Maki Yamaguchi

Invertebrate connectin spans as much as 3.5 μm in the giant sarcomeres of crayfish claw muscle

In crayfish claw closer muscle, the giant sarcomeres are 8.3 μm long at rest, four times longer than vertebrate striated muscle sarcomeres, and they are extensible up to 13 μm upon stretch. Invertebrate connectin (I‐connectin) is an elastic protein which holds the A band at the center of the sarcomere. The entire sequence of crayfish I‐connectin was predicted from cDNA sequences of 53 424 bp (17 352 residues; 1960 kDa). Crayfish I‐connectin contains two novel 68‐ and 71‐residue repeats, and also two PEVK domains and one kettin region. Kettin is a small isoform of I‐connectin. Immunoblot tests using antibody to the 68‐residue repeats revealed the presence of I‐connectin also in long sarcomeres of insect leg muscle and barnacle ventral muscle. Immunofluorescence microscopy demonstrated that the two repeats, the long spacer and the two PEVK domains contribute to sarcomere extension. These regions rich in charged amino acids, occupying 63% of the crayfish I‐connectin molecule, may allow a span of a 3.5 μm distance as a new class of composite spring.

Effects of adenosine diphosphate on the structure of myosin cross-bridges: an X-ray diffraction study on a single skinned frog muscle fibre

SummaryUsing a technique to obtain a detailed X-ray diffraction pattern from a single skinned frog muscle fibre, we studied the effects of ADP on the structure and arrangement of myosin heads. An imaging plate and a cooled-CCD X-ray detector were used to record the diffraction patterns. Addition of 1 mm ADP to a rigor fibre increased the intensity of the third-order meridional reflection of the myosin repeat by 50–85%. The intensity of the sixth-order meridional reflection also increased. After removing the ADP, these intensities decreased but did not return to the level before the ADP was added. No significant changes were observed in the intensities of the equatorial reflections and the actin layer-lines. These results suggest that, upon ADP binding, the conformation of a myosin head changes without detaching from actin. The structural change may involve a relative motion between domains of the myosin head by the closure of the cleft to which an ADP molecule binds.

Activating efficiency of Ca2+ and cross-bridges as measured by phosphate analog release.

To assess the activating efficiency of Ca2+ and cross-bridges, the release rates of phosphate analogs from skinned fibers were estimated from the recovery of contractility and that of stiffness. Estimations were performed based on the assumptions that contractility was indicative of the population of analog-free myosin heads and that stiffness reflected the population of formed cross-bridges. Aluminofluoride (AlFx) and orthovanadate (Vi) were used as phosphate analogs with mechanically skinned fibers from rabbit psoas muscle. The use of the analogs enabled the functional assessment of activation level in the total absence of ATP. Fibers loaded with the analogs gradually recovered contractility and stiffness in normal plain rigor solution. The addition of Ca2+ to the plain rigor solution significantly accelerated their recovery, whereas ADP had no appreciable effect. ATP plus Ca2+(contracting condition) accelerated the recovery by several tens of times. These results indicate that the cross-bridges formed during contraction have prominent activating efficiency, which is indispensable to attain full activation. A comparison between the activating efficiency evaluated from stiffness and that from contractility suggested that Ca2+ is more potent in accelerating the binding of actin to analog-bound myosin heads whereas cross-bridges mainly accelerate the subsequent analog-releasing step.

Protruding masticatory (superfast) myosin heads from staggered thick filaments of dog jaw muscle revealed by X-ray diffraction

To characterize the structure of jaw muscle fibres expressing masticatory (superfast) myosin, X-ray diffraction patterns of glycerinated fibres of dog masseter were compared with those of dog tibialis anterior in the relaxed state. Meridional reflections of masseter fibres were laterally broad, indicating that myosin filaments are staggered along the filament axis. Compared with tibialis anterior fibres, the peak of the first myosin layer line of masseter fibres was lower in intensity and shifted towards the meridian, while lattice spacings were larger at a similar sarcomere length. These suggest that the myosin heads of masticatory fibres are mobile, and tend to protrude from the filament shaft towards actin filaments. Lowering temperature or treating with N-phenylmaleimide shifted the peak of the first myosin layer line of tibialis anterior fibres towards the meridian and the resulting profile resembled that of masseter fibres. This suggests that the protruding mobile heads in the non-treated masticatory fibres are in the ATP-bound state. The increased population of weakly binding cross-bridges may contribute towards the high specific force of masticatory fibres during contraction. Electron micrographs confirmed the staggered alignment of thick filaments along the filament axis within sarcomeres of masticatory fibres, a feature that may confer efficient force development over a wide range of the sarcomere lengths.

Structure and Function of Masticatory (Superfast) Myosin

Abstract Masticatory (superfast) myosin is specifically expressed in jaw-closing muscles and is found in carnivorous lower vertebrates and several orders of mammals. In humans, this myosin is not expressed in those whose masticatory myosin heavy chain gene is defective due to a frame-shift deletion. This myosin has a very ancient phylogenetic origin, and is considered as a distinct subclass (labeled as II M) of vertebrate striated myosins, its heavy chain exhibiting a less than 70% sequence homology to other known striated muscle myosin heavy chains. ATPase activity of masticatory myosin is higher than that of fast myosin, and fibers expressing masticatory myosin exhibit a higher maximal tension than fast or slow fibers and intermediate shortening velocity between those of fast and slow limb fibers. These characteristics are considered very appropriate in carnivores for catching and grasping prey, and in folivores for the mastication of tough vegetable matter. Regarding the structural aspect, myosin heads of masticatory fibers are reported to be mobile and protrude more from the thick filaments toward actin by X-ray diffraction experiments at the synchrotron facility. Electron micrographs of masticatory fibres showed that the sarcomere structure of fibers is disordered and the M-line is barely discernible. These structural characteristics may explain the unique contracting properties.

X-ray diffraction analysis of the effects of myosin regulatory light chain phosphorylation and butanedione monoxime on skinned skeletal muscle fibers

The phosphorylation of the myosin regulatory light chain (RLC) is an important modulator of skeletal muscle performance and plays a key role in posttetanic potentiation and staircase potentiation of twitch contractions. The structural basis for these phenomena within the filament lattice has not been thoroughly investigated. Using a synchrotron radiation source at SPring8, we obtained X-ray diffraction patterns from skinned rabbit psoas muscle fibers before and after phosphorylation of myosin RLC in the presence of myosin light chain kinase, calmodulin, and calcium at a concentration below the threshold for tension development ([Ca(2+)] = 10(-6.8)M). After phosphorylation, the first myosin layer line slightly decreased in intensity at ∼0.05 nm(-1)along the equatorial axis, indicating a partial loss of the helical order of myosin heads along the thick filament. Concomitantly, the (1,1/1,0) intensity ratio of the equatorial reflections increased. These results provide a firm structural basis for the hypothesis that phosphorylation of myosin RLC caused the myosin heads to move away from the thick filaments towards the thin filaments, thereby enhancing the probability of interaction with actin. In contrast, 2,3-butanedione monoxime (BDM), known to inhibit contraction by impeding phosphate release from myosin, had exactly the opposite effects on meridional and equatorial reflections to those of phosphorylation. We hypothesize that these antagonistic effects are due to the acceleration of phosphate release from myosin by phosphorylation and its inhibition by BDM, the consequent shifts in crossbridge equilibria leading to opposite changes in abundance of the myosin-ADP-inorganic phosphate complex state associated with helical order of thick filaments.

Modulating factors of calcium-free contraction at low [MgATP]: a physiological study on the steady states of skinned fibres of frog skeletal muscle.

Factors that modulate Ca2+-free contraction at low [MgATP] were examined by analysing steady tension development in skinned fibres of frog skeletal muscle. The commonly accepted bell- shaped relationship between steady tension and log (1/[MgATP]) was found to be highly susceptible to subtle experimental conditions at the higher [MgATP] side (right limb). The limb shifted to the right with increased fibre thickness, interrupted stirring of the bathing solution, increased temperature and fibre extension, although the effects of temperature and extension were marked only in thick fibres (cross-sectional area >6000μm2). The shift of the right limb was reproduced by an addition of ADP to the bathing solution. These results, together with the extreme steepness of the right limb in thick fibres, suggest that a diffusion-dependent self- regenerative activation occurs in thick fibres in which ADP accumulation and ATP depletion positively feed back through further activation of the myofibrillar ATPase. Numerical simulation supported the hypothesis of the self-regenerative activation under poor diffusion conditions, and suggested that a small rise in temperature and fibre extension can trigger the self-regenerative process at the right limb. Consequently, ADP, temperature and fibre extension are deduced to be the primary potentiators of the activation at low [MgATP]. The high efficiency of ADP in shifting the limb suggests that the activating efficiency of the MgADP-bound actomyosin complex is higher than the nucleotide-free actomyosin complex.

Tension Recovery following Ramp-Shaped Release in High-Ca and Low-Ca Rigor Muscle Fibers: Evidence for the Dynamic State of AMADP Myosin Heads in the Absence of ATP.

During muscle contraction, myosin heads (M) bound to actin (A) perform power stroke associated with reaction, AMADPPi → AM + ADP + Pi. In this scheme, A • M is believed to be a high-affinity complex after removal of ATP. Biochemical studies on extracted protein samples show that, in the AM complex, actin-binding sites are located at both sides of junctional peptide between 50K and 20K segments of myosin heavy chain. Recently, we found that a monoclonal antibody (IgG) to the junctional peptide had no effect on both in vitro actin-myosin sliding and skinned muscle fiber contraction, though it covers the actin-binding sites on myosin. It follows from this that, during muscle contraction, myosin heads do not pass through the static rigor AM configuration, determined biochemically and electron microscopically using extracted protein samples. To study the nature of AM and AMADP myosin heads, actually existing in muscle, we examined mechanical responses to ramp-shaped releases (0.5% of Lo, complete in 5ms) in single skinned rabbit psoas muscle fibers in high-Ca (pCa, 4) and low-Ca (pCa, >9) rigor states. The fibers exhibited initial elastic tension drop and subsequent small but definite tension recovery to a steady level. The tension recovery was present over many minutes in high-Ca rigor fibers, while it tended to decrease quickly in low-Ca rigor fibers. EDTA (10mM, with MgCl2 removed) had no appreciable effect on the tension recovery in high-Ca rigor fibers, while it completely eliminated the tension recovery in low-Ca rigor fibers. These results suggest that the AMADP myosin heads in rigor muscle have long lifetimes and dynamic properties, which show up as the tension recovery following applied release. Possible AM linkage structure in muscle is discussed in connection with the X-ray diffraction pattern from contracting muscle, which is intermediate between resting and rigor muscles.