Tsuyoshi Akimoto

Direct demonstration of the cross-bridge recovery stroke in muscle thick filaments in aqueous solution by using the hydration chamber

Despite >50 years of research work since the discovery of sliding filament mechanism in muscle contraction, structural details of the coupling of cyclic cross-bridge movement to ATP hydrolysis are not yet fully understood. An example would be whether lever arm tilting on the myosin filament backbone will occur in the absence of actin. The most direct way to elucidate such movement is to record ATP-induced cross-bridge movement in hydrated thick filaments. Using the hydration chamber, with which biological specimens can be kept in an aqueous environment in an electron microscope, we have succeeded in recording ATP-induced cross-bridge movement in hydrated thick filaments consisting of rabbit skeletal muscle myosin, with gold position markers attached to the cross-bridges. The position of individual cross-bridges did not change appreciably with time in the absence of ATP, indicating stability of time-averaged cross-bridge mean position. On application of ATP, individual cross-bridges moved nearly parallel to the filament long axis. The amplitude of the ATP-induced cross-bridge movement showed a peak at 5–7.5 nm. At both sides of the filament bare region, across which the cross-bridge polarity was reversed, the cross-bridges were found to move away from, but not toward, the bare region. Application of ADP produced no appreciable cross-bridge movement. Because ATP reacts rapidly with the cross-bridges (M) to form complex (M·ADP·Pi) with an average lifetime >10 s, the observed cross-bridge movement is associated with reaction, M + ATP → M·ADP·Pi. The cross-bridges were observed to return to their initial position after exhaustion of ATP. These results constitute direct demonstration of the cross-bridge recovery stroke.

Electron microscopic evidence for the myosin head lever arm mechanism in hydrated myosin filaments using the gas environmental chamber

Muscle contraction results from an attachment-detachment cycle between the myosin heads extending from myosin filaments and the sites on actin filaments. The myosin head first attaches to actin together with the products of ATP hydrolysis, performs a power stroke associated with release of hydrolysis products, and detaches from actin upon binding with new ATP. The detached myosin head then hydrolyses ATP, and performs a recovery stroke to restore its initial position. The strokes have been suggested to result from rotation of the lever arm domain around the converter domain, while the catalytic domain remains rigid. To ascertain the validity of the lever arm hypothesis in muscle, we recorded ATP-induced movement at different regions within individual myosin heads in hydrated myosin filaments, using the gas environmental chamber attached to the electron microscope. The myosin head were position-marked with gold particles using three different site-directed antibodies. The amplitude of ATP-induced movement at the actin binding site in the catalytic domain was similar to that at the boundary between the catalytic and converter domains, but was definitely larger than that at the regulatory light chain in the lever arm domain. These results are consistent with the myosin head lever arm mechanism in muscle contraction if some assumptions are made.

Electron microscopic evidence for the thick filament interconnections associated with the catch state in the anterior byssal retractor muscle of Mytilus edulis.

The anterior byssal retractor muscle (ABRM) of a bivalve mollusc Mytilus edulis is known to exhibit catch state, i.e. a prolonged tonic contraction maintained with very little energy expenditure. Two different hypotheses have been put forward concerning the catch state; one assumes actin-myosin linkages between the thick and thin filaments that dissociate extremely slowly (linkage hypothesis), while the other postulates a load-bearing structure other than actin-myosin linkages (parallel hypothesis). We explored the possible load-bearing structure responsible for the catch state by examining the arrangement of the thick and thin filaments within the ABRM fibers, using techniques of quick freezing and freeze substitution. No thick filament aggregation was observed in the cross-section of the fibers quickly frozen not only in the relaxed and actively contracting states but also in the catch state. The thick filaments were, however, occasionally interconnected with each other either directly or by distinct projections in all the three states studied. The proportion of the interconnected thick filaments relative to the total thick filaments in a given cross-sectional area was much larger in the catch state than in the relaxed and actively contracting states, providing evidence that the thick filament interconnection is responsible for the catch state.

Electron microscopic recording of myosin head power stroke in hydrated myosin filaments.

Muscle contraction results from cyclic attachment and detachment between myosin heads and actin filaments, coupled with ATP hydrolysis. Despite extensive studies, however, the amplitude of myosin head power stroke still remains to be a mystery. Using the gas environmental chamber, we have succeeded in recording the power stroke of position-marked myosin heads in hydrated mixture of actin and myosin filaments in a nearly isometric condition, in which myosin heads do not produce gross myofilament sliding, but only stretch adjacent elastic structures. On application of ATP, individual myosin heads move by ~3.3 nm at the distal region, and by ~2.5 nm at the proximal region of myosin head catalytic domain. After exhaustion of applied ATP, individual myosin heads return towards their initial position. At low ionic strength, the amplitude of myosin head power stroke increases to >4 nm at both distal and proximal regions of myosin heads catalytic domain, being consistent with the report that the force generated by individual myosin heads in muscle fibers is enhanced at low ionic strength. The advantages of the present study over other in vitro motility assay systems, using myosin heads detached from myosin filaments, are discussed.

Structural origin of the series elastic component in horseshoe crab skeletal muscle fibers

Abstract The structural origin of the highly compliant series elastic component (SEC) in arthropod skeletal muscle was investigated with intact muscle fibers and glycerinated myofibril bundles from the telson depressor muscle of a horseshoe crab Tachypleus tridentatus . In electrically stimulated muscle fibers, the extension of SEC at the maximum isometric force P 0 was about 6% of the slack fiber length L 0 (sarcomere length, 7 μm), i.e. about 210 nm per half-sarcomere, being too large to be explained by the cross-bridge and the thin filament elasticities. High-speed cinematography of the fibers during a quick release showed uniform distribution of the SEC in each sarcomere. In Ca 2+ -activated myofibril bundles, the A-band length was observed to increase transiently by about 10% during Ca 2+ -activated contraction. The above elastic A-band lengthening in the myofibril bundles amounted to 5–6% of the initial sarcomere length in the isometric condition, being almost equal to the extension of SEC at P 0 in isometrically contracting muscle fibers. Since the A-band lengthening is known to result from misalignment of the thick filaments, these results indicate that the highly compliant SEC mostly originates from the thick filament misalignment in each A-band during isometric force generation, and titin network providing elastic restoring force.

Visualization and Recording of Structural Changes in Hydrated, Living Muscle Myofilaments using the Gas Environmental Chamber

Although more than 50 years have passed since the monumental discovery of ATP-dependent sliding filament mechanism in muscle contraction, the mechanism of cyclic attachment-detachment cycle between myosin heads extending from myosin filaments and corresponding sites on actin filaments still remains to be a matter for debate and speculation. Using the gas environmental chamber (EC) attached to an electron microscope, we have succeeded in recording images of hydrated myosin filaments, with gold particle position markers attached to individual myosin heads. In the absence of ATP, the position of individual myosin heads does not change appreciably with time, indicating stability of time-averaged myosin head mean position. On ATP application, individual myosin heads move parallel to the filament axis by ~6 nm. At both sides of the filament bare region, across which myosin head polarity is reversed, individual myosin heads move away from, but not towards, the bare region, indicating that the observed myosin head movement corresponds to the recovery stroke, associated with reaction, M + ATP → M·ADP·Pi. After exhaustion of applied ATP, individual myosin heads return towards their initial position. Recently, we have further succeeded in recording ATPinduced myosin head power stroke in the presence of actin filaments, and have found that the amplitude of power stroke in the isometric condition is ~3 nm, and increases to ~5 nm at low ionic strength, in accordance with the physiological experiments that Ca2+-activated force increases ~two fold at low ionic strength. These findings about the novel features of ATP-induced myosin head movement indicate that the EC is an extremely powerful tool, which enables us to visualize and investigate behavior of individual myosin heads in living, hydrated myosin filaments, retaining their physiological function. Finally, we emphasize that our EC work still remains to be the only attempt to investigate function of hydrated macromolecules.

Evidence for the involvement of myosin subfragment 2 in muscle contraction.

Muscle contraction results from alternate formation and breading of cross-links between the myosin head (subfragment 1; S-l), extending from the thick filament and a neighboring thin filament (Huxley A. F., 1957; Huxley H. E., 1969). The energy for contraction is supplied by ATP hydrolysis. Since the ATPase activity and actin binding site are localized in the S-l region of myosin, S-l is commonly believed to play a major role in muscle contraction. In fact, in vitro motility assay experiments have shown that S-l alone is sufficient to produce force and move actin filaments. However, the ATP-dependent actin-myosin sliding observed in the assay systems is not the same as that actually taking place in muscle.

Steady-state force–velocity relation of ATP-dependent sliding between slime mold myosin, arranged on paramyosin filaments, and algal cell actin cables

Abstract To investigate characteristics of ATP-dependent sliding of a non-muscle cell myosin, obtained from a cellular slime mold Dictyostelium discoideum , on actin filament, we prepared hybrid thick filaments, in which Dictyostelium myosin was regularly arranged around paramyosin filaments obtained from a molluscan smooth muscle. A single to a few hybrid filaments were attached to a polystyrene bead (diameter, 4.5 μ m; specific gravity, 1.5), and the filaments were made to slide on actin filament arrays (actin cables) in the internodal cell of an alga Chara corallina , mounted on the rotor of a centrifuge microscope. The filament-attached bead was observed to move with a constant velocity under a constant external load for many seconds. The steady-state force–velocity relation of Dictyostelium myosin sliding on actin cables was hyperbolic in shape except for large loads ≤0.7–0.8 P 0 , being qualitatively similar to that of skeletal muscle fibres, despite a considerable variation in the number of myosin molecules interacting with actin cables. Comparison of the P–V curves between Dictyostelium myosin and muscle myosins sliding on actin cables suggests that the time of attachment to actin in a single attachment–detachment cycle is much longer in Dictyostelium myosin than in muscle myosins.

Erratum to “Basic Properties of ATP-Induced Myosin Head Movement in Hydrated Myosin Filaments, Studied Using the Gas Environmental Chamber” [Micron 113 (2018) 48-60]

The Publisher regrets that this article is an accidental duplication of an article that has already been published in Micron Volume 113, October 2018, Pages 48-60, http://dx.doi.org/10.1016/j.micron.2018. 06.005. The duplicate article has therefore been withdrawn. The full Elsevier Policy on Article Withdrawal can be found at https://www.elsevier.com/about/our-business/policies/articlewithdrawal.

WITHDRAWN: Basic Properties of ATP-Induced Myosin Head Movement in Hydrated Myosin Filaments, Studied Using the Gas Environmental Chamber

Although more than 50 years have passed since the monumental discovery of Huxley and Hanson that muscle contraction results from relative sliding between actin and myosin filaments, coupled with ATP hydrolysis, the mechanism underlying the filament sliding still remains to be a mystery. It is generally believed that the myofilament sliding is caused by cyclic attachment-detachment between myosin heads in myosin filaments and myosin-binding sites in actin filaments. Attempts to prove the myosin head movement using techniques of X-ray diffraction and chemical probes attached to myosin heads have failed to obtain clear results because of the asynchronous nature of myosin head movement. Using the gas environmental chamber (EC) attached to an electron microscope, we succeeded in recording myosin head movement in hydrated myosin filaments, coupled with ATP hydrolysis with the following results: (1)In the absence of actin filaments, myosin heads fluctuate around a definite neutral position, so that their time-averaged position remains unchanged; (2) On ATP application, myosin heads bind with ATP to be in the charged-up state, M-ADP-Pi, and perform a recovery stroke in the direction away from the myosin filament central bare zone and stay in the post-recovery stroke position; (3) In the actin-myosin filament mixture, myosin heads form rigor linkages with actin, and bind with applied ATP to be in the charged-up state, M-ADP-Pi, and perform a power stroke in the direction towards the myosin filament bare zone, while releasing ADP and Pi to stay in the post-power stroke position; (4) In both recovery and power strokes, myosin heads in the non charged-up state return to the neutral position. These results indicate that the charged-up myosin heads decide their direction of movement without being guided by actin filaments.