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

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

Summary of fiber mechanics of iso-, str- and rls-HST.

1<p>from reference <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039422#pone.0039422-Taylor1" target="_blank">[22]</a>.</p>2<p>A repeat represents a 38.7 nm length of the thin filament.</p>3<p>Includes out-of-target-zone attachments on actin subunit G.</p>4<p>Based on 5.7×10⁸ thick filaments/fiber cross section and 7.1 myosin heads per thin filament half repeat <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039422#pone.0039422-Taylor1" target="_blank">[22]</a>.</p

Distribution of cross-bridges for each actin subunit in the 38.7 nm axial period.

<p>The actin subunit designations are the same as those used for iso-HST <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039422#pone.0039422-Wu2" target="_blank">[25]</a>. Occupancy is given as a frequency, which means the total number of myosin heads, both weak and strong-binding, is divided by the total number of repeats in the data set for each state. On the right side are the occupancies for strong-binding attachments; on the left side are the occupancies for weak-binding attachments. Actin subunits H-K are target zone subunits; actin subunits R and S are bound to the Tn head complex. Orientation has Z-line at the bottom, M-line at the top. (A) iso-HST; (B) str-HST; (C) rls-HST.</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

Typical mechanical traces.

<p>(A) Following a quick release of 4.5 nm/half sarcomere from isometric tension. (B) Following a quick stretch of 3 nm/half sarcomere from isometric tension. Adapted from reference <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039422#pone.0039422-Ford1" target="_blank">[2]</a>.</p

Projection images from each HST state.

<p>(A-C) Projection images of regions from each tomogram. (A) iso-HST state; (B) rls-HST;(C) str-HST. (D-F) Projection images from 15 reassembled primary mask class averages from each state. (D) iso-HST state; (E) rls-HST; (F) str-HST. In D-F, one mask motif structure has been outlined in each panel. Paired brackets show the location of the actin target zone; black arrows the Tn complex and white arrowheads the myosin head origins. Orientation has Z-line at the bottom, M-line at the top. Bar in the top panels is 50 nm.</p

Composite views of weak-binding cross-bridge models.

<p>Axial views are oriented with Z-band at the bottom; azimuthal views are looking down the filament towards the Z-band. All weak-binding models were built starting from the scallop transition state structure (magenta) docked in the strong-binding configuration. In all panels, gold  =  str-HST, gray  =  rls-HST. (A) Weak-binding cross-bridge models superimposed onto the scallop MD. (B) Weak-binding models superimposed onto actin subunit I. The largest azimuthal MD displacements for pre-stroke and TM-bridges (filled and open arrows, respectively) were both found in str-HST.</p

Angular ranges of lever arm angles for target zone bridges.

<p>iso-, str- and rls-HST are shown separately as well as the combined data from all three states. Panels A, C, E and G show axial lever arm angles, computed from the projection of the lever arm axis onto the fiber axis. Panels B, D, F and H show azimuthal lever arm angles after all primary mask class averages are transformed to thin filament actin subunit I. Sketches in the upper left hand of A and B show the angle convention. Vertical lines represent the initial structures used for the model building, red for rigor acto-S1 and magenta for the scallop transition state docked onto actin in the strong-binding configuration. Percentage value is calculated as: (number of attachments of this type in this range)/(total number of attachments of this type in this state) * 100. In this convention, the axial lever arm angle of the Holmes S1 structure is 70.5° and of the scallop transition state structure, 107°.</p