 15.2 nm diameter of the core structures. The internal density of the filament core appears solid medially and hollow at the poles. The differentiation of thick filament structure into supramolecular domains possessing specific substructures of characteristic stabilities suggests a sequential mode for thick filament assembly. In this model, the two myosin isoforms have distinct roles in assembly. The behavior of the myosins, including nucleation of assembly and determination of filament length, depend upon paramyosin and the core structure as well as their intrinsic molecular properties.     

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).     

Fine structure and assembly in vitro of nuclear lamina in plant cells

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Ultrastructure of the flagellar apparatus of Oxyrrhis marina

Summary— Oxyrrhis marina, like all dinoflagellates, possesses one transverse and one longitudinal flagellum, which show structural differences. The transverse flagellum contains a small fibre, 20 nm in diameter, associated with doublet no.7, whereas the longitudinal flagellum is substantially by a large (200–300 nm) hollows structure closely resembling the paraflagellar rod described by several authors in kinetoplastidae and in euglenoids. This structure is made up of a hemicylindrical network of filaments which are often linked on one side to the outer doublet no. 4, and on the other side to a dense plate. Another thinner filamentous network closes this hemicyclinder. In cross-section, the wall of this structure is made up of 8 filaments 2–4 nm in diameter that show a thicker periodic structure. In longitudinal section the same filaments appear arranged in periodic rhombus meshes or a helicoidal pattern, depending on the orientation of the section relative to the axoneme.