Electron Tomography of Cryofixed, Isometrically Contracting Insect Flight Muscle Reveals Novel Actin-Myosin Interactions

BACKGROUND: Isometric muscle contraction, where force is generated without muscle shortening, is a molecular traffic jam in which the number of actin-attached motors is maximized and all states of motor action are trapped with consequently high heterogeneity. This heterogeneity is a major limitation to deciphering myosin conformational changes in situ. METHODOLOGY: We used multivariate data analysis to group repeat segments in electron tomograms of isometrically contracting insect flight muscle, mechanically monitored, rapidly frozen, freeze substituted, and thin sectioned. Improved resolution reveals the helical arrangement of F-actin subunits in the thin filament enabling an atomic model to be built into the thin filament density independent of the myosin. Actin-myosin attachments can now be assigned as weak or strong by their motor domain orientation relative to actin. Myosin attachments were quantified everywhere along the thin filament including troponin. Strong binding myosin attachments are found on only four F-actin subunits, the "target zone", situated exactly midway between successive troponin complexes. They show an axial lever arm range of 77°/12.9 nm. The lever arm azimuthal range of strong binding attachments has a highly skewed, 127° range compared with X-ray crystallographic structures. Two types of weak actin attachments are described. One type, found exclusively in the target zone, appears to represent pre-working-stroke intermediates. The other, which contacts tropomyosin rather than actin, is positioned M-ward of the target zone, i.e. the position toward which thin filaments slide during shortening. CONCLUSION: We present a model for the weak to strong transition in the myosin ATPase cycle that incorporates azimuthal movements of the motor domain on actin. Stress/strain in the S2 domain may explain azimuthal lever arm changes in the strong binding attachments. The results support previous conclusions that the weak attachments preceding force generation are very different from strong binding attachments

Structural Changes in Isometrically Contracting Insect Flight Muscle Trapped following a Mechanical Perturbation

The application of rapidly applied length steps to actively contracting muscle is a classic method for synchronizing the response of myosin cross-bridges so that the average response of the ensemble can be measured. Alternatively, electron tomography (ET) is a technique that can report the structure of the individual members of the ensemble. We probed the structure of active myosin motors (cross-bridges) by applying 0.5% changes in length (either a stretch or a release) within 2 ms to isometrically contracting insect flight muscle (IFM) fibers followed after 5–6 ms by rapid freezing against a liquid helium cooled copper mirror. ET of freeze-substituted fibers, embedded and thin-sectioned, provides 3-D cross-bridge images, sorted by multivariate data analysis into ∼40 classes, distinct in average structure, population size and lattice distribution. Individual actin subunits are resolved facilitating quasi-atomic modeling of each class average to determine its binding strength (weak or strong) to actin. ∼98% of strong-binding acto-myosin attachments present after a length perturbation are confined to “target zones” of only two actin subunits located exactly midway between successive troponin complexes along each long-pitch helical repeat of actin. Significant changes in the types, distribution and structure of actin-myosin attachments occurred in a manner consistent with the mechanical transients. Most dramatic is near disappearance, after either length perturbation, of a class of weak-binding cross-bridges, attached within the target zone, that are highly likely to be precursors of strong-binding cross-bridges. These weak-binding cross-bridges were originally observed in isometrically contracting IFM. Their disappearance following a quick stretch or release can be explained by a recent kinetic model for muscle contraction, as behaviour consistent with their identification as precursors of strong-binding cross-bridges. The results provide a detailed model for contraction in IFM that may be applicable to contraction in other types of muscle

Cross-Bridge Number, Position, and Angle in Target Zones of Cryofixed Isometrically Active Insect Flight Muscle

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°). 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

Tomographic 3D reconstruction of quick-frozen, Ca2+ activated 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 crossbridges are usually single myosin heads, bound preferentially to actin target zones sited midway between troponins. Active attached bridges (˜30% of all heads) depart markedly in axial and azimuthal angles from Rayments 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 ˜105° to ˜70°. The results suggest the MD tilts and slews on actin from weak to strong binding, followed by swinging of the LCD through an ˜35° axial angle, giving an ˜13 nm interaction distance and an ˜4–6 nm working stroke

Summary of 2-headed & mask motif structures.

<p>Values are calculated as: (number of structures)/(number of repeats) * 100. Because a single repeat might have two 2-headed bridges, the theoretical maximum is 200% (as seen in rigor). The same argument applies to mask motifs.</p>1<p>Includes all two-headed or mask motif attachments.</p>2<p>Means both heads are strong attachments.</p>3<p>The mean lever arm axial angle of heads bound to target zone actin subunits H and I on the M-ward side compared with that on actin subunits J and K on the Z-ward side.</p>4<p>Indicates both attachments occur on target zone actins H-K.</p

Reassembled averaged repeats for str-HST.

<p>Each reassembled repeat is a combination of one left-side and one right-side primary class and if necessary, between one and four troponin bridge class averages. Three repeats are included in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039422#pone-0039422-g007" target="_blank">Figure 7</a> and two, similar in appearance to 150 and having no myosin head attachments have been removed for considerations of space. These plus the three in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039422#pone-0039422-g007" target="_blank">Figure 7</a> include all the repeats to which quasiatomic models were built. The number in the upper right hand corner is the number assigned to the repeat. These numbers are referred to in the text and correspond to one of the raw repeats. Along the top two rows are those repeats with bridging density to the Tn complex. Quasiatomic models were not built for Tn bridges. Row 2 also contains all the mask motifs. Row 3 and the first two repeats in row 4 contain all the two-headed bridges. The rest are one-headed attachments of one form or another. The last two rows and the two repeats not included contain all the repeats that have no bridging density of any kind on one or more sides. Color scheme: strong-binding cross-bridges are red, weak-binding cross-bridges are magenta. Actin subunits are light green and light blue respectively with the target zone actins colored darker shades. Tn is colored orange, TM is colored yellow.</p

Quasiatomic models built for three str- and three rls-HST reassembled repeats.

<p>The top row contains mask motifs; the bottom row contains two-headed bridges. The three rls-HST models also have troponin bridge density, which is lacking in the str-HST models. Color scheme and labeling is same as for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039422#pone-0039422-g005" target="_blank">Figures 5</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039422#pone-0039422-g006" target="_blank">6</a> except that the ELC is colored dark blue and the RLC is colored cyan. Each of these is shown as <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039422#pone.0039422.s001" target="_blank">Movies S1</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039422#pone.0039422.s002" target="_blank">S2</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039422#pone.0039422.s003" target="_blank">S3</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039422#pone.0039422.s004" target="_blank">S4</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039422#pone.0039422.s005" target="_blank">S5</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039422#pone.0039422.s006" target="_blank">S6</a>.</p