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Shigeru Chaen - One of the best experts on this subject based on the ideXlab platform.
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basic properties of atp induced Myosin Head movement in hydrated Myosin filaments studied using the gas environmental chamber
Micron, 2018Co-Authors: Haruo Sugi, Tsuyoshi Akimoto, Shigeru ChaenAbstract: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.
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Electron Microscopic Recording of the Power and Recovery Strokes of Individual Myosin Heads Coupled with ATP Hydrolysis: Facts and Implications
MDPI AG, 2018Co-Authors: Haruo Sugi, Shigeru Chaen, Tsuyoshi AkimotoAbstract:The most straightforward way to get information on the performance of individual Myosin Heads producing muscle contraction may be to record their movement, coupled with ATP hydrolysis, electron-microscopically using the gas environmental chamber (EC). The EC enables us to visualize and record ATP-induced Myosin Head movement in hydrated skeletal muscle Myosin filaments. When actin filaments are absent, Myosin Heads fluctuate around a definite neutral position, so that their time-averaged mean position remains unchanged. On application of ATP, Myosin Heads are found to move away from, but not towards, the bare region, indicating that Myosin Heads perform a recovery stroke (average amplitude, 6 nm). After exhaustion of ATP, Myosin Heads return to their neutral position. In the actin–Myosin filament mixture, Myosin Heads form rigor actin Myosin linkages, and on application of ATP, they perform a power stroke by stretching adjacent elastic structures because of a limited amount of applied ATP ≤ 10 µM. The average amplitude of the power stroke is 3.3 nm and 2.5 nm at the distal and the proximal regions of the Myosin Head catalytic domain (CAD), respectively. The power stroke amplitude increases appreciably at low ionic strength, which is known to enhance Ca2+-activated force in muscle. In both the power and recovery strokes, Myosin Heads return to their neutral position after exhaustion of ATP
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Electron microscopic recording of Myosin Head power stroke in hydrated Myosin filaments.
Scientific Reports, 2015Co-Authors: Haruo Sugi, Hiroki Minoda, Takuya Miyakawa, Yumiko Miyauchi, Masaru Tanokura, Tsuyoshi Akimoto, Shigeru Chaen, Seiryo SugiuraAbstract: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.
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definite differences between in vitro actin Myosin sliding and muscle contraction as revealed using antibodies to Myosin Head
PLOS ONE, 2014Co-Authors: Shigeru Chaen, Takuya Miyakawa, Masaru Tanokura, Yasutake Saeki, Takakazu Kobayashi, Takahiro Abe, Yoshiki Ohnuki, Kazushige Kimura, Seiryo SugiuraAbstract:Muscle contraction results from attachment-detachment cycles between Myosin Heads extending from Myosin filaments and actin filaments. It is generally believed that a Myosin Head first attaches to actin, undergoes conformational changes to produce force and motion in muscle, and then detaches from actin. Despite extensive studies, the molecular mechanism of Myosin Head conformational changes still remains to be a matter for debate and speculation. The Myosin Head consists of catalytic (CAD), converter (CVD) and lever arm (LD) domains. To give information about the role of these domains in the Myosin Head performance, we have examined the effect of three site-directed antibodies to the Myosin Head on in vitro ATP-dependent actin-Myosin sliding and Ca2+-activated contraction of muscle fibers. Antibody 1, attaching to junctional peptide between 50K and 20K heavy chain segments in the CAD, exhibited appreciable effects neither on in vitro actin-Myosin sliding nor muscle fiber contraction. Since antibody 1 covers actin-binding sites of the CAD, one interpretation of this result is that rigor actin-Myosin linkage is absent or at most a transient intermediate in physiological actin-Myosin cycling. Antibody 2, attaching to reactive lysine residue in the CVD, showed a marked inhibitory effect on in vitro actin-Myosin sliding without changing actin-activated Myosin Head (S1) ATPase activity, while it showed no appreciable effect on muscle contraction. Antibody 3, attaching to two peptides of regulatory light chains in the LD, had no significant effect on in vitro actin-Myosin sliding, while it reduced force development in muscle fibers without changing MgATPase activity. The above definite differences in the effect of antibodies 2 and 3 between in vitro actin-Myosin sliding and muscle contraction can be explained by difference in experimental conditions; in the former, Myosin Heads are randomly oriented on a glass surface, while in the latter Myosin Heads are regularly arranged within filament-lattice structures.
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Effect of Antibodies to Myosin Head Reveals Definite Difference between In Vitro Actin-Myosin Sliding and Muscle Contraction
Biophysical Journal, 2013Co-Authors: Takahiro Abe, Takuya Miyakawa, Takakazu Kobayashi, Shigeru Chaen, Suguru Tanokura, Yoshiki OhnukiAbstract:Mechanism of Myosin Head power stroke, responsible for muscle contraction, still remains to be a matter of debate and speculation. Despite considerable progress in studying actin filament sliding over Myosin fixed on a glass surface, it is not clear whether the in vitro actin-Myosin sliding takes place by a mechanism similar to contraction in muscle, consisting of three-dimensional myofilament lattice structures. To make this point clear, we prepared two different monoclonal antibodies, one directed to reactive lysine residue close to the Myosin Head converter region (anti-RLR antibody) while the other directed to two peptides of regulatory light chain in the Myosin Head lever arm region (anti-LD antibody). We compared the effect of these antibodies on in vitro actin- Myosin sliding and contraction of skinned rabbit psoas muscle fibers with the following results: (1) anti-RLR antibody completely inhibited in vitro actin-Myosin sliding without changing actin-activated Myosin Head ATPase activity, while it showed no effect on Ca2+-activated contraction of muscle fibers; (2) anti-LD antibody had no effect on in vitro actin-Myosin sliding, but suppressed Ca2+-activated muscle fiber contraction without changing Mg-ATPase activity. These results indicate definite difference between in vitro actin-Myosin sliding and muscle contraction.
Dmitrii I Levitsky - One of the best experts on this subject based on the ideXlab platform.
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the effect of gly126arg substitution in alpha tropoMyosin on interaction of Myosin with actin in the atp hydrolysis cycle
Cell and Tissue Biology, 2018Co-Authors: Nikita A Rysev, I A Nevzorov, S V Avrova, Dmitrii I Levitsky, Yu. S. BorovikovAbstract:It is known that the regulation of muscle contraction is carried out by tropoMyosin and calcium-sensitive protein troponin, which form thin filament with the F-actin. The noncanonical glycine residue at position 126 of the central part of the skeletal alpha-tropoMyosin destabilizes the structure of this protein. The substitution of glycine residue by arginine residue stabilizes the central region of tropoMyosin, displaces tropoMyosin to the open position and activates the switching actin monomers on during the ATP hydrolysis cycle. To investigate how Gly126Arg substitution affects the interaction of the Myosin Head with F-actin in the ATP hydrolysis cycle, the Myosin subfragment-1 (S1) was modified with a 1,5-IAEDANS fluorescent probe and AEDANS-S1 was incorporated into the ghost muscle fiber. Multistage changes in the mobility and spatial organization of the Myosin Head during simulation of different stages of the ATP hydrolysis cycle were studied by polarization fluorescence microscopy. It was shown that, in the regulated thin filaments of the ghost muscle fiber at high concentrations of Ca2+, Gly126Arg substitution significantly increases the number of Myosin Heads strongly associated with F-actin when simulating strong binding of Myosin to actin, but reduces the number of such Heads when imitating weak binding of Myosin. Such changes in the behavior of Myosin in the ATP hydrolysis cycle indicate an increase in the efficiency of Myosin cross-bridges. A significant increase in the relative amount of Myosin strongly bound to actin was also observed at low Са2+ concentrations. This indicates an increase in Са2+-sensitivity of a thin filament initiated by Gly126Arg substitution. The obtained data suggest that the stabilization effects of the central part of tropoMyosin by Gly126Arg substitution are realized through the abnormal behavior of tropoMyosin and troponin, which leads to a change in the nature of the interaction of Myosin with actin and tropoMyosin in the ATP hydrolysis cycle.
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transient interaction between the n terminal extension of the essential light chain 1 and motor domain of the Myosin Head during the atpase cycle
Biochemical and Biophysical Research Communications, 2018Co-Authors: Dmitrii I Levitsky, Daria S. Logvinova, Olga P. Nikolaeva, Alexander M MatyushenkoAbstract:The molecular mechanism of muscle contraction is based on the ATP-dependent cyclic interaction of Myosin Heads with actin filaments. Myosin Head (Myosin subfragment-1, S1) consists of two major domains, the motor domain responsible for ATP hydrolysis and actin binding, and the regulatory domain stabilized by light chains. Essential light chain-1 (LC1) is of particular interest since it comprises a unique N-terminal extension (NTE) which can bind to actin thus forming an additional actin-binding site on the Myosin Head and modulating its motor activity. However, it remains unknown what happens to the NTE of LC1 when the Head binds ATP during ATPase cycle and dissociates from actin. We assume that in this state of the Head, when it undergoes global ATP-induced conformational changes, the NTE of LC1 can interact with the motor domain. To test this hypothesis, we applied fluorescence resonance energy transfer (FRET) to measure the distances from various sites on the NTE of LC1 to S1 active site in the motor domain and changes in these distances upon formation of S1-ADP-BeFx complex (stable analog of S1∗-AТP state). For this, we produced recombinant LC1 cysteine mutants, which were first fluorescently labeled with 1,5-IAEDANS (donor) at different positions in their NTE and then introduced into S1; the ADP analog (TNP-ADP) bound to the S1 active site was used as an acceptor. The results show that formation of S1-ADP-BeFx complex significantly decreases the distances from Cys residues in the NTE of LC1 to TNP-ADP in the S1 active site; this effect was the most pronounced for Cys residues located near the LC1 N-terminus. These results support the concept of the ATP-induced transient interaction of the LC1 N-terminus with the S1 motor domain.
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RESEARCH ARTICLE Does Interaction between the Motor and Regulatory Domains of the Myosin Head Occur during ATPase Cycle? Evidence from Thermal Unfolding Studies on Myosin
2016Co-Authors: Daria S. Logvinova, Denis I. Markov, Olga P. Nikolaeva, Nikolai N. Sluchanko, Dmitry S. Ushakov, Dmitrii I LevitskyAbstract:Myosin Head (Myosin subfragment 1, S1) consists of two major structural domains, the motor (or catalytic) domain and the regulatory domain. Functioning of the Myosin Head as a molecular motor is believed to involve a rotation of the regulatory domain (lever arm) relative to the motor domain during the ATPase cycle. According to predictions, this rotation can be accompanied by an interaction between the motor domain and the C-terminus of the essen-tial light chain (ELC) associated with the regulatory domain. To check this assumption, we applied differential scanning calorimetry (DSC) combined with temperature dependences of fluorescence to study changes in thermal unfolding and the domain structure of S1, which occur upon formation of the ternary complexes S1-ADP-AlF4- and S1-ADP-BeFx that mimic S1 ATPase intermediate states S1**-ADP-Pi and S1*-ATP, respectively. To identify the thermal transitions on the DSC profiles (i.e. to assign them to the structural domains of S1), we compared the DSC data with temperature-induced changes in fluorescence of either tryptophan residues, located only in the motor domain, or recombinant ELC mutants (light chain 1 isoform), which were first fluorescently labeled at different positions in their C-termi
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does interaction between the motor and regulatory domains of the Myosin Head occur during atpase cycle evidence from thermal unfolding studies on Myosin subfragment 1
PLOS ONE, 2015Co-Authors: Daria S. Logvinova, Denis I. Markov, Olga P. Nikolaeva, Nikolai N. Sluchanko, Dmitry S. Ushakov, Dmitrii I LevitskyAbstract:Myosin Head (Myosin subfragment 1, S1) consists of two major structural domains, the motor (or catalytic) domain and the regulatory domain. Functioning of the Myosin Head as a molecular motor is believed to involve a rotation of the regulatory domain (lever arm) relative to the motor domain during the ATPase cycle. According to predictions, this rotation can be accompanied by an interaction between the motor domain and the C-terminus of the essential light chain (ELC) associated with the regulatory domain. To check this assumption, we applied differential scanning calorimetry (DSC) combined with temperature dependences of fluorescence to study changes in thermal unfolding and the domain structure of S1, which occur upon formation of the ternary complexes S1-ADP-AlF4- and S1-ADP-BeFx that mimic S1 ATPase intermediate states S1**-ADP-Pi and S1*-ATP, respectively. To identify the thermal transitions on the DSC profiles (i.e. to assign them to the structural domains of S1), we compared the DSC data with temperature-induced changes in fluorescence of either tryptophan residues, located only in the motor domain, or recombinant ELC mutants (light chain 1 isoform), which were first fluorescently labeled at different positions in their C-terminal half and then introduced into the S1 regulatory domain. We show that formation of the ternary complexes S1-ADP-AlF4- and S1-ADP-BeFx significantly stabilizes not only the motor domain, but also the regulatory domain of the S1 molecule implying interdomain interaction via ELC. This is consistent with the previously proposed concepts and also adds some new interesting details to the molecular mechanism of the Myosin ATPase cycle.
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thermal denaturation and aggregation of Myosin subfragment 1 isoforms with different essential light chains
International Journal of Molecular Sciences, 2010Co-Authors: Denis I. Markov, Olga P. Nikolaeva, Eugene O Zubov, B I Kurganov, Dmitrii I LevitskyAbstract:We compared thermally induced denaturation and aggregation of two isoforms of the isolated Myosin Head (Myosin subfragment 1, S1) containing different “essential” (or “alkali”) light chains, A1 or A2. We applied differential scanning calorimetry (DSC) to investigate the domain structure of these two S1 isoforms. For this purpose, a special calorimetric approach was developed to analyze the DSC profiles of irreversibly denaturing multidomain proteins. Using this approach, we revealed two calorimetric domains in the S1 molecule, the more thermostable domain denaturing in two steps. Comparing the DSC data with temperature dependences of intrinsic fluorescence parameters and S1 ATPase inactivation, we have identified these two calorimetric domains as motor domain and regulatory domain of the Myosin Head, the motor domain being more thermostable. Some difference between the two S1 isoforms was only revealed by DSC in thermal denaturation of the regulatory domain. We also applied dynamic light scattering (DLS) to analyze the aggregation of S1 isoforms induced by their thermal denaturation. We have found no appreciable difference between these S1 isoforms in their aggregation properties under ionic strength conditions close to those in the muscle fiber (in the presence of 100 mM KCl). Under these conditions kinetics of this process was independent of protein concentration, and the aggregation rate was limited by irreversible denaturation of the S1 motor domain.
Haruo Sugi - One of the best experts on this subject based on the ideXlab platform.
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basic properties of atp induced Myosin Head movement in hydrated Myosin filaments studied using the gas environmental chamber
Micron, 2018Co-Authors: Haruo Sugi, Tsuyoshi Akimoto, Shigeru ChaenAbstract: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.
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Electron Microscopic Recording of the Power and Recovery Strokes of Individual Myosin Heads Coupled with ATP Hydrolysis: Facts and Implications
MDPI AG, 2018Co-Authors: Haruo Sugi, Shigeru Chaen, Tsuyoshi AkimotoAbstract:The most straightforward way to get information on the performance of individual Myosin Heads producing muscle contraction may be to record their movement, coupled with ATP hydrolysis, electron-microscopically using the gas environmental chamber (EC). The EC enables us to visualize and record ATP-induced Myosin Head movement in hydrated skeletal muscle Myosin filaments. When actin filaments are absent, Myosin Heads fluctuate around a definite neutral position, so that their time-averaged mean position remains unchanged. On application of ATP, Myosin Heads are found to move away from, but not towards, the bare region, indicating that Myosin Heads perform a recovery stroke (average amplitude, 6 nm). After exhaustion of ATP, Myosin Heads return to their neutral position. In the actin–Myosin filament mixture, Myosin Heads form rigor actin Myosin linkages, and on application of ATP, they perform a power stroke by stretching adjacent elastic structures because of a limited amount of applied ATP ≤ 10 µM. The average amplitude of the power stroke is 3.3 nm and 2.5 nm at the distal and the proximal regions of the Myosin Head catalytic domain (CAD), respectively. The power stroke amplitude increases appreciably at low ionic strength, which is known to enhance Ca2+-activated force in muscle. In both the power and recovery strokes, Myosin Heads return to their neutral position after exhaustion of ATP
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Electron microscopic recording of Myosin Head power stroke in hydrated Myosin filaments.
Scientific Reports, 2015Co-Authors: Haruo Sugi, Hiroki Minoda, Takuya Miyakawa, Yumiko Miyauchi, Masaru Tanokura, Tsuyoshi Akimoto, Shigeru Chaen, Seiryo SugiuraAbstract: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.
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electron microscopic evidence for the Myosin Head lever arm mechanism in hydrated Myosin filaments using the gas environmental chamber
Biochemical and Biophysical Research Communications, 2011Co-Authors: Hiroki Minoda, Tatsuhiro Okabe, Yuhri Inayoshi, Takuya Miyakawa, Yumiko Miyauchi, Masaru Tanokura, Eisaku Katayama, Takeyuki Wakabayashi, Tsuyoshi Akimoto, Haruo SugiAbstract: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.
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dynamic electron microscopy of atp induced Myosin Head movement in living muscle thick filaments
Proceedings of the National Academy of Sciences of the United States of America, 1997Co-Authors: Haruo Sugi, Tsuyoshi Akimoto, Shigeru Chaen, Kazuo Sutoh, Noboru Oishi, Suechika SuzukiAbstract:Although muscle contraction is known to result from movement of the Myosin Heads on the thick filaments while attached to the thin filaments, the Myosin Head movement coupled with ATP hydrolysis still remains to be investigated. Using a gas environmental (hydration) chamber, in which biological specimens can be kept in wet state, we succeeded in recording images of living muscle thick filaments with gold position markers attached to the Myosin Heads. The position of individual Myosin Heads did not change appreciably with time in the absence of ATP, indicating stability of the Myosin Head mean position. On application of ATP, the position of individual Myosin Heads was found to move by ≈20 nm along the filament axis, whereas no appreciable movement of the filaments was detected. The ATP-induced Myosin Head movement was not observed in filaments in which ATPase activity of the Myosin Heads was eliminated. Application of ADP produced no appreciable Myosin Head movement. These results show that the ATP-induced Myosin Head movement takes place in the absence of the thin filaments. Because ATP reacts rapidly with the Myosin Head (M) to form the complex (M⋅ADP⋅Pi) with an average lifetime of >10 s, the observed Myosin Head movement may be mostly associated with reaction, M + ATP → M⋅ADP⋅Pi. This work will open a new research field to study dynamic structural changes of individual biomolecules, which are kept in a living state in an electron microscope.
Malcolm Irving - One of the best experts on this subject based on the ideXlab platform.
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phosphorylation of Myosin regulatory light chain controls Myosin Head conformation in cardiac muscle
Journal of Molecular and Cellular Cardiology, 2015Co-Authors: Thomas Kampourakis, Malcolm IrvingAbstract:The effect of phosphorylation on the conformation of the regulatory light chain (cRLC) region of Myosin in ventricular trabeculae from rat heart was determined by polarized fluorescence from thiophosphorylated cRLCs labelled with bifunctional sulforhodamine (BSR). Less than 5% of cRLCs were endogenously phosphorylated in this preparation, and similarly low values of basal cRLC phosphorylation were measured in fresh intact ventricle from both rat and mouse hearts. BSR-labelled cRLCs were thiophosphorylated by a recombinant fragment of human cardiac Myosin light chain kinase, which was shown to phosphorylate cRLCs specifically at serine 15 in a calcium- and calmodulin-dependent manner, both in vitro and in situ. The BSR-cRLCs were exchanged into demembranated trabeculae, and polarized fluorescence intensities measured for each BSR-cRLC in relaxation, active isometric contraction and rigor were combined with RLC crystal structures to calculate the orientation distribution of the C-lobe of the cRLC in each state. Only two of the four C-lobe orientation populations seen during relaxation and active isometric contraction in the unphosphorylated state were present after cRLC phosphorylation. Thus cRLC phosphorylation alters the equilibrium between defined conformations of the cRLC regions of the Myosin Heads, rather than simply disordering the Heads as assumed previously. cRLC phosphorylation also changes the orientation of the cRLC C-lobe in rigor conditions, showing that the orientation of this part of the Myosin Head is determined by its interaction with the thick filament even when the Head is strongly bound to actin. These results suggest that cRLC phosphorylation controls the contractility of the heart by modulating the interaction of the cRLC region of the Myosin Heads with the thick filament backbone.
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the contributions of filaments and cross bridges to sarcomere compliance in skeletal muscle
The Journal of Physiology, 2014Co-Authors: Elisabetta Brunello, Marco Linari, Malcolm Irving, Vincenzo Lombardi, Gabriella Piazzesi, Theyencheri Narayanan, Marco Caremani, Luca Melli, Manuel Fernandezmartinez, Massimo ReconditiAbstract:Force generation in the muscle sarcomere is driven by the Head domain of the Myosin molecule extending from the thick filament to form cross-bridges with the actin-containing thin filament. Following attachment, a structural working stroke in the Head pulls the thin filament towards the centre of the sarcomere, producing, under unloaded conditions, a filament sliding of ∼ 11 nm. The mechanism of force generation by the Myosin Head depends on the relationship between cross-bridge force and movement, which is determined by compliances of the cross-bridge (C(cb)) and filaments. By measuring the force dependence of the spacing of the high-order Myosin- and actin-based X-ray reflections from sartorius muscles of Rana esculenta we find a combined filament compliance (Cf) of 13.1 ± 1.2 nm MPa(-1), close to recent estimates from single fibre mechanics (12.8 ± 0.5 nm MPa(-1)). C(cb) calculated using these estimates is 0.37 ± 0.12 nm pN(-1), a value fully accounted for by the compliance of the Myosin Head domain, 0.38 ± 0.06 nm pN(-1), obtained from the intensity changes of the 14.5 nm Myosin-based X-ray reflection in response to 3 kHz oscillations imposed on single muscle fibres in rigor. Thus, a significant contribution to C(cb) from the Myosin tail that joins the Head to the thick filament is excluded. The low C(cb) value indicates that the Myosin Head generates isometric force by a small sub-step of the 11 nm stroke that drives filament sliding at low load. The implications of these results for the mechanism of force generation by Myosins have general relevance for cardiac and non-muscle Myosins as well as for skeletal muscle.
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mechanics of Myosin function in white muscle fibres of the dogfish scyliorhinus canicula
The Journal of Physiology, 2012Co-Authors: S Parkholohan, Marco Linari, Massimo Reconditi, Luca Fusi, Elisabetta Brunello, Malcolm Irving, Mario Dolfi, Vincenzo Lombardi, Timothy G WestAbstract:The contractile properties of muscle fibres have been extensively investigated by fast perturbation in sarcomere length to define the mechanical characteristics of myofilaments and Myosin Heads that underpin refined models of the acto-Myosin cycle. Comparison of published data from intact fast-twitch fibres of frog muscle and demembranated fibres from fast muscle of rabbit shows that stiffness of the rabbit Myosin Head is only ∼62% of that in frog. To clarify if and how much the mechanical characteristics of the filaments and Myosin Heads vary in muscles of different animals we apply the same high resolution mechanical methods, in combination with X-ray diffraction, to fast-twitch fibres from the dogfish (Scyliorhinus canicula). The values of equivalent filament compliance (Cf) measured by X-ray diffraction and in mechanical experiments are not significantly different; the best estimate from combining these values is 17.1 ±1.0 nm MPa −1 . This value is larger than Cf in frog, 13.0 ±0.4 nm MPa −1 . The longer thin filamentsindogfishaccountforonlypartofthisdifference.Theaverageisometricforceexertedby eachattachedMyosinHeadat5 ◦ C,4.5 pN,andthemaximumslidingdistanceaccountedforbythe Myosinworkingstroke,11 nm,aresimilartothoseinfrog,whiletheaverageMyosinHeadstiffness of dogfish (1.98 ±0.31pNnm −1 ) is smaller than that of frog (2.78 ±0.30pNnm −1 ). Taken together these results indicate that the working stroke responsible for the generation of isometric force is a larger fraction of the total Myosin Head working stroke in the dogfish than in the frog.
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motion of Myosin Head domains during activation and force development in skeletal muscle
Proceedings of the National Academy of Sciences of the United States of America, 2011Co-Authors: Massimo Reconditi, Marco Linari, Elisabetta Brunello, Vincenzo Lombardi, Gabriella Piazzesi, Pasquale Bianco, Theyencheri Narayanan, Pierre Panine, Malcolm IrvingAbstract:Muscle contraction is driven by a change in the structure of the Head domain of Myosin, the “working stroke” that pulls the actin filaments toward the midpoint of the Myosin filaments. This movement of the Myosin Heads can be measured very precisely in intact muscle cells by X-ray interference, but until now this technique has not been applied to physiological activation and force generation following electrical stimulation of muscle cells. By using this approach, we show that the long axes of the Myosin Head domains are roughly parallel to the filaments in resting muscle, with their center of mass offset by approximately 7 nm from the C terminus of the Head domain. The observed mass distribution matches that seen in electron micrographs of isolated Myosin filaments in which the Heads are folded back toward the filament midpoint. Following electrical stimulation, the Heads move by approximately 10 nm away from the filament midpoint, in the opposite direction to the working stroke. The time course of this motion matches that of force generation, but is slower than the other structural changes in the Myosin filaments on activation, including the loss of helical and axial order of the Myosin Heads and the change in periodicity of the filament backbone. The rate of force development is limited by that of attachment of Myosin Heads to actin in a conformation that is the same as that during steady-state isometric contraction; force generation in the actin-attached Head is fast compared with the attachment step.
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the conformation of Myosin Head domains in rigor muscle determined by x ray interference
Biophysical Journal, 2003Co-Authors: Massimo Reconditi, Marco Linari, Vincenzo Lombardi, Gabriella Piazzesi, Theyencheri Narayanan, Natalia A Koubassova, Ian M Dobbie, Olivier Diat, Malcolm IrvingAbstract: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).
Takuya Miyakawa - One of the best experts on this subject based on the ideXlab platform.
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Electron microscopic recording of Myosin Head power stroke in hydrated Myosin filaments.
Scientific Reports, 2015Co-Authors: Haruo Sugi, Hiroki Minoda, Takuya Miyakawa, Yumiko Miyauchi, Masaru Tanokura, Tsuyoshi Akimoto, Shigeru Chaen, Seiryo SugiuraAbstract: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.
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definite differences between in vitro actin Myosin sliding and muscle contraction as revealed using antibodies to Myosin Head
PLOS ONE, 2014Co-Authors: Shigeru Chaen, Takuya Miyakawa, Masaru Tanokura, Yasutake Saeki, Takakazu Kobayashi, Takahiro Abe, Yoshiki Ohnuki, Kazushige Kimura, Seiryo SugiuraAbstract:Muscle contraction results from attachment-detachment cycles between Myosin Heads extending from Myosin filaments and actin filaments. It is generally believed that a Myosin Head first attaches to actin, undergoes conformational changes to produce force and motion in muscle, and then detaches from actin. Despite extensive studies, the molecular mechanism of Myosin Head conformational changes still remains to be a matter for debate and speculation. The Myosin Head consists of catalytic (CAD), converter (CVD) and lever arm (LD) domains. To give information about the role of these domains in the Myosin Head performance, we have examined the effect of three site-directed antibodies to the Myosin Head on in vitro ATP-dependent actin-Myosin sliding and Ca2+-activated contraction of muscle fibers. Antibody 1, attaching to junctional peptide between 50K and 20K heavy chain segments in the CAD, exhibited appreciable effects neither on in vitro actin-Myosin sliding nor muscle fiber contraction. Since antibody 1 covers actin-binding sites of the CAD, one interpretation of this result is that rigor actin-Myosin linkage is absent or at most a transient intermediate in physiological actin-Myosin cycling. Antibody 2, attaching to reactive lysine residue in the CVD, showed a marked inhibitory effect on in vitro actin-Myosin sliding without changing actin-activated Myosin Head (S1) ATPase activity, while it showed no appreciable effect on muscle contraction. Antibody 3, attaching to two peptides of regulatory light chains in the LD, had no significant effect on in vitro actin-Myosin sliding, while it reduced force development in muscle fibers without changing MgATPase activity. The above definite differences in the effect of antibodies 2 and 3 between in vitro actin-Myosin sliding and muscle contraction can be explained by difference in experimental conditions; in the former, Myosin Heads are randomly oriented on a glass surface, while in the latter Myosin Heads are regularly arranged within filament-lattice structures.
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Effect of Antibodies to Myosin Head Reveals Definite Difference between In Vitro Actin-Myosin Sliding and Muscle Contraction
Biophysical Journal, 2013Co-Authors: Takahiro Abe, Takuya Miyakawa, Takakazu Kobayashi, Shigeru Chaen, Suguru Tanokura, Yoshiki OhnukiAbstract:Mechanism of Myosin Head power stroke, responsible for muscle contraction, still remains to be a matter of debate and speculation. Despite considerable progress in studying actin filament sliding over Myosin fixed on a glass surface, it is not clear whether the in vitro actin-Myosin sliding takes place by a mechanism similar to contraction in muscle, consisting of three-dimensional myofilament lattice structures. To make this point clear, we prepared two different monoclonal antibodies, one directed to reactive lysine residue close to the Myosin Head converter region (anti-RLR antibody) while the other directed to two peptides of regulatory light chain in the Myosin Head lever arm region (anti-LD antibody). We compared the effect of these antibodies on in vitro actin- Myosin sliding and contraction of skinned rabbit psoas muscle fibers with the following results: (1) anti-RLR antibody completely inhibited in vitro actin-Myosin sliding without changing actin-activated Myosin Head ATPase activity, while it showed no effect on Ca2+-activated contraction of muscle fibers; (2) anti-LD antibody had no effect on in vitro actin-Myosin sliding, but suppressed Ca2+-activated muscle fiber contraction without changing Mg-ATPase activity. These results indicate definite difference between in vitro actin-Myosin sliding and muscle contraction.
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Definite Difference Between In Vitro Actin-Myosin Sliding and Muscle Contraction Revealed by the Effect of Antibody to Myosin Head Converter Domain
Biophysical Journal, 2012Co-Authors: Takakazu Kobayashi, Takuya Miyakawa, Masaru Tanokura, Yasutake Saeki, Shigeru Chaen, Hiroki MinodaAbstract:Myofilament sliding in muscle is believed to result from rotation of the Myosin Head catalytic domain (CAD) around the converter domain (CD). To explore the validity of this mechanism, we compared the effect of antibody to Myosin Head converter domain (IgG, anti-CD antibody) between in vitro actin-Myosin sliding and muscle fiber contraction. In agreement with the expectation that binding of massive antibody to the CD impairs rotation of the CAD around the CD, the ATP-dependent sliding of actin filaments over Myosin Heads on a glass surface was inhibited by the antibody (0.14mg/ml). Meanwhile, the antibody (up to 1.5mg/ml) showed no appreciable effect on the actin-activated Myosin Head ATPase activity, indicating that the antibody has no effect on the ATPase activity in the CAD. Unexpectedly, the antibody (up to 3mg/ml) showed no appreciable effect on the maximum Ca2+-activated isometric force, the maximum shortening velocity, and the Mg-activated ATPase activity in glycerol-extracted rabbit psoas muscle fibers. The possibility that the antibody does not diffuse into muscle fibers can be excluded by our published results that other antibodies readily inhibit muscle fiber contraction. These findings therefore suggest that the antibody binding to the Myosin Head CD does not impair performance of Myosin Heads producing force and motion in muscle fibers.
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electron microscopic evidence for the Myosin Head lever arm mechanism in hydrated Myosin filaments using the gas environmental chamber
Biochemical and Biophysical Research Communications, 2011Co-Authors: Hiroki Minoda, Tatsuhiro Okabe, Yuhri Inayoshi, Takuya Miyakawa, Yumiko Miyauchi, Masaru Tanokura, Eisaku Katayama, Takeyuki Wakabayashi, Tsuyoshi Akimoto, Haruo SugiAbstract: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.
