Tomographic 3D reconstruction of quick-frozen, Ca2+-activated contracting insect flight muscle.

Motor actions of myosin were directly visualized by electron tomography of insect flight muscle quick-frozen during contraction. In 3D images, active cross-bridges are usually single myosin heads, bound preferentially to actin target zones sited midway between troponins. Active attached bridges (approximately 30% of all heads) depart markedly in axial and azimuthal angles from Rayment's rigor acto-S1 model, one-third requiring motor domain (MD) tilting on actin, and two-thirds keeping rigor contact with actin while the light chain domain (LCD) tilts axially from approximately 105 degrees to approximately 70 degrees. The results suggest the MD tilts and slews on actin from weak to strong binding, followed by swinging of the LCD through an approximately 35 degrees axial angle, giving an approximately 13 nm interaction distance and an approximately 4-6 nm working stroke.

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.

Experiments on rigor crossbridge action and filament sliding in insect flight muscle.

We have explored three aspects of rigor crossbridge action: 1. Under rigor conditions, slow stretching (2% per hour) of insect flight muscle (IFM) from Lethocerus causes sarcomere ruptures but never filament sliding. However, in 1 mM AMPPNP, slow stretching (5%/h) causes filament sliding but no sarcomere ruptures, although stiffness equals rigor values. Thus loaded rigor attachments in IFM show no strain relief over several hours, but near-rigor states that allow short-term strain relief indicate different grades of strongly bound bridges, and suggest approaches to annealing the rigor lattice. 2. Sarcomeres of Lethocerus flight muscle, stretched 20-60% and then rigorized, show "hybrid" crossbridge patterns, with overlap zones in rigor, but H-bands relaxed and revealing four-stranded R-hand helical thick filament structure. The sharp boundary exhibits precise phasing between relaxed and rigor arrays along each thick filament. Extrapolating one lattice into the other should allow detailed modeling of the action of each myosin head as it enters rigor. 3. The "A-(bee)-Z problem" exposes a conflict about actin rotational alignment between A-bands and Z-bands of bee IFM, raising the possibility that rigor induction might rotate actins forcefully from one pattern to the other. As Squire noted, 3-D reconstructions of Z-bands in relaxed bee IFM2) imply A-bands where actin target zones form rings rather than helices around thick filaments. However, we confirm Trombitás et al. that rigor crossbridges in bee IFM mark helically arrayed target zones. Moreover, we find that loose crossbridge interactions in relaxed bee IFM mark the same helical pattern. Thus no change of actin rotational alignment by rigor crossbridges seems necessary, but 3-D structure of IFM Z-bands should be re-evaluated regarding the apparent contradiction with A-band symmetry.

Insect crossbridges, relaxed by spin-labeled nucleotide, show well-ordered 90 degrees state by X-ray diffraction and electron microscopy, but spectra of electron paramagnetic resonance probes report disorder.

The structure of glycerinated Lethocerus insect flight muscle fibers, relaxed by spin-labeled ATP and vanadate (Vi), was examined using X-ray diffraction, electron microscopy and electron paramagnetic resonance (e.p.r.) spectra. We obtained excellent relaxation of MgATP quality as determined by mechanical criteria, using vanadate trapping of 2' spin-labeled 3' deoxyATP at 3 degree C. In rigor fibers, when the diphosphate analog is bound in the absence of Vi, the probes on myosin heads are well-ordered, in agreement with electron microscopic and X-ray patterns showing that myosin heads are ordered when attached strongly to actin. In relaxed muscle, however, e.p.r. spectra report orientational disorder of bound (Vi-trapped) spin-labeled nucleotide, while electron microscopic and X-ray patterns both show well-ordered bridges at a uniform 90 degrees angle to the filament axis. The spin-labeled nucleotide orientation is highly disordered, but not completely isotropic; the slight anisotropy observed in probe spectra is consistent with a shift of approximately 10% of probes from angles close to 0 degrees to angles close to 90 degrees. Measurements of probe mobility suggest that the interaction between probe and protein remains as tight in relaxed fibers as in rigor, and thus that the disorder in relaxed fibers arises from disorders of (or within) the protein and not from disorder of the probe relative to the protein. Fixation of the relaxed fibers with glutaraldehyde did not alter any aspect of the spectrum of the Vi-trapped analog, including the slight order observed, showing that the extensive inter- and intra-molecular cross-linking of the first step of sample preparation for electron microscopy had not altered relaxed crossbridge orientations. Two models that may reconcile the apparently disparate results obtained on relaxed fibers are presented: (1) a rigid myosin head could possess considerable disorder in the regular array about the thick filament; or (2) the nucleotide site could be on a disordered, probably distal, domain of myosin, while a more proximal region is well ordered on the thick filament backbone. Our findings suggest that when e.p.r. probes signal disorder of a local site or domain, this is complementary, not contradictory, to signals of general order. The e.p.r. spectra show that a portion of the myosin molecule can be disordered at the same time as the X-ray diffraction and electron microscopy show the bulk of myosin head mass to be uniformly oriented and regularly arrayed.

Connecting filaments, core filaments, and side-struts: a proposal to add three new load-bearing structures to the sliding filament model.

This report concerns structural forces in resting muscle and proposes three additions to the sliding filament model to account for these mechanical properties. The proposal includes: connecting filaments (C-filaments) which connect the ends of each thick filament to the neighboring Z-lines, core filaments which support the myosin of the thick filament and which attach to the C-filaments, and side-struts which bind the thick filaments together along their length and restrict their radial movement. C-filaments would act as the parallel elastic element and transmit the passive tension to the thick filaments. Isolated myofibrils (mechanically-skinned and detergent-treated frog semitendinosus fibers) when stretched progressively showed exponentially-increasing passive tension which did not disappear when filament overlap was exceeded, but continued to rise. SL was monitored with a HeNe laser. Passive tension phasically exceeded 3 X 10(5) N/M2. Electron microscopy (thin-sectioned and freeze-fracture/deep-etch specimens) of non-overlap fibers showed orderly fibril structure with clear separation of A- and I-bands. In the gap between them could be seen filaments, 40-50 A in diameter, connected to the thick filament ends. Unlike actin, these filaments did not become decorated by myosin S-1. Equatorial X-ray measurements showed that stretching relaxed skinned muscles squeezed the thick filaments closer; this radial compression continued beyond filament overlap. Extreme stretch of fibers caused the thick filaments to strain several-fold. Treatment of non-overlap fibers with a high ionic strength pyrophosphate myosin solvent caused a large drop in passive tension and stiffness, but no change in SL was detected nor was myofibril continuity detectably affected. Non-overlap fibrils, when treated with elastase, released A-segments which retain three-dimensional coherency . Deep-etch EM's of non-overlap fibers disclosed abundant structures (about 75 A) wide attaching adjacent thick filaments.

A-band mass exceeds mass of its filament components by 30-45%.

We have measured filament lattice spacing in fibrils using X-ray diffraction, and find that STEM-determined mass/length values reported for myofilaments should give 13% w/v as the filament protein concentration in the lattice of the AO-band (filament overlap zone) of both insect flight muscle (IFM) and vertebrate skeletal muscle ( VSkM ). This is well below the actual mass concentration of AO-bands as measured by immersion refractometry of detergent-washed rigor myofibrils under the phase microscope. This technique identifies an immersion fluid of suitable refractive index (RI) for matching out all image contrast between background and the selected cross-band. We used RI fluids in which the effective RI matching component is a large-particle solute (Percoll or hemocyanin) excluded by the filament lattice. The measured RI indicates that protein concentration in AO-bands is 16-17% in Lethocerus and other IFM fibrils including CAF-digested fibrils, and is 18-19% in rabbit VSkM fibrils. On IFM fibrils we also measured absolute buoyant density in Percoll as greater than or equal to 1.042; this supports the value for mass concentration as determined by RI. The mass discrepancy between fibrils and filaments does not seem to arise from faults in the methods used. We therefore accept the STEM-determined mass/length values for thick filaments which indicate 4 myosins/crown in IFM and 3 in VSkM , and we believe there is considerable extra nonfilament material (concentration: 30-50 mg/ml) between the filaments in fibrils. In stretched VSkM , it is the H-bands, not the I-bands, which have an excess over filament mass content. The extra mass has not been identified.