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1.
Passive tension and stiffness of vertebrate skeletal and insect flight muscles: the contribution of weak cross-bridges and elastic filaments. 总被引:10,自引:3,他引:7
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Tension and dynamic stiffness of passive rabbit psoas, rabbit semitendinosus, and waterbug indirect flight muscles were investigated to study the contribution of weak-binding cross-bridges and elastic filaments (titin and minititin) to the passive mechanical behavior of these muscles. Experimentally, a functional dissection of the relative contribution of actomyosin cross-bridges and titin and minititin was achieved by 1) comparing mechanically skinned muscle fibers before and after selective removal of actin filaments with a noncalcium-requiring gelsolin fragment (FX-45), and 2) studying passive tension and stiffness as a function of sarcomere length, ionic strength, temperature, and the inhibitory effect of a carboxyl-terminal fragment of smooth muscle caldesmon. Our data show that weak bridges exist in both rabbit skeletal muscle and insect flight muscle at physiological ionic strength and room temperature. In rabbit psoas fibers, weak bridge stiffness appears to vary with both thin-thick filament overlap and with the magnitude of passive tension. Plots of passive tension versus passive stiffness are multiphasic and strikingly similar for these three muscles of distinct sarcomere proportions and elastic proteins. The tension-stiffness plot appears to be a powerful tool in discerning changes in the mechanical behavior of the elastic filaments. The stress-strain and stiffness-strain curves of all three muscles can be merged into one, by normalizing strain rate and strain amplitude of the extensible segment of titin and minititin, further supporting the segmental extension model of resting tension development. 相似文献
2.
The stiffness of skeletal muscle in isometric contraction and rigor: the fraction of myosin heads bound to actin. 总被引:14,自引:2,他引:12
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M Linari I Dobbie M Reconditi N Koubassova M Irving G Piazzesi V Lombardi 《Biophysical journal》1998,74(5):2459-2473
Step changes in length (between -3 and +5 nm per half-sarcomere) were imposed on isolated muscle fibers at the plateau of an isometric tetanus (tension T0) and on the same fibers in rigor after permeabilization of the sarcolemma, to determine stiffness of the half-sarcomere in the two conditions. To identify the contribution of actin filaments to the total half-sarcomere compliance (C), measurements were made at sarcomere lengths between 2.00 and 2.15 microm, where the number of myosin cross-bridges in the region of overlap between the myosin filament and the actin filament remains constant, and only the length of the nonoverlapped region of the actin filament changes with sarcomere length. At 2.1 microm sarcomere length, C was 3.9 nm T0(-1) in active isometric contraction and 2.6 nm T0(-1) in rigor. The actin filament compliance, estimated from the slope of the relation between C and sarcomere length, was 2.3 nm microm(-1) T0(-1). Recent x-ray diffraction experiments suggest that the myosin filament compliance is 1.3 nm microm(-1) T0(-1). With these values for filament compliance, the difference in half-sarcomere compliance between isometric contraction and rigor indicates that the fraction of myosin cross-bridges attached to actin in isometric contraction is not larger than 0.43, assuming that cross-bridge elasticity is the same in isometric contraction and rigor. 相似文献
3.
Microscopic analysis of the elastic properties of nebulin in skeletal myofibrils. 总被引:1,自引:1,他引:0
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The elastic properties of nebulin were studied by measuring the elasticity of single skeletal myofibrils, from which the portion of the thin filament located at the I band had been selectively removed by treatment with plasma gelsolin under rigor conditions. In this myofibril model, a portion of each nebulin molecule at the I band was expected to be free of actin filaments and exposed. The length of the exposed portion of the nebulin molecule was controlled by performing the gelsolin treatment at various sarcomere lengths. The relation between the passive tension and extension of the exposed portion of the nebulin showed a convex curve starting from a slack length, apparently in a fashion similar to that of wool. The slack sarcomere length shifted depending on the length of the exposed portion of the nebulin, however, the relation being represented by a single master curve. The elastic modulus of nebulin was estimated to be two to three orders of magnitude smaller than that of an actin filament. Based on these results, we conclude that nebulin attaches to an actin filament in a side-by-side fashion and that it does not significantly contribute to the elastic modulus of thin filaments. The relation between the passive tension and extension of connectin (titin) was obtained for a myofibril from which thin filaments had been completely removed with gelsolin under contracting conditions; this showed a concave curve, consistent with the previous results obtained in single fibers. 相似文献
4.
The properties of some models of the actin filament are compared with those of the thin filament in muscle. The greater stiffness of thin filaments ex vivo with respect to F-actin in vitro is attributed to the effect of both protein osmotic pressure and the attached cross-bridges. By comparing the stiffness of thin filaments in vitro and in isometric and rigor muscles the stiffness of thin filaments in relaxed muscle is computed. The upper limit of thin filament stretching is deduced to approach approximately 10 nm microm(-1). It is also calculated that, on stretching by 2.02 nm of the fully non-overlapped thin filament or by 1.59 nm of the thin filament on isometric contraction, the energy released on the hydrolysis of one molecule of ATP is fully used up. 相似文献
5.
C. David Williams Mary K. Salcedo Thomas C. Irving Michael Regnier Thomas L. Daniel 《Proceedings. Biological sciences / The Royal Society》2013,280(1766)
Classic interpretations of the striated muscle length–tension curve focus on how force varies with overlap of thin (actin) and thick (myosin) filaments. New models of sarcomere geometry and experiments with skinned synchronous insect flight muscle suggest that changes in the radial distance between the actin and myosin filaments, the filament lattice spacing, are responsible for between 20% and 50% of the change in force seen between sarcomere lengths of 1.4 and 3.4 µm. Thus, lattice spacing is a significant force regulator, increasing the slope of muscle''s force–length dependence. 相似文献
6.
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 Reedy's (1968) concept that cross-bridges in rigor attach only to certain azimuths on the actin filaments (“target areas”); the 145 Å 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. 相似文献
7.
Three-dimensional image reconstruction of insect flight muscle. II. The rigor actin layer 总被引:3,自引:1,他引:2
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The averaged structure of rigor cross-bridges in insect flight muscle is further revealed by three-dimensional reconstruction from 25-nm sections containing a single layer of thin filaments. These exhibit two thin filament orientations that differ by 60 degrees from each other and from myac layer filaments. Data from multiple tilt views (to +/- 60 degrees) was supplemented by data from thick sections (equivalent to 90 degrees tilts). In combination with the reconstruction from the myac layer (Taylor et al., 1989), the entire unit cell is reconstructed, giving the most complete view of in situ cross-bridges yet obtained. All our reconstructions show two classes of averaged rigor cross-bridges. Lead bridges have a triangular shape with leading edge angled at approximately 45 degrees and trailing edge angled at approximately 90 degrees to the filament axis. We propose that the lead bridge contains two myosin heads of differing conformation bound along one strand of F-actin. The lead bridge is associated with a region of the thin filament that is apparently untwisted. We suggest that the untwisting may reflect the distribution of strain between myosin and actin resulting from two-headed, single filament binding in the lead bridge. Rear bridges are oriented at approximately 90 degrees to the filament axis, and are smaller and more cylindrical, suggesting that they consist of single myosin heads. The rear bridge is associated with a region of apparently normal thin filament twist. We propose that differing myosin head angles and conformations consistently observed in rigor embody different stages of the power stroke which have been trapped by a temporal sequence of rigor cross-bridge formation under the constraints of the intact filament lattice. 相似文献
8.
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. 相似文献
9.
Cross-bridge arrangements in Limulus muscle 总被引:9,自引:0,他引:9
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. 相似文献
10.
D M Warshaw J M Desrosiers S S Work K M Trybus 《The Journal of biological chemistry》1991,266(36):24339-24343
To test the idea that the in vitro motility assay is a simplified model system for muscle contraction, the MgATP-dependent movement of actin filaments by thiophosphorylated smooth muscle myosin was characterized in the presence of the products MgADP and inorganic phosphate. The dependence of actin filament velocity on MgATP concentration was hyperbolic with a maximum velocity of 0.6 micron/s and an apparent Km = 40 microM (30 degrees C). MgADP competitively inhibited actin movement by MgATP with a Ki = 0.25 mM. Inorganic phosphate did not affect actin filament velocity in the presence of 1 mM MgATP, but competitively inhibited movement in the presence of 50 microM MgATP with a Ki = 9.5 mM. The effects of ADP and Pi on velocity agree with fiber mechanical studies, confirming that the motility assay is an excellent system to investigate the molecular mechanisms of force generation and shortening in smooth muscle. The rate at which rigor cross-bridges can be recruited to move actin filaments was observed by initiating cross-bridge cycling from rigor by flash photolysis of caged MgATP. Following the flash, which results in a rapid increase in MgATP concentration, actin filaments experienced a MgATP-dependent delay prior to achieving steady state velocity. The delay at low MgATP concentrations was interpreted as evidence that motion generating cross-bridges are slowed by a load due to a transiently high percentage of rigor cross-bridges immediately following MgATP release. 相似文献
11.
Modeling rigor cross-bridge patterns in muscle I. Initial studies of the rigor lattice of insect flight muscle. 总被引:3,自引:2,他引:1
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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. 相似文献
12.
Non-specific termination of simian virus 40 DNA replication. 总被引:4,自引:0,他引:4
Axial X-ray diffraction patterns have been studied from relaxed, contracted and rigor vertebrate striated muscles at different sarcomere lengths to determine which features of the patterns depend on the interaction of actin and myosin. The intensity of the myosin layer lines in a live, relaxed muscle is sometimes less in a stretched muscle than in the muscle at rest-length; the intensity depends not only on the sarcomere length but on the time that has elapsed since dissection of the muscle. The movement of cross-bridges giving rise to these intensity changes are not caused solely by the withdrawal of actin from the A-band.When a muscle contracts or passes into rigor many changes occur that are independent of the sarcomere length: the myosin layer lines decrease in intensity to about 30% of their initial value when the muscle contracts, and disappear completely when the muscle passes into rigor. Both in contracting and rigor muscles at all sarcomere lengths the spacings of the meridional reflections at 143 Å and 72 Å are 1% greater than from a live relaxed muscle at rest-length. It is deduced that the initial movement of cross-bridges from their positions in resting muscle does not depend on the interaction of each cross-bridge with actin, but on a conformational change in the backbone of the myosin filament: occurring as a result of activation. The possibility is discussed that the conformational change occurs because the myosin filament, like the actin filament, has an activation control mechanism. Finally, all the X-ray diffraction patterns are interpreted on a model in which the myosin filament can exist in one of two possible states: a relaxed state which gives a diffraction pattern with strong myosin layer lines and an axial spacing of 143.4 Å, and an activated state which gives no layer lines but a meridional spacing of 144.8 Å. 相似文献
13.
The cross-bridge stiffness can be used to estimate the number of S1 that are bound to actin during contraction, which is a critical parameter for elucidating the fundamental mechanism of the myosin motor. At present, the development of active tension and the increase in muscle stiffness due to S1 binding to actin are thought to be linearly related to the number of cross-bridges formed upon activation. The nonlinearity of total stiffness with respect to active force is thought to arise from the contribution of actin and myosin filament stiffness to total sarcomere elasticity. In this work, we reexamined the relation of total stiffness to tension during activation and during exposure to N-benzyl-p-toluene sulphonamide, an inhibitor of cross-bridge formation. In addition to filament and cross-bridge elasticity, our findings are best accounted for by the inclusion of an extra elasticity in parallel with the cross-bridges, which is formed upon activation but is insensitive to the subsequent level of cross-bridge formation. By analyzing the rupture tension of the muscle (an independent measure of cross-bridge formation) at different levels of activation, we found that this additional elasticity could be explained as the stiffness of a population of no-force-generating cross-bridges. These findings call into question the assumption that active force development can be taken as directly proportional to the cross-bridge number. 相似文献
14.
X-ray diffraction evidence for the extensibility of actin and myosin filaments during muscle contraction. 总被引:34,自引:11,他引:23
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K Wakabayashi Y Sugimoto H Tanaka Y Ueno Y Takezawa Y Amemiya 《Biophysical journal》1994,67(6):2422-2435
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. 相似文献
15.
Cross-bridge number, position, and angle in target zones of cryofixed isometrically active insect flight muscle
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Tregear RT Reedy MC Goldman YE Taylor KA Winkler H Franzini-Armstrong C Sasaki H Lucaveche C Reedy MK 《Biophysical journal》2004,86(5):3009-3019
Electron micrographic tomograms of isometrically active insect flight muscle, freeze substituted after rapid freezing, show binding of single myosin heads at varying angles that is largely restricted to actin target zones every 38.7 nm. To quantify the parameters that govern this pattern, we measured the number and position of attached myosin heads by tracing cross-bridges through the three-dimensional tomogram from their origins on 14.5-nm-spaced shelves along the thick filament to their thin filament attachments in the target zones. The relationship between the probability of cross-bridge formation and axial offset between the shelf and target zone center was well fitted by a Gaussian distribution. One head of each myosin whose origin is close to an actin target zone forms a cross-bridge most of the time. The probability of cross-bridge formation remains high for myosin heads originating within 8 nm axially of the target zone center and is low outside 12 nm. We infer that most target zone cross-bridges are nearly perpendicular to the filaments (60% within 11 degrees ). The results suggest that in isometric contraction, most cross-bridges maintain tension near the beginning of their working stroke at angles near perpendicular to the filament axis. Moreover, in the absence of filament sliding, cross-bridges cannot change tilt angle while attached nor reach other target zones while detached, so may cycle repeatedly on and off the same actin target monomer. 相似文献
16.
Kulke M Neagoe C Kolmerer B Minajeva A Hinssen H Bullard B Linke WA 《The Journal of cell biology》2001,154(5):1045-1057
Kettin is a high molecular mass protein of insect muscle that in the sarcomeres binds to actin and alpha-actinin. To investigate kettin's functional role, we combined immunolabeling experiments with mechanical and biochemical studies on indirect flight muscle (IFM) myofibrils of Drosophila melanogaster. Micrographs of stretched IFM sarcomeres labeled with kettin antibodies revealed staining of the Z-disc periphery. After extraction of the kettin-associated actin, the A-band edges were also stained. In contrast, the staining pattern of projectin, another IFM-I-band protein, was not altered by actin removal. Force measurements were performed on single IFM myofibrils to establish the passive length-tension relationship and record passive stiffness. Stiffness decreased within seconds during gelsolin incubation and to a similar degree upon kettin digestion with mu-calpain. Immunoblotting demonstrated the presence of kettin isoforms in normal Drosophila IFM myofibrils and in myofibrils from an actin-null mutant. Dotblot analysis revealed binding of COOH-terminal kettin domains to myosin. We conclude that kettin is attached not only to actin but also to the end of the thick filament. Kettin along with projectin may constitute the elastic filament system of insect IFM and determine the muscle's high stiffness necessary for stretch activation. Possibly, the two proteins modulate myofibrillar stiffness by expressing different size isoforms. 相似文献
17.
The oval myosin filament profiles in transverse sections through the M-band of Lethocerus flight muscle are arranged in one of three orientations 60 degrees apart and point along the 11 directions of the hexagonal filament lattice. Relative orientations are not systematically related to give a superlattice structure, but neither are the orientations arranged completely randomly. In fact there is a nearly random structure with a slight bias towards adjacent filaments being identically oriented. This form of M-band structure is explained in terms of interactions between quasi-equivalent M-bridges. Its implications with regard to myosin crossbridge arrangement depend on the rotational symmetry of the crossbridge helix. For 6-stranded helices, 60 degrees rotations have no noticeable effect. However, in the case of the more likely 4-stranded structure, our results show that the crossbridge origins in the insect flight muscle A-band would be highly disordered. This disorder must be accounted for in interpreting both the flared-X crossbridge interactions seen in transverse sections of rigor insect flight muscle and the beautiful X-ray diffraction patterns from the same preparation. It is likely that in rigor insect muscle, some flared-Xs have the two heads of single myosin molecules interacting with two different actin filaments, whereas other flared-Xs have both of the myosin heads in one molecule interacting with the same actin filament. 相似文献
18.
Compliance of thin filaments in skinned fibers of rabbit skeletal muscle. 总被引:19,自引:7,他引:12
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The mechanical compliance (reciprocal of stiffness) of thin filaments was estimated from the relative compliance of single, skinned muscle fibers in rigor at sarcomere lengths between 1.8 and 2.4 micron. The compliance of the fibers was calculated as the ratio of sarcomere length change to tension change during imposition of repetitive cycles of small stretches and releases. Fiber compliance decreased as the sarcomere length was decreased below 2.4 micron. The compliance of the thin filaments could be estimated from this decrement because in this range of lengths overlap between the thick and thin filaments is complete and all of the myosin heads bind to the thin filament in rigor. Thus, the compliance of the overlap region of the sarcomere is constant as length is changed and the decrease in fiber compliance is due to decrease of the nonoverlap length of the thin filaments (the I band). The compliance value obtained for the thin filaments implies that at 2.4-microns sarcomere length, the thin filaments contribute approximately 55% of the total sarcomere compliance. Considering that the sarcomeres are approximately 1.25-fold more compliant in active isometric contractions than in rigor, the thin filaments contribute approximately 44% to sarcomere compliance during isometric contraction. 相似文献
19.
Actin-myosin interaction: The role of myosin in determining the actin pattern in self-assembled ‘hybrid’ contractile units 总被引:3,自引:0,他引:3
Self-assembly of actin-myosin filamentous complexes was assayed by polymerizing rabbit G-ADP actin on formed filaments of lobster myosin. The resulting contractile units indicate a 12-member actin orbital rather than the six-member orbital obtained previously using rabbit myosin and actin. Furthermore, the pattern of actin distribution surrounding the myosin filament is similar to that of the lobster tonic muscle sarcomere rather than the trigonal actin position characteristic of vertebrate muscle. The results show that the pattern and mode of actin complexing is determined by the specific myosin and the arrangement of the cross-bridges on the organized filament. 相似文献
20.
Symmetry and three-dimensional arrangement of filaments in vertebrate striated muscle 总被引:2,自引:0,他引:2
J M Squire 《Journal of molecular biology》1974,90(1):153-160