Rigor Insect Flight Muscle Research Articles

The Origins of Cross-Bridges in Active and Rigor Insect Flight Muscle are not as Predicted from Acto-S1 and X-Ray Crystallography

In fast-frozen, actively contracting insect flight muscle (IFM) fibers we noted a surprising azimuthal slew in the myosin lever arms that was wholly unexpected from the currently available crystal structures of isolated myosin S1 and which indicates the crystal structures may not reflect the normal in situ constraints within the sarcomere where myosin cross-bridges are restricted to originate from a well defined zone on the thick filament [Wu et al., 2010, PLoS-ONE, 5(9): e12643]. However, previous studies did not reveal the S2 domains, which most accurately define the myosin head origin. Here, we have used electron tomography (ET) of Lethocerus IFM fibers in rigor in which the filament lattice has been swollen in low ionic strength buffer to view the S2 origins of rigor “lead bridges”. These rigor lead bridges bind to the same region of the thin filament as myosin heads of active muscle so their origins should reflect the same thick filament positions as active heads. We examined 80 nm thick transverse sections cut with a vibrating knife to minimize section compression and shearing artifacts, and imaged with < 60 e-/A2 total exposure during each tilt series to minimize radiation-induced section thinning. This analysis shows two different distributions of lead bridge origins depending on the presence of the rear bridges. However, the origins are consistent with target zone accessibility of strong binding myosin heads being limited to two successive 14.5 nm crowns. Subvolume averages of both thick filaments as well as actin filaments are being pursued with the goal of reassembling a region of the filament lattice using high S/N averages. Supported by NIGMS and NIAMSD.

Electron Tomography of Swollen Rigor Fibers of Insect Flight Muscle Reveals a Short and Variably Angled S2 Domain

Subfragment 2 (S2), the segment that links the two myosin heads to the thick filament backbone, may serve as a swing-out adapter allowing crossbridge access to actin, as the elastic component of crossbridges and as part of a phosphorylation-regulated on-off switch for crossbridges in smooth muscle. Low-salt expansion increases interfilament spacing (from 52 nm to 67 nm) of rigor insect flight muscle fibers and exposes a tethering segment of S2 in many crossbridges. Docking an actoS1 atomic model into EM tomograms of swollen rigor fibers identifies in situ for the first time the location, length and angle assignable to a segment of S2. Correspondence analysis of 1831 38.7 nm crossbridge repeats grouped self-similar forms from which class averages could be computed. The full range of the variability in angles and lengths of exposed S2 was displayed by using class averages for atomic fittings of acto-S1, while S2 was modeled by fitting a length of coiled-coil to unaveraged individual repeats. This hybrid modeling shows that the average length of S2 tethers along the thick filament (except near the tapered ends) is ∼10 nm, or 16% of S2′s total length, with an angular range encompassing 90° axially and 120° azimuthally. The large range of S2 angles indicates that some rigor bridges produce positive force that must be balanced by others producing drag force. The short tethering segment clarifies constraints on the function of S2 in accommodating variable myosin head access to actin. We suggest that the short length of S2 may also favor intermolecular head−head interactions in IFM relaxed thick filaments.

Real Space Refinement of Acto-myosin Structures from Sectioned Muscle

We have adapted a real space refinement protocol originally developed for high-resolution crystallographic analysis for use in fitting atomic models of actin filaments and myosin subfragment 1 (S1) to 3-D images of thin-sectioned, plastic-embedded whole muscle. The rationale for this effort is to obtain a refinement protocol that will optimize the fit of the model to the density obtained by electron microscopy and correct for poor geometry introduced during the manual fitting of a high-resolution atomic model into a lower resolution 3-D image. The starting atomic model consisted of a rigor acto-S1 model obtained by X-ray crystallography and helical reconstruction of electron micrographs. This model was rebuilt to fit 3-D images of rigor insect flight muscle at a resolution of 7 nm obtained by electron tomography and image averaging. Our highly constrained real space refinement resulted in modest improvements in the agreement of model and reconstruction but reduced the number of conflicting atomic contacts by 70% without loss of fit to the 3-D density. The methodology seems to be well suited to the derivation of stereochemically reasonable atomic models that are consistent with experimentally determined 3-D reconstructions computed from electron micrographs.

Tomographic three-dimensional reconstruction of insect flight muscle partially relaxed by AMPPNP and ethylene glycol.

Rigor insect flight muscle (IFM) can be relaxed without ATP by increasing ethylene glycol concentration in the presence of adenosine 5'-[beta'gamma- imido]triphosphate (AMPPNP). Fibers poised at a critical glycol concentration retain rigor stiffness but support no sustained tension ("glycol-stiff state"). This suggests that many crossbridges are weakly attached to actin, possibly at the beginning of the power stroke. Unaveraged three-dimensional tomograms of "glycol-stiff" sarcomeres show crossbridges large enough to contain only a single myosin head, originating from dense collars every 14.5 nm. Crossbridges with an average 90 degrees axial angle contact actin midway between troponin subunits, which identifies the actin azimuth in each 38.7-nm period, in the same region as the actin target zone of the 45 degrees angled rigor lead bridges. These 90 degrees "target zone" bridges originate from the thick filament and approach actin at azimuthal angles similar to rigor lead bridges. Another class of glycol-PNP crossbridge binds outside the rigor actin target zone. These "nontarget zone" bridges display irregular forms and vary widely in axial and azimuthal attachment angles. Fitting the acto-myosin subfragment 1 atomic structure into the tomogram reveals that 90 degrees target zone bridges share with rigor a similar contact interface with actin, while nontarget crossbridges have variable contact interfaces. This suggests that target zone bridges interact specifically with actin, while nontarget zone bridges may not. Target zone bridges constitute only approximately 25% of the myosin heads, implying that both specific and nonspecific attachments contribute to the high stiffness. The 90 degrees target zone bridges may represent a preforce attachment that produces force by rotation of the motor domain over actin, possibly independent of the regulatory domain movements.

Electron Tomography of Insect Flight Muscle in Rigor and AMPPNP at 23°C

Treatment of rigor fibers of insect flight muscle (IFM) with AMPPNP at 23°C causes a 70% drop in tension with little change in stiffness. In order to visualize the changes in crossbridge conformation and distribution that give rise to the mechanical response, we have produced three-dimensional reconstructions by tomography of both rigor and AMPPNP-treated muscle that do not average the repeating motifs of crossbridges, and thereby retain information on variability of crossbridge structure and distribution. Tomograms can be averaged when display of only the regular features is wanted. Tomograms of rigor IFM show double-headed lead and single-headed rear crossbridges. Tomograms of IFM treated with AMPPNP at 23°C reveal many double-headed and some single-headed “lead” bridges but few crossbridges corresponding to the rear bridges of rigor. Instead, new non-rigor forms of variably angled crossbridges are found bound to actin sites not labeled with myosin heads in rigor. This indicates that the rear bridges of rigor have redistributed during the transition from rigor to the AMPPNP state, which could explain the maintenance of rigor stiffness despite the loss of tension. Comparison of in situcrossbridges in tomograms of rigor with atomic model of acto-S1, the complex formed by myosin subfragment 1 and actin, reveals that the regulatory domain of S1 would require significant bending and realignment to fit into both types of rigor crossbridges. The modifications are particularly significant for the rear bridges and suggest that differential strain in the regulatory domain of rear bridges may be the basis for their detachment and redistribution upon binding AMPPNP. Similar compari son using lead-type crossbridges in AMPPNP reveals departures from the rigor acto-S1 atomic model that include azimuthal straightening and a slight M-ward bending in the regulatory domain. Both the motor and regulatory domains of the new non-rigor crossbridges differ from those in the atomic model of acto-S1. A new crossbridge motif identified in AMPPNP-treated muscle consists of paired rigor-like and non-rigor crossbridges and suggests possible transitions in the myosin working stroke.

Three-dimensional Structure of Nucleotide-bearing Crossbridges in Situ: Oblique Section Reconstruction of Insect Flight Muscle in AMPPNP at 23°C

We have explored the three-dimensional structure of myosin crossbridges in situin order to define the structural changes that occur when nucleotide binds to the myosin motor. When AMPPNP binds to rigor insect flight muscle, each half sarcomere lengthens by ∼2.0 nm and tension is reduced by ∼70% without a reduction in stiffness, suggesting partial reversal of the power stroke. We have obtained averaged oblique section three-dimen sional reconstructions of mechanically monitored insect flight muscle in AMPPNP that permit simultaneous examination of all myosin crossbridges within the unit cell and direct comparison of calculated transforms with X-ray diagrams of the native fibers. Transforms calculated from the oblique section reconstruction of AMPPNP insect flight muscle at 23 °C show good agreement with native X-ray diagrams, suggesting that the average crossbridge forms in the reconstruction reflect the native structure. In contrast to the rigor lead and rear crossbridges in the double chevrons, the averaged reconstruction of AMPPNP fibers show only one crossbridge class, in the position of the rigor lead bridge. The portion of the crossbridge close to the thick filament appears broader than in rigor, and shows a small 0.5 to 1.0 nm M-ward shift of the regulatory domain region of myosin. In transverse view, AMPPNP “lead” crossbridges are less azimuthally bent than rigor. Fitting the atomic model of actomyosin subfragment 1 to the averaged crossbridges shows that the detectable differences between rigor bridges and between rigor and AMPPNP bridges occur in the alignment and angles of the regulatory domains and suggests that rear bridges are more strained than lead crossbridges. The apparent absence of rear bridges in AMPPNP in averaged reconstructions indicates detachment of a number of force-bearing bridges, which conflicts with the maintained stiffness of the fibers used for the reconstruction. This conflict may be explained if rigor rear bridges become distributed irregularly over more actin sites in AMPPNP, so that their average density is too low to appear in the averaged reconstructions. The reconstructions indicate that in insect flight muscle the response of in siturigor crossbridges to AMPPNP binding is not uniform. Lead bridges persist but have altered structure in the light chain domain, whereas rear bridges detach and possibly redistribute. Shape changes in attached myosin heads within the myofibrillar lattice are in the appropriate direction and of the appropriate magnitude needed to explain the sarcomere lengthening. This could be a direct response to nucleotide binding, a passive response to rear bridge detachment, or a combination of both.

Crossbridges in the Complete Unit Cell of Rigor Insect Flight Muscle Imaged by Three-dimensional Reconstruction from Oblique Sections

We have computed two types of 3-D reconstructions from single images of oblique transverse sections through rigor insect flight muscle (IFM) that permit simultaneous examination of all myosin crossbridges within the unit cell. One type, crystallographic serial section reconstruction (CSSR), utilizes primarily real space image manipulations of the periodic crossbridge lattice to obtain a 3-D reconstruction from a single image. The CSSRs, which do not average successive unit cells along the filament axis, reveal variations in the rigor double chevrons within the 116 nm long axial repeat and in particular show that specific crossbridges are absent. CSSRs establish that in rigor, the 116 nm period contains nine 12·9 nm repeats of attached crossbridges rather than the eight 14·5 nm repeats of myosin head origins observed in the relaxed state. This indicates that dominance of the actin repeat on myosin head form enforces axial and azimuthal changes on the crossbridge origins on the thick filament. The second type, superlattice reconstruction (SLR), is carried out entirely in Fourier space and produces air averaged reconstruction with the symmetry of the unit cell enforced. SLRs measure the 3-D transform of the complete unit, cell, permitting direct comparison with X-ray diagrams from native muscle. Averaging several SLRs together has produced the highest resolution reconstruction of IFM to date. Oblique section reconstructions made by both methods confirm in greater detail the presence of two-headed lead crossbridges and single-headed rear crossbridges implying heads with differing angles and conformation. Reduced twist in the thin filament coincident with the lead crossbridge is also apparent. We have modeled several interpretations of this reduced twist in terms of structural changes in both myosin and actin at the lead bridge. In addition, these 3-D images resolve a feature just. Z-ward of the rear crossbridge where antibody labeling has identified part of the large troponin complex of IFM.

3D reconstruction from the Fourier transform of a single superlattice image of an oblique section

An image of a thin oblique section through a 3D crystal exhibits superlattice periods much larger than the unit cell dimensions of the crystal. Within a superlattice period the contents of theunit cell of the 3D crystal are sampled at different levels, so that a 2D image of the section contains 3D information about the crystal. The 2D Fourier transform of an electron micrograph of such an oblique section thus exhibits superlattice spots, which provide an estimate of the 3D transform of the original crystal. The strengths of the observed spots are reduced from their true values by convolution with a weighting function that depends on section thickness. A method is described that uses phase relationships among symmetry-related structure factors to determine the section thickness and hence the weighting function. Wiener filter deconvolution of the section thickness is performed, in which the filter level is set by the ratio of diffraction spot intensity to background intensity. From the deconvoluted set of structure factors a 3D map of the unit cell can be computed by a standard crystallographic Fourier program. The approach is illustrated with images of oblique sections through rigor insect flight muscle.

Computation of a three dimensional image of a periodic specimen from a single view of an oblique section

We describe here a method for computing a three dimensional map of a periodic specimen from a single electron micrograph of an obliquely cut section. Neighbouring areas of such an image display successively the contents of the unit cell of the structure. The reconstruction procedure can be considered in two steps. The first step involves restacking of successive areas to produce an image akin to that produced by serial section reconstruction. The resolution normal to the section would, at this stage, be limited by the thickness of the section, since the micrograph represents a projection of the density in the section. However, because of the periodic nature of the specimen, the image contains redundant information, which can be used in an attempt to deconvolute the section thickness and thus produce improved resolution normal to the section. The computation can be carried out directly with the densities or more conveniently, particularly for three dimensional crystals, by using Fourier transforms. The approach, which is most powerful when the section is thin, is insensitive to the collapse of the section caused by electron irradiation. Striated muscle provides particularly suitable specimens for such analysis and we present, as examples, computed maps of the M-band of fish muscle and of insect flight muscle in rigor.

Three-dimensional reconstructions from single oblique sections of rigor insect flight muscle

The oblique section 3-D image reconstruction method offers some advantages over techniques which combine multiple tilt views. A complete reconstruction, limited only by section thickness and geometry, is possible from a single image. Moreover, the reconstruction is insensitive to section thinning caused by prolonged electron irradiation, and the missing cone problem due to physical restrictions in specimen tilt is overcome. A major limitation is that sections must be thin relative to the unit cell dimension running approximately normal to the section plane.

Assembly of Actin Filaments Studied by Laser Light Scattering and Fluorescence Photobleaching Recovery

Barnett, V. A., and D. D. Thomas. 1984. Saturation transfer EPR of spin-labeled muscle fibers: dependence on sarcomere length. J. Mol. Biol. 179:83-102. Cooke, R., M. S. Crowder, and D. D. Thomas. 1982. Orientation of spin-labeled myosin heads in contracting muscle fibers. Nature (Lond.). 300:776-778. Heuser, J. G. 1983. Direct visualisation of the myosin cross-bridge helices on relaxed rabbit psoas thick filaments. J. Mol Biol. 171:105-109. Huxley, H. E., and W. Brown. 1967. The low angle x-ray diagram of vertebrate striated muscle and its behaviour during contraction and rigor. J. Mol. Biol. 30:383-434. Polnaszek, C. F., and J. H. Freed. 1975. ESR studies of anisotropic ordering, spin relaxation and slow tumbling in liquid crystalline solvents. J. Phys. Chem. 79:2283-2306. Poulsen, F. R., and J. Lowy. 1983. Small-angle x-ray scattering from myosin heads in relaxed and rigor frog skeletal muscles. Nature (Lond.). 207:146-152. Taylor, K. A., M. C. Reedy, L. Cordova, and M. K. Reedy. 1984. Three-dimensional reconstruction of rigor insect flight muscle from tilted thin sections. Nature (Lond. ). 310:285-291. Thomas, D. D., and R. Cooke. 1980. Orientation of spin-labeled myosin heads in glycerinated muscle fibers. Biophys. J. 32:891-906. Thomas, D. D., S. Ishiwata, J. C. Seidel, and J. Gergely. 1980. Submillisecond rotational dynamics of spin-labeled cross-bridges in myofibrils. Biophys. J. 32:873-890.

Three-dimensional reconstruction of rigor insect flight muscle from tilted thin sections.

Rigor cross-bridges show two conformations paired within each 38.7-nm axial repeat. The two forms may express two stages of the cross-bridge cycle during contraction. Differing numbers of myosin heads per cross-bridge and associated helical changes in the thin filament distinguish the two forms and suggest that both myosin heads usually bind to a single thin filament.

Three-dimensional structure of the insect ( Lethocerus) flight muscle M-band

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.

Orientation and rotational mobility of spin-labelled myosin heads in insect flight muscle in rigor.

We have performed electron paramagnetic resonance (e.p.r.) experiments on spin-labelled myosin heads in glycerinated insect flight muscle fibres and myofibrils in rigor. Conventional e.p.r. was used to determine the orientation distribution of spin labels relative to the fibre axis, and saturation transfer e.p.r. was used to determine the submillisecond rotational mobility. An iodoacetamide spin label has previously been shown to react selectively with a reactive SH group on the myosin heads of rabbit skeletal fibres, and this label appeared to react with a similar group in insect fibres. Although selective labelling of this group was achieved in insect fibres and myofibrils, the reaction proceeded more slowly than in skeletal muscle, making it more difficult to label myosin heads selectively in insect muscle. The fraction of spin labels bound to myosin was 0.88±0.07 in insect fibres and 0.97±0.05 in rabbit. The fraction of myosin heads labelled was 0.65±0.15 for insect and 0.81±0.10 for rabbit. Both conventional and saturation transfer e.p.r. spectra of insect myosin, myofibrils and fibres were very similar to those of rabbit. The orientation distribution of spin labels relative to the fibre axis in rigor was narrow (16° for rabbit, 22° for insect), and the centre of the angular distribution was essentially the same for insect as for rabbit (68°–69°). This high degree of orientation was accompanied by strong immobilization of the probe on the microsecond time scale. The same immobilization was observed for rigor myofibrils as for purified myosin in the presence of excess actin, but considerable microsecond rotational motion was observed in myosin filaments free of actin. Thus, in insect as well as rabbit, assuming that the labelled heads are representative of all heads, more than 80% of the myosin heads appear to bind to actin in rigor, and the actomyosin bonds are rotationally rigid and oriented within a narrow angular range with respect to the fibre axis.

Arrangement of cross-bridges in insect flight muscle in rigor

We have previously proposed that in insect flight muscle in rigor, the cross-bridges are formed by the attachment of one head of a myosin molecule to one actin filament and the other head to a neighbouring filament (Offer & Elliott, 1978). We have now analysed this type of model in more detail and considered the geometrical conditions that have to be fulfilled for such two-filament interaction to be possible. We find that two labelling patterns are possible. In both, only 2 7 of the actin subunits are labelled. In one type there is an unlabelled subunit between two labelled subunits of the long-pitched strands of the actin filament; in the other type, a similar grouping occurs in one long-pitched strand, while in the other strand there is a gap of two unlabelled subunits. We have shown that if the myosin molecules on the insect thick filament are arranged on a right-handed, four-stranded helix (Wray, 1979 a,b), it is possible to find conditions where only two-filament interaction and no single-filament interaction would be expected. In this case, approximately 9 16 of the myosin heads attach, while the remainder are unattached. With other thick filament symmetries a mixture of single-filament and two-filament interactions would be expected. The two-filament interaction type of model can, with a suitable choice of actin-myosin binding geometry, explain the appearance of transverse and longitudinal sections of insect flight muscle in rigor (Reedy, 1968). The Fourier transforms of many variations of the two-filament interaction model have been calculated. We find several detailed models, differing principally in the slew angle of attachment of myosin heads and in the position of troponin, which are able to account satisfactorily for the observed X-ray diffraction patterns of this muscle in rigor (Miller & Tregear, 1972; Holmes et al., 1980).

Interpretation of the low angle X-ray diffraction from insect flight muscle in rigor

The structure of insect flight muscle is formally described in terms of actin-based cross-bridges upon which successive symmetry operations are performed, in combination with a modulation function. The Fourier transform of the structure is generated by means of these steps. The model transform is fitted to the observed diffraction pattern from insect flight muscle in rigor and the position of the rigor cross-bridges deduced; they are found to lie across the long helix of actin monomers and to project away from the thin filament. The cross-bridges interact with approximately one-third of the actin monomers, and show a strong preference for a particular orientation between the thick and thin filaments.

Can a myosin molecule bind to two actin filaments?

It is suggested that in striated muscles the two heads of one myosin molecule are able to interact with different actin filaments. This would provide a simple explanation for the appearance and arrangement of cross-bridges in insect flight muscle in rigor.

Symmetry and three-dimensional arrangement of filaments in vertebrate striated muscle

A theoretical analysis is given of the three-dimensional geometry of the arrangement of actin and myosin filaments in vertebrate striated muscle. Three notable points arise out of this analysis: 1. (i) Only one of the possible models for the symmetry of the myosin filaments, that with a three-stranded helical arrangement of myosin heads, seems to provide an obvious explanation for the occurrence of the observed myosin filament superlattice. 2. (ii) In the almost square Z-line lattice only two alternative regular arrangements of equivalent actin filaments are possible. One of these, that with all the actin filaments identically oriented, seems the more likely to be correct. The same two arrangements are also the only two actin arrangements which could fit regularly into the myosin superlattice. 3. (iii) Taking the myosin filaments to be three-stranded for reasons given in (i) and taking the possible actin arrangements to be as in (ii), then the geometry of myosin cross-bridge interactions with actin can be analysed. On this basis, by analogy with previous results from rigor insect flight muscle, it is likely that in rigor vertebrate striated muscle, only about two thirds of the myosin cross-bridges are attached to actin.

General model of myosin filament structure: II. Myosin filaments and cross-bridge interactions in vertebrate striated and insect flight muscles

Details are given of a proposed geometry of cross-bridge interaction in rigor insect flight muscle based on the general model of myosin filament structure (Squire, 1971). It is shown that on the basis of this model the symmetry of the myosin filaments in insect muscle must be that of a six-stranded helix of pitch 2310 Å and repeat 1155 Å. The flared-X appearances of cross-bridges in thin transverse sections of rigor insect muscle (Reedy, 1967) are explained in terms of the proposed geometry of interaction. The mass transference involved in the change from a relaxed to a rigor state of a vertebrate striated muscle is also considered in terms of the probable geometry of cross-bridge interaction in this muscle. It is shown that there are three types of filament symmetry which satisfy all the data available at present about myosin filaments in vertebrate striated muscle.

Myofilaments and cross bridges as demonstrated by freeze-fracturing and etching

Replicas of frozen-fractured, glycerinated, unfixed insect flight muscle in rigor were studied. Both etched and unetched fractures were examined. A clear differentiation of thick filament profiles either side of the M band in oblique fractures was seen. Transverse fractures of thick filaments revealed a differentiation between cortex and core. Etched longitudinal fractures demonstrate that thick filaments are readily split. Where thick and thin filaments lie side by side, they are linked by cross bridges at 36-nm spacings, and which lie at an angle to the filaments. Large areas of such fractures show overall linear arrays at 25°, 60°, and 90° resulting from bridge alignment. Optical transforms of these micrographs show strong off-meridional spots. Unetched longitudinal fractures of thin filaments reveal a right-handed double helix with a 33-nm repeat and rows of 10-nm diameter beads, the heads of myosin cross bridges in linear arrays with a 37-nm repeat.