Robert J. Perz-Edwards

X-ray diffraction evidence for myosin-troponin connections and tropomyosin movement during stretch activation of insect flight muscle

Stretch activation is important in the mechanical properties of vertebrate cardiac muscle and essential to the flight muscles of most insects. Despite decades of investigation, the underlying molecular mechanism of stretch activation is unknown. We investigated the role of recently observed connections between myosin and troponin, called “troponin bridges,” by analyzing real-time X-ray diffraction “movies” from sinusoidally stretch-activated Lethocerus muscles. Observed changes in X-ray reflections arising from myosin heads, actin filaments, troponin, and tropomyosin were consistent with the hypothesis that troponin bridges are the key agent of mechanical signal transduction. The time-resolved sequence of molecular changes suggests a mechanism for stretch activation, in which troponin bridges mechanically tug tropomyosin aside to relieve tropomyosin’s steric blocking of myosin–actin binding. This enables subsequent force production, with cross-bridge targeting further enhanced by stretch-induced lattice compression and thick-filament twisting. Similar linkages may operate in other muscle systems, such as mammalian cardiac muscle, where stretch activation is thought to aid in cardiac ejection.

Reverse actin sliding triggers strong myosin binding that moves tropomyosin

Actin/myosin interactions in vertebrate striated muscles are believed to be regulated by the “steric blocking” mechanism whereby the binding of calcium to the troponin complex allows tropomyosin (TM) to change position on actin, acting as a molecular switch that blocks or allows myosin heads to interact with actin. Movement of TM during activation is initiated by interaction of Ca2+ with troponin, then completed by further displacement by strong binding cross-bridges. We report x-ray evidence that TM in insect flight muscle (IFM) moves in a manner consistent with the steric blocking mechanism. We find that both isometric contraction, at high [Ca2+], and stretch activation, at lower [Ca2+], develop similarly high x-ray intensities on the IFM fourth actin layer line because of TM movement, coinciding with x-ray signals of strong-binding cross-bridge attachment to helically favored “actin target zones.” Vanadate (Vi), a phosphate analog that inhibits active cross-bridge cycling, abolishes all active force in IFM, allowing high [Ca2+] to elicit initial TM movement without cross-bridge attachment or other changes from relaxed structure. However, when stretched in high [Ca2+], Vi-“paralyzed” fibers produce force substantially above passive response at pCa ∼ 9, concurrent with full conversion from resting to active x-ray pattern, including x-ray signals of cross-bridge strong-binding and TM movement. This argues that myosin heads can be recruited as strong-binding “brakes” by backward-sliding, calcium-activated thin filaments, and are as effective in moving TM as actively force-producing cross-bridges. Such recruitment of myosin as brakes may be the major mechanism resisting extension during lengthening contractions.

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.

Myosin S2 Origins Track Evolution of Strong Binding on Actin by Azimuthal Rolling of Motor Domain

Myosin crystal structures have given rise to the swinging lever arm hypothesis, which predicts a large axial tilt of the lever arm domain during the actin-attached working stroke. Previous work imaging the working stroke in actively contracting, fast-frozen Lethocerus muscle confirmed the axial tilt; but strongly bound myosin heads also showed an unexpected azimuthal slew of the lever arm around the thin filament axis, which was not predicted from known crystal structures. We hypothesized that an azimuthal reorientation of the myosin motor domain on actin during the weak-binding to strong-binding transition could explain the lever arm slew provided that myosins α-helical coiled-coil subfragment 2 (S2) domain emerged from the thick filament backbone at a particular location. However, previous studies did not adequately resolve the S2 domain. Here we used electron tomography of rigor muscle swollen by low ionic strength to pull S2 clear of the thick filament backbone, thereby revealing the azimuth of its point of origin. The results show that the azimuth of S2 origins of those rigor myosin heads, bound to the actin target zone of actively contracting muscle, originate from a restricted region of the thick filament. This requires an azimuthal reorientation of the motor domain on actin during the weak to strong transition.

The basketweave form of the Z-band is expanded relative to the small-square form.

A review of the vertebrate muscle Z-disk (Luther 2009) contains a small but critical error. In discussing the transverse appearance of the Z-band (pp. 175–176) the author states that the transformation from small-square to basketweave form is accompanied by a 20% reduction in lattice spacing, which the author also represents visually with schematic diagrams in Fig. 5c and d. Unfortunately, that is exactly backwards. The results of Goldstein et al. (1986, 1987, 1989) indicate that the basketweave form is expanded by 20%, relative to the small-square form, as can be appreciated readily from the electron micrographs in Fig. 5a and b of the review. With the correct view of the lattice spacing changes in mind, the transition between the small-square and basketweave forms can be more easily understood, as shown in the diagram below. The models, based on three-dimensional reconstructions from the Squire lab (Luther et al. 2002; Morris et al. 1990), show parallel actin filaments (light grey) from one sarcomere with their neighboring, oppositely oriented, parallel filaments (dark grey) from the adjacent sarcomere. Each dark-light grey, anti-parallel pair is connected by a Z-link (black). In the full Z-band structure, Z-links are repeated with 4-fold screw symmetry along each filament, but for clarity only two Z-links are shown in the diagram. Each Z-link is presumably composed of an a-actinin homodimer (Luther 2009). In the relaxed, small-square form (a), the smaller lattice spacing causes the Z-links to kink sharply so that they are close to, or touching one another in the central portion of Z-link. Side to side association between a-actinin dimers have been previously observed by negative stain (Suzuki et al. 1976), and a-actinin is well known to self-associate and form aggregates (Masaki and Takaiti 1969). In the activated, basketweave form (b), the larger lattice spacing allows the Z-links to curve smoothly from one filament to the adjacent one. Although for simplicity I have described the lattice spacing change as driving the conformational change in the Z-links, as has been previously suggested (Yamaguchi et al. 1985; Goldstein et al. 1986), the reverse is equally plausible, that a conformational change in the Z-link drives the lattice spacing change, and at present one can only say that the lattice form and spacing are correlated. The significance of the structural transition in the Z-band is but one unanswered question among many

Electron Tomography of Thick Sections of Insect Flight Muscle

Insect flight muscle (IFM) is a good model system within which to visualize actin-myosin interactions due to its highly ordered lattice of actin and myosin filaments. Lethocerus flight muscle is perhaps the best ordered muscle in nature. Electron tomography (ET) of Lethocerus IFM has recently resulted in a model for the weak to strong transition that incorporates large azimuthal changes in the position of the lever arm compared to that predicted from crystal structures of myosin subfragment 1 in both the nucleotide free and transition states (Wu et al. PLoS-ONE, Sept. 2010). Those studies did not visualize the S2 domain in either the raw tomogram or in subvolume averages which would clarify the crossbridge origin. Here we have used ET of IFM fibers in rigor in which the filament lattice has been swollen in low ionic strength buffer to view where S2 emerges from the thick filament backbone as a test of the weak to strong transition. Previous ET on myac layers (single filament layers containing alternating myosin and actin filaments) of these same swollen rigor fibers revealed the S2 domain with clarity. In the present work, we are examining 80 nm thick transverse and longitudinal sections of swollen rigor IFM fibers in order to visualize all of the crossbridges originating from each 14.5 nm crown on the thick filament, but especially the so-called lead bridges, which bind the thin filament within the same target zone of isometric contraction. Class averages of both thick filaments as well as myac layers are being pursued. The thick filaments show subfilaments in the backbone and many of the myac layer raw repeat subvolumes show S2. Progress on this study will be presented. Supported by NIGMS and NIAMSD.