Mass Measurements of Lobster Muscle Thick Filaments by Scanning Transmission Electron Microscopy
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The structure of muscle thick filaments has been elucidated by a combination of electron microscopic and X-ray diffraction methods. Although X-ray diffraction provides much detailed information about the structure of the filaments, a long-standing problem is the number of myosin molecules per repeating unit along the filament. Direct measurements using STEM have recently established that in vertebrate skeletal muscle and insect flight muscle there are respectively 3 and 4 myosin molecules in each 145Å. period along the filament.Keywords:
Myofibril
Although more than 50 yeares have passed since the monumental discovery of sliding filament mechanism in muscle contraction, the moleculare mechanism of myosin head movement, coupled with ATP hydrolysis, is still a matter for debate and speculation. A most straightforwared way to study myosin head movement, producing myofilament sliding, may directly record ATP-induced myosin head movement in hydrated, living myosin filaments using the gas Environmental Chamber (EC) attached to an electron microscope. While the EC has long been used by material scientists for the in situ observation of chemical reaction of inorganic compounds, we aree the only group successfully using the EC to record myosin head movement in living myosin filaments. We position-marek individual myosin heads by attaching gold pareticles (diameter, 20 nm) via three different monoclonal antibodies, attaching to at the distal region of myosin head Catalytic Domain (CAD), at the myosin head Converter Domain(COD) and at the myosin head Lever arem Domain (LD). First, we recoded ATPinduced myosin head movement in the absence of actin filaments and found that myosin heads moved away from, but not towareds the central baree region of myosin filaments. We also succeeded in recording ATP-induced myosin head power stroke in actin-myosin filament mixture. Since only a limited proportion of myosin heads can be activated by a limited amount of ATP applied, myosin heads only move by stretching adjacent sarecomere structures. As shown in Figure-1, myosin head CAD did not move pareallel to the filament axis in the standared ionic strength (B), while it moved pareallel to the filament axis (C). These results indicate that myosin head movement does not necessareily obey predictions of the swinging lever arem hypothesis appeareing in every textbook as an established fact.
Meromyosin
Myofilament
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Myofibril
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Of all the myosin filaments in muscle, the most important in terms of human health, and so far the least studied, are those in the human heart. Here we report a 3D single-particle analysis of electron micrograph images of negatively stained myosin filaments isolated from human cardiac muscle in the normal (undiseased) relaxed state. The resulting 28-Å resolution 3D reconstruction shows axial and azimuthal (no radial) myosin head perturbations within the 429-Å axial repeat, with rotations between successive 132 Å-, 148 Å-, and 149 Å-spaced crowns of heads close to 60°, 35°, and 25° (all would be 40° in an unperturbed three-stranded helix). We have defined the myosin head atomic arrangements within the three crown levels and have modeled the organization of myosin subfragment 2 and the possible locations of the 39 Å-spaced domains of titin and the cardiac isoform of myosin-binding protein-C on the surface of the myosin filament backbone. Best fits were obtained with head conformations on all crowns close to the structure of the two-headed myosin molecule of vertebrate chicken smooth muscle in the dephosphorylated relaxed state. Individual crowns show differences in head-pair tilts and subfragment 2 orientations, which, together with the observed perturbations, result in different intercrown head interactions, including one not reported before. Analysis of the interactions between the myosin heads, the cardiac isoform of myosin-binding protein-C, and titin will aid in understanding of the structural effects of mutations in these proteins known to be associated with human cardiomyopathies.
Meromyosin
Human heart
Cardiac muscle
Electron micrographs
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We have used electron microscopy and solubility measurements to investigate the assembly and structure of purified human platelet myosin and myosin rod into filaments. In buffers with ionic strengths of less than 0.3 M, platelet myosin forms filaments which are remarkable for their small size, being only 320 nm long and 10-11 nm wide in the center of the bare zone. The dimensions of these filaments are not affected greatly by variation of the pH between 7 and 8, variation of the ionic strength between 0.05 and 0.2 M, the presence or absence of 1 mM Mg++ or ATP, or variation of the myosin concentration between 0.05 and 0.7 mg/ml. In 1 mM Ca++ and at pH 6.5 the filaments grow slightly larger. More than 90% of purified platelet myosin molecules assemble into filaments in 0.1 M KC1 at pH 7. Purified preparations of the tail fragment of platelet myosin also form filaments. These filaments are slightly larger than myosin filaments formed under the same conditions, indicating that the size of the myosin filaments may be influenced by some interaction between the head and tail portions of myosin molecules. Calculations based on the size and shape of the myosin filaments, the dimensions of the myosin molecule and analysis of the bare zone reveal that the synthetic platelet myosin filaments consists of 28 myosin molecules arranged in a bipolar array with the heads of two myosin molecules projecting from the backbone of the filament at 14-15 nm intervals. The heads appear to be loosely attached to the backbone by a flexible portion of the myosin tail. Given the concentration of myosin in platelets and the number of myosin molecules per filament, very few of these thin myosin filaments should be present in a thin section of a platelet, even if all of the myosin molecules are aggregated into filaments.
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Abstract 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.
Myofilament
Meromyosin
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Myosin plays a fundamental role in muscle contraction. Approximately 300 myosins form a bipolar thick filament, in which myosin is continuously replaced by protein turnover. However, it is unclear how rapidly this process occurs and whether the myosin exchange rate differs depending on the region of the thick filament. To answer this question, we first measured myosin release and insertion rates over a short period and monitored myotubes expressing a photoconvertible fluorescence protein-tagged myosin, which enabled us to monitor myosin release and insertion simultaneously. About 20% of myosins were replaced within 10 min, while 70% of myosins were exchanged over 10 h with symmetrical and biphasic alteration of myosin release and insertion rates. Next, a fluorescence pulse-chase assay was conducted to investigate whether myosin is incorporated into specific regions in the thick filament. Newly synthesized myosin was located at the tip of the thick filament rather than the center in the first 7 min of pulse-chase labeling and was observed in the remainder of the thick filament by 30 min. These results suggest that the myosin replacement rate differs depending on the regions of the thick filament. We concluded that myosin release and insertion occur concurrently and that myosin is more frequently exchanged at the tip of the thick filament.
Meromyosin
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The current data and concepts of the structural organization of the head of myosin, one of the major muscle proteins, are reviewed. The primary structure of the isolated myosin head (myosin subfragment-1) heavy chain and localization in it of sites and groups responsible for the binding and hydrolysis of ATP and myosin interaction with actin, are considered. Evidence is given of reciprocal spatial distribution of these sites and their localization on the myosin head surface. Some present-day concepts on the domain organization of the myosin head and its changes occurring during binding and hydrolysis of ATP, are discussed. A model describing the folding of the heavy and light chains in the myosin head is proposed.
Meromyosin
Heavy chain
Folding (DSP implementation)
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Biochemical changes of myosin in chicken myofibrils exposed to nonenzymatic, hydroxyl radical generation systems (HRGS) were investigated by means of cross-linking reaction, ATPase activity, salt solubility, and 40% saturated ammonium sulfate (AS) extractability. HRGS treatment of myofibrils caused cross-linking of myosin heavy chains (MHC) via disulfide bonding, an increase in Ca-ATPase activity, and a decrease in K-ATPase activity, suggesting that thiol groups of myosin including those at the active site were modified. The specific changes depended on the concentrations of H(2)O(2) in HRGS as well as the weight ratio of H(2)O(2) to myofibrils. On the other hand, the decrease in salt solubility or AS extractability of myosin in HRGS-treated samples proceeded slowly when compared with the cross-linking reaction of MHC, indicating that considerable amounts of myosin biopolymers remained hydrophilic in the ionic solutions. The results demonstrated that initial cross-linking of MHC occurred inside the myosin molecule, and this was followed by progressive aggregation of myosin molecules through intermolecular cross-linking. Oxidation under the current experimental condition decreased the gel-forming ability of myofibrillar proteins, which coincided with the progress of the intra- and intermolecular cross-linking reactions as well as with ATPase activity changes.
Myofibril
Thiol
Myosin ATPase
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Rabbit (cipher)
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Helix (gastropod)
Molecular motor
Adductor muscles
Meromyosin
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