Active supercontraction in rolling-up muscles of Glomeris marginata (myriapoda,diplopoda)

The lateroventral muscles of Glomeris marginata keep the animal rolled up and are able to develop and maintain great tension. Their fibers are not equipped with a particularly strong contractile apparatus but can super-contract. The sarcomere shortens its resting length by up 60% and in a typical supercontraction the thick filaments pass through the Z-line into adjacent sarcomeres. The Z-line structure changes according to the contraction state: It passes from a homogeneous, dense zig-zag line in decontracted fibers to a rarified, vaguely outlined Z-band in supercontracted fibers, in which it is possible to see actin and myosin filaments. The Z-line is thus involved in an active expanding process and is functionally very different from the fragmented and discontinuous Z-line of “classical” supercontracting muscles. The different meaning of the two cases of supercontraction is discussed.     

A Nebulin Ruler Does Not Dictate Thin Filament Lengths

To generate force, striated muscle requires overlap between uniform-length actin and myosin filaments. The hypothesis that a nebulin ruler mechanism specifies thin filament lengths by targeting where tropomodulin (Tmod) caps the slow-growing, pointed end has not been rigorously tested. Using fluorescent microscopy and quantitative image analysis, we found that nebulin extended 1.01-1.03 μm from the Z-line, but Tmod localized 1.13-1.31 μm from the Z-line, in seven different rabbit skeletal muscles. Because nebulin does not extend to the thin filament pointed ends, it can neither target Tmod capping nor specify thin filament lengths. We found instead a strong correspondence between thin filament lengths and titin isoform sizes for each muscle. Our results suggest the existence of a mechanism whereby nebulin specifies the minimum thin filament length and sarcomere length regulates and coordinates pointed-end dynamics to maintain the relative overlap of the thin and thick filaments during myofibril assembly.     

Forces measured with micro-fabricated cantilevers during actomyosin interactions produced by filaments containing different myosin isoforms and loop 1 structures

Background

There is evidence that the actin-activated ATP kinetics and the mechanical work produced by muscle myosin molecules are regulated by two surface loops, located near the ATP binding pocket (loop 1), and in a region that interfaces with actin (loop 2). These loops regulate force and velocity of contraction, and have been investigated mostly in single molecules. There is a lack of information of the work produced by myosin molecules ordered in filaments and working cooperatively, which is the actual muscle environment.

Methods

We use micro-fabricated cantilevers to measure forces produced by myosin filaments isolated from mollusk muscles, skeletal muscles, and smooth muscles containing variations in the structure of loop 1 (tonic and phasic myosins). We complemented the experiments with in-vitro assays to measure the velocity of actin motility.

Results

Smooth muscle myosin filaments produced more force than skeletal and mollusk myosin filaments when normalized per filament overlap. Skeletal muscle myosin propelled actin filaments in a higher sliding velocity than smooth muscle myosin. The values for force and velocity were consistent with previous studies using myosin molecules, and suggest a close correlation with the myosin isoform and structure of surface loop 1.

General significance

The technique using micro-fabricated cantilevers to measure force of filaments allows for the investigation of the relation between myosin structure and contractility, allowing experiments to be conducted with an array of different myosin isoforms. Using the technique we observed that the work produced by myosin molecules is regulated by amino-acid sequences aligned in specific loops.     

The contractile apparatus of striated muscle in the course of atrophy and regeneration

Summary Changes in the contractile apparatus of denervated rat soleus muscles were investigated during the course of reinnervation.As observed earlier, in the course of denervation atrophy the ratio of myosin to actin filaments decreases because myosin filaments disappear faster than actin filaments (Jakubiec-Puka et al. 1981 a). After reinnervation the amount of myosin filaments and myosin heavy chains (myosin HC) in the muscle increased during the first few days; the increment of actin content was negligible. The proportion of myosin HC to actin remained lower than normal for about 30 days. The excess of actin filaments frequently observed in the newly-formed myofibrils reflects this disproportion.The results show a lability of myosin and suggest some cytoskeletal role for actin filaments.     

The use of cryomethods for research on the sarcomere ultrastructure of rabbit skeletal muscles

The ultrastructure of sarcomeres of glycerinated rabbit psoas muscle was studied using freeze-fracture-etching, freeze-drying and optical diffraction techniques in comparison with the investigation of this muscle by plastic sections and negative staining methods. In frozen and dried myofibrils isolated from the above muscle the stripes of minor proteins location in A- and I-disks were clearly seen. The pivot structure in thick filaments was revealed in longitudinal fractures of the muscle. The ordered arrangement of myosin heads (crossbridges) associated with actin filaments was preserved in frozen longitudinal fractures as evidenced by optical diffraction. Freeze etching technique allowed to revealed some details of Z-line structure: alpha-actinin bridges connecting the ends of actin filaments of neighbouring sarcomeres and to preserve the lateral struts between actin filaments in I-disks.     

Thin-filament length correlates with fiber type in human skeletal muscle

Force production in skeletal muscle is proportional to the amount of overlap between the thin and thick filaments, which, in turn, depends on their lengths. Both thin- and thick-filament lengths are precisely regulated and uniform within a myofibril. While thick-filament lengths are essentially constant across muscles and species (～1.65 μm), thin-filament lengths are highly variable both across species and across muscles of a single species. Here, we used a high-resolution immunofluorescence and image analysis technique (distributed deconvolution) to directly test the hypothesis that thin-filament lengths vary across human muscles. Using deltoid and pectoralis major muscle biopsies, we identified thin-filament lengths that ranged from 1.19 ± 0.08 to 1.37 ± 0.04 μm, based on tropomodulin localization with respect to the Z-line. Tropomodulin localized from 0.28 to 0.47 μm further from the Z-line than the NH(2)-terminus of nebulin in the various biopsies, indicating that human thin filaments have nebulin-free, pointed-end extensions that comprise up to 34% of total thin-filament length. Furthermore, thin-filament length was negatively correlated with the percentage of type 2X myosin heavy chain within the biopsy and shorter in type 2X myosin heavy chain-positive fibers, establishing the existence of a relationship between thin-filament lengths and fiber types in human muscle. Together, these data challenge the widely held assumption that human thin-filament lengths are constant. Our results also have broad relevance to musculoskeletal modeling, surgical reattachment of muscles, and orthopedic rehabilitation.     

Influence of dispersion in myosin filament orientation and anisotropic filament contractions in smooth muscle

A new constitutive model for the biomechanical behaviour of smooth muscle tissue is proposed. The active muscle contraction is accomplished by the relative sliding between actin and myosin filaments, comprising contractile units in the smooth muscle cells. The orientation of the myosin filaments, and thereby the contractile units, are taken to exhibit a statistical dispersion around a preferred direction. The number of activated cross-bridges between the actin and myosin filaments governs the contractile force generated by the muscle and also the contraction speed. A strain-energy function is used to describe the mechanical behaviour of the smooth muscle tissue. Besides the active contractile apparatus, the mechanical model also incorporates a passive elastic part. The constitutive model was compared to histological and isometric tensile test results for smooth muscle tissue from swine carotid artery. In order to be able to predict the active stress at different muscle lengths, a filament dispersion significantly larger than the one observed experimentally was required. Furthermore, a comparison of the predicted active stress for a case of uniaxially oriented myosin filaments and a case of filaments with a dispersion based on the experimental histological data shows that the difference in generated stress is noticeable but limited. Thus, the results suggest that myosin filament dispersion alone cannot explain the increase in active muscle stress with increasing muscle stretch.     

X-ray diffraction evidence for the extensibility of actin and myosin filaments during muscle contraction.

To clarify the extensibility of thin actin and thick myosin filaments in muscle, we examined the spacings of actin and myosin filament-based reflections in x-ray diffraction patterns at high resolution during isometric contraction of frog skeletal muscles and steady lengthening of the active muscles using synchrotron radiation as an intense x-ray source and a storage phosphor plate as a high sensitivity, high resolution area detector. Spacing of the actin meridional reflection at approximately 1/2.7 nm-1, which corresponds to the axial rise per actin subunit in the thin filament, increased about 0.25% during isometric contraction of muscles at full overlap length of thick and thin filaments. The changes in muscles stretched to approximately half overlap of the filaments, when they were scaled linearly up to the full isometric tension, gave an increase of approximately 0.3%. Conversely, the spacing decreased by approximately 0.1% upon activation of muscles at nonoverlap length. Slow stretching of a contracting muscle increased tension and increased this spacing over the isometric contraction value. Scaled up to a 100% tension increase, this corresponds to a approximately 0.26% additional change, consistent with that of the initial isometric contraction. Taken together, the extensibility of the actin filament amounts to 3-4 nm of elongation when a muscle switches from relaxation to maximum isometric contraction. Axial spacings of the layer-line reflections at approximately 1/5.1 nm-1 and approximately 1/5.9 nm-1 corresponding to the pitches of the right- and left-handed genetic helices of the actin filament, showed similar changes to that of the meridional reflection during isometric contraction of muscles at full overlap. The spacing changes of these reflections, which also depend on the mechanical load on the muscle, indicate that elongation is accompanied by slight changes of the actin helical structure possibly because of the axial force exerted by the actomyosin cross-bridges. Additional small spacing changes of the myosin meridional reflections during length changes applied to contracting muscles represented an increase of approximately 0.26% (scaled up to a 100% tension increase) in the myosin periodicity, suggesting that such spacing changes correspond to a tension-related extension of the myosin filaments. Elongation of the myosin filament backbone amounts to approximately 2.1 nm per half sarcomere. The results indicate that a large part (approximately 70%) of the sarcomere compliance of an active muscle is caused by the extensibility of the actin and myosin filaments; 42% of the compliance resides in the actin filaments, and 27% of it is in the myosin filaments.     

Fine structure of the homologous tergo-coxal muscles of flying and flightless beetles

The flight-related tergo-coxal muscles of flying and flightless beetles are compared. In the flying beetle, Pachynoda sinuata, the myofibrils and cylindrical and the myofilaments packed in double hexagonal arrays. The sarcomeres are short (2.8 micrometer) and wide with many large, closely packed adjacent mitochondria but the sarcoplasmic reticulum is poorly developed in this fibrillar (asynchronous) muscle. Sarcoplasmic glycogen in rosette form is abundant. In the flightless beetle, Anthia thoracica, the myofibrils are lamellar-like with sarcomeres of 5.3 micrometer. The myosin filaments form a single hexagonal array each thick filament having an orbital of 11 to 12 thin filaments. The width of the Z-line (120 nm) of A. thoracia muscle was twice that of the Z-line of P. sinuata muscle. The sarcoplasmic reticulum and T-system are well-developed in this afibrillar (synchronous) muscle. Few glycogen granules are present. Triangular projections of the sarcolemma occur regularly opposite the Z-lines in A. thoracica and they appear to extend into the Z-lines. Membranous connections joint adjacent Z-lines in A. thoracica and occasionally in P. sinuata.     

Fine structure of wide and narrow vertebrate muscle Z-lines. A proposed model and computer simulation of Z-line architecture

A model of the structure of vertebrate Z-lines and Z-line analogs is introduced and supported by evidence from electron microscope studies of wide Z-lines (rat and feline soleus, and feline and canine cardiac muscles), narrow Z-lines (guppy, newt and frog skeletal muscles), and Z-rods (from a patient with nemaline myopathy and from cardiac muscles of aged dog). The model is based on a pair of Z-filaments (termed a Z-unit), which are linked near their centers at a 90 degrees angle and form bridges between neighboring antipolar thin (actin) filaments. A square lattice of four Z-filament pairs (the basic structure of the Z-line, termed a Z-line unit) defines the geometrical position of the I-square unit. In this native state of the Z-line, small square and large square net forms appear in cross-section. Other cross-sectional patterns of Z-lines, including basket-weave and diagonal-square net patterns, can be explained by detachment of the Z-filament from the Z-filament binding region within each Z-filament pair due to chemical or physical stress. Dissection of Z-lines and Z-line analogs with calcium-activated neutral protease provides evidence that the width of all wide Z-line structures is determined by the amount of overlap of antipolar thin filaments from adjacent sarcomeres. Longitudinal patterns of narrow and wide Z-lines are shown and described in relation to the model. To test the proposed model, the dynamics of the Z-line unit structure were computer-simulated. An attempt was made to correlate longitudinal (z direction) and cross-sectional (x and y directions) patterns and to determine the amount of movement of thin or Z-filaments that is required to explain the diversity observed in cross-sectional patterns of Z-lines. The computer simulations demonstrated that the structural transitions among the small square, and therefore large square net, as well as basket-weave and diagonal-square net forms seen in cross-sections could be caused by movements of thin filaments less than 10 nm in any direction (x, y or z).(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

LOCUS AND STATE OF AGGREGATION OF MYOSIN IN TISSUE SECTIONS OF VERTEBRATE SMOOTH MUSCLE

Structures with the characteristics of molecular myosin were identified by electron microscopy in tissue sections of vertebrate smooth muscle. No thick filaments of myosin were found regardless of preparative procedures, which included fixation at rest and in contraction, glycerine extraction, and storage at low pH prior to fixation. Absence of thick myosin filaments and presence of what appear to be myosin molecules is in accord with conclusions based on X-ray diffraction (3, 12) and birefringence data (4) from living smooth muscles at rest and in contraction. Explanations are provided for appearances thought by others (6, 20, 21) to represent thick myosin filaments. Our present observations are in accord with the model for smooth muscle contraction which we have previously proposed (1).     

FILAMENT ULTRASTRUCTURE AND ORGANIZATION IN VERTEBRATE SMOOTH MUSCLE : Contraction Hypothesis Based on Localization of Actin and Myosin

Using a variety of preparative techniques for electron microscopy, we have obtained evidence for the disposition of actin and myosin in vertebrate smooth muscle. All longitudinal myofilaments seen in sections appear to be actin. Previous reports of two types of longitudinal filaments in sections are accounted for by technical factors, and by differentiated areas of opacity along individual filaments. Dense bodies with actin emerging from both ends have been identified in homogenates, and resemble Z discs from skeletal muscle (Huxley, 1963). In sections, short, dark-staining lateral filaments 15–25 A in diameter link adjacent actin filaments within dense bodies and in membrane dense pataches. They appear homologous with Z-disc filaments. Similar lateral filaments connect actin to plasma membrane. Dense bodies and dense patches, therefore, are attachment points and denote units analogous to sarcomeres. In glycerinated, methacrylate-embedded sections, lateral processes different in length and staining characteristics from lateral filaments in dense bodies exist at intervals along actin filaments. These processes are about 30 A wide and resemble heavy meromyosin from skeletal muscle. They also resemble heads of whole molecules of myosin in negatively stained material from gizzard homogenates. Intact single myosin molecules and dimers have been found, both free and attached to actin, even in media of very low ionic strength. Myosin can, therefore, exist in relatively disaggregated form. Models of the contraction mechanism of smooth muscle are proposed. The unique features are: (1) Myosin exists as small functional units. (2) Movement occurs by interdigitation and sliding of actin filaments.     

Study of regulatory effect of tropomyosin on actin-myosin interaction in skeletal muscle by in vitro motility assay

The interaction between myosin and actin in striated muscle tissue is regulated by Ca²⁺ via thin filament regulatory proteins. Skeletal muscle possesses a whole pattern of myosin and tropomyosin isoforms. The regulatory effect of tropomyosin on actin-myosin interaction was investigated by measuring the sliding velocity of both actin and actin-tropomyosin filaments over fast and slow skeletal myosins using the in vitro motility assay. The actin-tropomyosin filaments were reconstructed with tropomyosin isoforms from striated muscle tissue. It was found that tropomyosins with different content of α-, β-, and γ-chains added to actin filaments affect the sliding velocity of filaments in different ways. On the other hand, the sliding velocity of filaments with the same content of α-, β-, and Γ-chains depends on myosin isoforms of striated muscle. The reciprocal effects of myosin and tropomyosin on actin-myosin interaction in striated muscle may play a significant role in maintenance of effective work of striated muscle both during ontogenesis and under pathological conditions.     

The contractile proteins of the human myometrium

The contractile proteins of the human myometrium were quantified and characterized in order to investigate their possible modifications during pregnancy. The myosin concentration was found to be 1 to 5 mg/g of tissue, while actin concentration ranged from 16 to 60 mg/g, leading to an actin/myosin ratio higher (Mean = 14) than in other smooth muscles. Purified myosin submitted to two-dimensional gel electrophoresis exhibited two isoelectric forms for the 17 KD Light Chain, the more acidic being predominant in the pregnant organ, the more basic in the non gravid one. The mobility of myosin of form filaments was studied using electron microscopy. Only the myosin purified from gravid uteri in its phosphorylated form did aggregate in long bipolar filaments. Actin was characterized in crude muscle extracts using two-dimensional gel electrophoresis. It appeared in three forms differing by their isoelectric points. The more basic form (gamma) predominates in the pregnant organ, as soon as 17 weeks of pregnancy, while in the non-pregnant uterus it is the intermediate (beta) form which is predominant.     

Purification of native myosin filaments from muscle

Analysis of the structure and function of native thick (myosin-containing) filaments of muscle has been hampered in the past by the difficulty of obtaining a pure preparation. We have developed a simple method for purifying native myosin filaments from muscle filament suspensions. The method involves severing thin (actin-containing) filaments into short segments using a Ca(2+)-insensitive fragment of gelsolin, followed by differential centrifugation to purify the thick filaments. By gel electrophoresis, the purified thick filaments show myosin heavy and light chains together with nonmyosin thick filament components. Contamination with actin is below 3.5%. Electron microscopy demonstrates intact thick filaments, with helical cross-bridge order preserved, and essentially complete removal of thin filaments. The method has been developed for striated muscles but can also be used in a modified form to remove contaminating thin filaments from native smooth muscle myofibrils. Such preparations should be useful for thick filament structural and biochemical studies.     

ATP-dependent movement of myosin in vitro: characterization of a quantitative assay

Sheetz and Spudich (1983, Nature (Lond.), 303:31-35) showed that ATP- dependent movement of myosin along actin filaments can be measured in vitro using myosin-coated beads and oriented actin cables from Nitella. To establish this in vitro movement as a quantitative assay and to understand better the basis for the movement, we have defined the factors that affect the myosin-bead velocity. Beads coated with skeletal muscle myosin move at a rate of 2-6 micron/s, depending on the myosin preparation. This velocity is independent of myosin concentration on the bead surface for concentrations above a critical value (approximately 20 micrograms myosin/2.5 X 10(9) beads of 1 micron in diameter). Movement is optimal between pH 6.8 and 7.5, at KCl concentrations less than 70 mM, at ATP concentrations greater than 0.1 mM, and at Mg2+ concentrations between 2 and 6 mM. From the temperature dependence of bead velocity, we calculate activation energies of 90 kJ/mol below 22 degrees C and 40 kJ/mol above 22 degrees C. Different myosin species move at their own characteristic velocities, and these velocities are proportional to their actin-activated ATPase activities. Further, the velocities of beads coated with smooth or skeletal muscle myosin correlate well with the known in vivo rates of myosin movement along actin filaments in these muscles. This in vitro assay, therefore, provides a rapid, reproducible method for quantitating the ATP- dependent movement of myosin molecules on actin.     

Integrin-mediated adhesion maintains sarcomeric integrity

Integrin-mediated adhesion to the ECM is essential for normal development of animal tissues. During muscle development, integrins provide the structural stability required to construct such a highly tensile, force generating tissue. Mutations that disrupt integrin-mediated adhesion in skeletal muscles give rise to a myopathy in humans and mice. To determine if this is due to defects in formation or defects in maintenance of muscle tissue, we used an inducible, targeted RNAi based approach to disrupt integrin-mediated adhesion in fully formed adult fly muscles. A decrease in integrin-mediated adhesion in adult muscles led to a progressive loss of muscle function due to a failure to maintain normal sarcomeric cytoarchitecture. This defect was due to a gradual, age dependent disorganization of the sarcomeric actin, Z-line, and M-line. Electron microscopic analysis showed that reduction in integrin-mediated adhesion resulted in detachment of actin filaments from the Z-lines, separation of the Z-lines from the membrane, and eventually to disintegration of the Z-lines. Our results show that integrin-mediated adhesion is essential for maintaining sarcomeric integrity and illustrate that the seemingly stable adhesive contacts underlying sarcomeric architecture are inherently dynamic.     

Light chain phosphorylation regulates the movement of smooth muscle myosin on actin filaments

In smooth muscles there is no organized sarcomere structure wherein the relative movement of myosin filaments and actin filaments has been documented during contraction. Using the recently developed in vitro assay for myosin-coated bead movement (Sheetz, M.P., and J.A. Spudich, 1983, Nature (Lond.)., 303:31-35), we were able to quantitate the rate of movement of both phosphorylated and unphosphorylated smooth muscle myosin on ordered actin filaments derived from the giant alga, Nitella. We found that movement of turkey gizzard smooth muscle myosin on actin filaments depended upon the phosphorylation of the 20-kD myosin light chains. About 95% of the beads coated with phosphorylated myosin moved at velocities between 0.15 and 0.4 micron/s, depending upon the preparation. With unphosphorylated myosin, only 3% of the beads moved and then at a velocity of only approximately 0.01-0.04 micron/s. The effects of phosphorylation were fully reversible after dephosphorylation with a phosphatase prepared from smooth muscle. Analysis of the velocity of movement as a function of phosphorylation level indicated that phosphorylation of both heads of a myosin molecule was required for movement and that unphosphorylated myosin appears to decrease the rate of movement of phosphorylated myosin. Mixing of phosphorylated smooth muscle myosin with skeletal muscle myosin which moves at 2 microns/s resulted in a decreased rate of bead movement, suggesting that the more slowly cycling smooth muscle myosin is primarily determining the velocity of movement in such mixtures.