The conformation of myosin head domains in rigor muscle determined by X-ray interference.

In the absence of adenosine triphosphate, the head domains of myosin cross-bridges in muscle bind to actin filaments in a rigor conformation that is expected to mimic that following the working stroke during active contraction. We used x-ray interference between the two head arrays in opposite halves of each myosin filament to determine the rigor head conformation in single fibers from frog skeletal muscle. During isometric contraction (force T(0)), the interference effect splits the M3 x-ray reflection from the axial repeat of the heads into two peaks with relative intensity (higher angle/lower angle peak) 0.76. In demembranated fibers in rigor at low force (<0.05 T(0)), the relative intensity was 4.0, showing that the center of mass of the heads had moved 4.5 nm closer to the midpoint of the myosin filament. When rigor fibers were stretched, increasing the force to 0.55 T(0), the heads' center of mass moved back by 1.1-1.6 nm. These motions can be explained by tilting of the light chain domain of the head so that the mean angle between the Cys(707)-Lys(843) vector and the filament axis increases by approximately 36 degrees between isometric contraction and low-force rigor, and decreases by 7-10 degrees when the rigor fiber is stretched to 0.55 T(0).

Fluorescence localization after photobleaching (FLAP): a new method for studying protein dynamics in living cells.

FLAP is a new method for localized photo-labelling and subsequent tracking of specific molecules within living cells. It is simple in principle, easy to implement and has a wide potential application. The molecule to be located carries two fluorophores: one to be photobleached and the other to act as a reference label. Unlike the related methods of fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP), the use of a reference fluorophore permits the distribution of the photo-labelled molecules themselves to be tracked by simple image differencing. In effect, FLAP is therefore comparable with methods of photoactivation. Its chief advantage over the method of caged fluorescent probes is that it can be used to track chimaeric fluorescent proteins directly expressed by the cells. Although methods are being developed to track fluorescent proteins by direct photoactivation, these still have serious drawbacks. In order to demonstrate FLAP, we have used nuclear microinjection of cDNA fusion constructs of beta-actin with yellow (YFP) and cyan (CFP) fluorescent proteins to follow both the fast relocation dynamics of monomeric (globular) G-actin and the much slower dynamics of filamentous F-actin simultaneously in living cells.

Conformation of the myosin motor during force generation in skeletal muscle.

Myosin motors drive muscle contraction, cytokinesis and cell locomotion, and members of the myosin superfamily have been implicated in an increasingly diverse range of cell functions. Myosin can displace a bound actin filament several nanometers in a single interaction. Crystallographic studies suggest that this 'working stroke' involves bending of the myosin head between its light chain and catalytic domains. Here we used X-ray fiber diffraction to test the crystallographic model and measure the interdomain bending during force generation in an intact single muscle fiber. The observed bending has two components: an elastic distortion and an active rotation that generates force. The average bend of the force-generating myosin heads in a muscle fiber is intermediate between those in crystal structures with different bound nucleotides, and the C-terminus of the head is displaced by 7 nm along the actin filament axis compared with the in vitro conformation seen in the absence of nucleotide.

Interference fine structure and sarcomere length dependence of the axial x-ray pattern from active single muscle fibers.

Axial x-ray diffraction patterns from single intact fibers of frog skeletal muscle were recorded by using a highly collimated x-ray beam at the European Synchrotron Radiation Facility. During isometric contraction at sarcomere lengths 2.2-3.2 microm, the M3 x-ray reflection, associated with the repeat of myosin heads along the filaments, was resolved into two peaks. The total M3 intensity decreased linearly with increasing sarcomere length and was directly proportional to the degree of overlap between myosin and actin filaments, showing that it comes from myosin heads in the overlap region. The separation between the M3 peaks was smaller at longer sarcomere length and was quantitatively explained by x-ray interference between myosin heads in the two overlap regions of each sarcomere. The relative intensity of the M3 peaks was independent of sarcomere length, showing that the axial periodicities of the nonoverlap and overlap regions of the myosin filament have the same value, 14.57 nm, during active contraction. In resting fibers the periodicity is 14.34 nm, so muscle activation produces a change in myosin filament structure in the nonoverlap as well as the overlap part of the filament. The results establish x-ray interferometry as a new tool for studying the motions of myosin heads during muscle contraction with unprecedented spatial resolution.

Changes in conformation of myosin heads during the development of isometric contraction and rapid shortening in single frog muscle fibres.

1. Two-dimensional X-ray diffraction patterns were recorded at the European Synchrotron Radiation Facility from central segments of intact single muscle fibres of Rana temporaria with 5 ms time resolution during the development of isometric contraction. Shortening at ca 0.8 times the maximum velocity was also imposed at the isometric tetanus plateau. 2. The first myosin-based layer line (ML1) and the second myosin-based meridional reflection (M2), which are both strong in resting muscle, were completely abolished at the plateau of the isometric tetanus. The third myosin-based meridional reflection (M3), arising from the axial repeat of the myosin heads along the filaments, remained intense but its spacing changed from 14.34 to 14.56 nm. The intensity change of the M3 reflection, IM3, could be explained as the sum of two components, I14.34 and I14.56, arising from myosin head conformations characteristic of rest and isometric contraction, respectively. 3. The amplitudes (A) of the X-ray reflections, which are proportional to the fraction of myosin heads in each conformation, changed with half-times that were similar to that of isometric force development, which was 33.5 +/- 2. 0 ms (mean +/- s.d., 224 tetani from three fibres, 4 C), measured from the end of the latent period. We conclude that the myosin head conformation changes synchronously with force development, at least within the 5 ms time resolution of these measurements. 4. The changes in the X-ray reflections during rapid shortening have two temporal components. The rapid decrease in intensity of the 14.56 nm reflection at the start of shortening is likely to be due to tilting of myosin heads attached to actin. The slower changes in the other reflections were consistent with a return to the resting conformation of the myosin heads that was about 60 % complete after shortening of 70 nm per half-sarcomere.

Elastic bending and active tilting of myosin heads during muscle contraction.

Muscle contraction is driven by a change in shape of the myosin head region that links the actin and myosin filaments. Tilting of the light-chain domain of the head with respect to its actin-bound catalytic domain is thought to be coupled to the ATPase cycle. Here, using X-ray diffraction and mechanical data from isolated muscle fibres, we characterize an elastic bending of the heads that is independent of the presence of ATP. Together, the tilting and bending motions can explain force generation in isometric muscle, when filament sliding is prevented. The elastic strain in the head is 2.0-2.7 nm under these conditions, contributing 40-50% of the compliance of the muscle sarcomere. We present an atomic model for changes in head conformation that accurately reproduces the changes in the X-ray diffraction pattern seen when rapid length changes are applied to muscle fibres both in active contraction and in the absence of ATP. The model predictions are relatively independent of which parts of the head are assumed to bend or tilt, but depend critically on the measured values of filament sliding and elastic strain.

The stiffness of skeletal muscle in isometric contraction and rigor: the fraction of myosin heads bound to actin.

Step changes in length (between -3 and +5 nm per half-sarcomere) were imposed on isolated muscle fibers at the plateau of an isometric tetanus (tension T0) and on the same fibers in rigor after permeabilization of the sarcolemma, to determine stiffness of the half-sarcomere in the two conditions. To identify the contribution of actin filaments to the total half-sarcomere compliance (C), measurements were made at sarcomere lengths between 2.00 and 2.15 microm, where the number of myosin cross-bridges in the region of overlap between the myosin filament and the actin filament remains constant, and only the length of the nonoverlapped region of the actin filament changes with sarcomere length. At 2.1 microm sarcomere length, C was 3.9 nm T0(-1) in active isometric contraction and 2.6 nm T0(-1) in rigor. The actin filament compliance, estimated from the slope of the relation between C and sarcomere length, was 2.3 nm microm(-1) T0(-1). Recent x-ray diffraction experiments suggest that the myosin filament compliance is 1.3 nm microm(-1) T0(-1). With these values for filament compliance, the difference in half-sarcomere compliance between isometric contraction and rigor indicates that the fraction of myosin cross-bridges attached to actin in isometric contraction is not larger than 0.43, assuming that cross-bridge elasticity is the same in isometric contraction and rigor.

Myosin head movements during isometric contraction studied by X-ray diffraction of single frog muscle fibres.

Time resolved X-ray diffraction experiments in single muscle fibres of the frog at 2.15 microns sarcomere length and 4 degrees C were performed at ID2 (SAXS), ESRF, Grenoble (France) to investigate the structural aspects of cross-bridge action during the development of the isometric tetanic tension (T0). Changes in the low angle myosin-based reflections were measured with 5 ms time resolution by signal averaging data collected with a 10 m camera length and a 2D gas-filled detector. Upon activation the intensity of the first order myosin layer line reflection, I(M1), and the intensity of the second order meridional reflection, I(M2), reduced practically to zero with a half-time which leads the tension rise by 15-20 ms. The complex changes of the intensity of the third order myosin meridional reflection, I(M3), and the increase of its axial spacing from 14.34 nm (at rest) to 14.57 nm (at T0) could be analysed by assuming that they were the result of the combination of the time dependent modulation in intensity of two closely spaced periodicities, one at 14.34 nm, characteristics of the myosin molecule at rest and the other at 14.57 nm, assumed by the myosin as a consequence of the activation and force production. I(14.34) drops monotonically in advance to isometric tension development with a half-time similar to that of I(M1) and I(M2), while I(14.57) rises from zero to a maximum in parallel with tension.

Elastic distortion of myosin heads and repriming of the working stroke in muscle.

Muscle contraction is driven by a cyclical interaction between the globular head domain of myosin and the actin filaments. We used quick stretches of 5 nm per half sarcomere to synchronize the movements of myosin heads in active single muscle fibres. The intensity of the 14.5 nm X-ray reflection decreased during the stretch, showing that the instantaneous elasticity of muscle involves distortion of myosin heads. Head movement continued at about 1,500 s-1 after the stretch, accompanied by partial force recovery. This indicates a reversal of the force-generating 'working stroke' in the myosin heads that is smaller and faster than assumed previously. By 50 ms after the stretch, myosin heads have regained both their original conformation and the ability to execute a normal working stroke. This 'repriming' process is slower than that following shortening but much faster than the ATP turnover rate per myosin head.

Changes in the x-ray diffraction pattern from single, intact muscle fibers produced by rapid shortening and stretch.

Changes in the x-ray diffraction patterns produced by 100-microseconds-length steps imposed during tetanic stimulation were recorded from single intact fibers of frog tibialis anterior muscle. For shortening steps, a staircase length change was applied, with a 20-ms interval between steps. For stretches, each 20-ms cycle started with a stretch, followed after 4 ms by shortening to the original length. Each shortening step in the staircase and each stretch in the stretch/shortening protocol produced a response similar to that of a single step from the isometric steady state. The intensity of the 14.5-nm x-ray reflection arising from the axial repeat of the myosin heads along their filaments decreased after both shortening and stretch; this decrease was not accompanied by broadening along or across the meridian. The relationship between the intensity after the length step and step amplitude was approximately linear for both stretches and shortening steps, extrapolating to zero intensity for 11-nm stretches and 13-nm shortening steps, but there was no significant intensity change for the first approximately 2 nm of shortening. These results are broadly consistent with conventional models of muscle contraction in which myosin heads move through about 10 nm during the working stroke in the shortening direction, but an additional distortion of the myosin heads may be produced by a stretch.