Direct visualization of human myosin II force generation using DNA origami-based thick filaments

AbstractThe sarcomere, the minimal mechanical unit of muscle, is composed of myosins, which self-assemble into thick filaments that interact with actin-based thin filaments in a highly-structured lattice. This complex imposes a geometric restriction on myosin in force generation. However, how single myosins generate force within the restriction remains elusive and conventional synthetic filaments do not recapitulate the symmetric bipolar filaments in sarcomeres. Here we engineered thick filaments using DNA origami that incorporate human muscle myosin to directly visualize the motion of the heads during force generation in a restricted space. We found that when the head diffuses, it weakly interacts with actin filaments and then strongly binds preferentially to the forward region as a Brownian ratchet. Upon strong binding, the two-step lever-arm swing dominantly halts at the first step and occasionally reverses direction. Our results illustrate the usefulness of our DNA origami-based assay system to dissect the mechanistic details of motor proteins.

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Direct visualization of human myosin II force generation using DNA origami-based thick filaments

10.1101/634287 ◽

2019 ◽

Author(s):

Keisuke Fujita ◽

Masashi Ohmachi ◽

Keigo Ikezaki ◽

Toshio Yanagida ◽

Mitsuhiro Iwaki

Keyword(s):

Muscle Contraction ◽

Single Molecule ◽

Dna Origami ◽

Force Generation ◽

Dynamic Features ◽

Forward Region ◽

Arm Swing ◽

Lever Arm ◽

Contraction Speed ◽

Thick Filaments

AbstractMuscle contraction can be explained by the swinging lever-arm model. However, the dynamic features of how the myosin head swings the lever-arm and its initial interactions with actin are not well understood even though they are essential for the muscle force generation, contraction speed, heat production, and response to mechanical perturbations. This is because myosin heads during force generation have not been directly visualized. Here, we engineered thick filaments composed of DNA origami and recombinant human muscle myosin, and directly visualized the heads during force generation using nanometer-precision single-molecule imaging. We found that when the head diffuses, it weakly interacts with actin filaments and then strongly binds preferentially to the forward region as a Brownian ratchet. Upon strong binding, the head two-step lever-arm swing dominantly halts at the first step and occasionally reverses direction. These results can explain all mechanical characteristics of muscle contraction and suggest that our DNA origami-based assay system can be used to dissect the mechanistic details of motor proteins.

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LOCALIZATION OF MYOSIN FILAMENTS IN SMOOTH MUSCLE

The Journal of Cell Biology ◽

10.1083/jcb.37.1.105 ◽

1968 ◽

Vol 37 (1) ◽

pp. 105-116 ◽

Cited By ~ 72

Author(s):

Robert E. Kelly ◽

Robert V. Rice

Keyword(s):

Smooth Muscle ◽

Cross Section ◽

Striated Muscle ◽

Thick Filament ◽

Longitudinal Axis ◽

Thin Filaments ◽

Chicken Gizzard ◽

Thick Filaments ◽

Myosin Filaments ◽

Muscle Myosin

Thick myosin filaments, in addition to actin filaments, were found in sections of glycerinated chicken gizzard smooth muscle when fixed at a pH below 6.6. The thick filaments were often grouped into bundles and run in the longitudinal axis of the smooth muscle cell. Each thick filament was surrounded by a number of thin filaments, giving the filament arrangement a rosette appearance in cross-section. The exact ratio of thick filaments to thin filaments could not be determined since most arrays were not so regular as those commonly found in striated muscle. Some rosettes had seven or eight thin filaments surrounding a single thick filament. Homogenates of smooth muscle of chicken gizzard also showed both thick and thin filaments when the isolation was carried out at a pH below 6.6, but only thin filaments were found at pH 7.4. No Z or M lines were observed in chicken gizzard muscle containing both thick and thin filaments. The lack of these organizing structures may allow smooth muscle myosin to disaggregate readily at pH 7.4.

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Plasticity in smooth muscle, a hypothesis

Canadian Journal of Physiology and Pharmacology ◽

10.1139/y94-190 ◽

1994 ◽

Vol 72 (11) ◽

pp. 1320-1324 ◽

Cited By ~ 41

Author(s):

Lincoln E. Ford ◽

Chun Y. Seow ◽

Victor R. Pratusevich

Keyword(s):

Smooth Muscle ◽

Preliminary Finding ◽

Muscle Length ◽

Shortening Velocity ◽

Thin Filaments ◽

Thick Filaments ◽

Initial Hypothesis ◽

In Series ◽

Cross Bridges ◽

Muscle Myosin

The controversial finding that the thick filaments of smooth muscle can be evanescent leads to the hypothesis that the large functional range of this muscle is accommodated by plastic rearrangements that place more thick filaments in series at longer lengths. Our preliminary finding that the shortening velocity and compliance of dog tracheal muscle were strongly dependent on adapted muscle length, while force was much less length dependent, supports this hypothesis (V.R. Pratusevich, C.Y. Seow, and L.E. Ford. Biophys. J. 66: A139, 1994). The hypothesis leads to two further corollaries. The first is that the lengthening of the thick filaments that must accompany their reformation will cause a series to parallel transition: fewer long filaments span the muscle length, but the longer filaments have more cross bridges acting in parallel. The second is that there is more than one activating mechanism in smooth muscle. It is known that myosin light chain phosphorylation activates the actomyosin ATPase, but this same phosphorylation also causes a structural change that facilitates filament formation. The consideration that the unaggregated, phosphorylated myosin must be prevented from competing with myosin in thick filaments and hydrolyzing ATP suggests that there must be a second mechanism that must allow the thin filaments to interact selectively with filamentous myosin. This need for a second activating mechanism may explain the presence of tropomyosin, calponin, and caldesmon on thin filaments. Although the two corollaries follow from the initial hypothesis, it should be emphasized that the three are not mutually dependent, and that the proof or disproof of any one of them would not prove or disprove the others.Key words: smooth muscle, myosin, thick filaments, contraction.

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The Central Role of the F-Actin Surface in Myosin Force Generation

Biology ◽

10.3390/biology10121221 ◽

2021 ◽

Vol 10 (12) ◽

pp. 1221

Author(s):

Matthew H. Doran ◽

William Lehman

Keyword(s):

Critical Role ◽

Structural Features ◽

Force Generation ◽

Actin Binding ◽

Myosin Isoforms ◽

Thin Filaments ◽

Cellular Processes ◽

Muscle Myosin ◽

Kinetics Of

Actin is one of the most abundant and versatile proteins in eukaryotic cells. As discussed in many contributions to this Special Issue, its transition from a monomeric G-actin to a filamentous F-actin form plays a critical role in a variety of cellular processes, including control of cell shape and cell motility. Once polymerized from G-actin, F-actin forms the central core of muscle-thin filaments and acts as molecular tracks for myosin-based motor activity. The ATP-dependent cross-bridge cycle of myosin attachment and detachment drives the sliding of myosin thick filaments past thin filaments in muscle and the translocation of cargo in somatic cells. The variation in actin function is dependent on the variation in muscle and non-muscle myosin isoform behavior as well as interactions with a plethora of additional actin-binding proteins. Extensive work has been devoted to defining the kinetics of actin-based force generation powered by the ATPase activity of myosin. In addition, over the past decade, cryo-electron microscopy has revealed the atomic-evel details of the binding of myosin isoforms on the F-actin surface. Most accounts of the structural interactions between myosin and actin are described from the perspective of the myosin molecule. Here, we discuss myosin-binding to actin as viewed from the actin surface. We then describe conserved structural features of actin required for the binding of all or most myosin isoforms while also noting specific interactions unique to myosin isoforms.

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Introductory Remarks

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s1431927600002646 ◽

1980 ◽

Vol 38 ◽

pp. 598-599

Author(s):

H. Mohri

Keyword(s):

Muscle Contraction ◽

Experimental Evidence ◽

Sea Urchin ◽

Constant Length ◽

Thin Filaments ◽

Bridge Formation ◽

Thick Filaments ◽

Sliding Mechanism ◽

Radial Spokes ◽

Cilia And Flagella

In 1959, Afzelius observed the presence of two rows of arms projecting from each outer doublet microtubule of the so-called 9 + 2 pattern of cilia and flagella, and suggested a possibility that the outer doublet microtubules slide with respect to each other with the aid of these arms during ciliary and flagellar movement. The identification of the arms as an ATPase, dynein, by Gibbons (1963)strengthened this hypothesis, since the ATPase-bearing heads of myosin molecules projecting from the thick filaments pull the thin filaments by cross-bridge formation during muscle contraction. The first experimental evidence for the sliding mechanism in cilia and flagella was obtained by examining the tip patterns of molluscan gill cilia by Satir (1965) who observed constant length of the microtubules during ciliary bending. Further evidence for the sliding-tubule mechanism was given by Summers and Gibbons (1971), using trypsin-treated axonemal fragments of sea urchin spermatozoa. Upon the addition of ATP, the outer doublets telescoped out from these fragments and the total length reached up to seven or more times that of the original fragment. Thus, the arms on a certain doublet microtubule can walk along the adjacent doublet when the doublet microtubules are disconnected by digestion of the interdoublet links which connect them with each other, or the radial spokes which connect them with the central pair-central sheath complex as illustrated in Fig. 1. On the basis of these pioneer works, the sliding-tubule mechanism has been established as one of the basic mechanisms for ciliary and flagellar movement.

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Urinary Titin N-Fragment as a Biomarker of Muscle Atrophy, Intensive Care Unit-Acquired Weakness, and Possible Application for Post-Intensive Care Syndrome

Journal of Clinical Medicine ◽

10.3390/jcm10040614 ◽

2021 ◽

Vol 10 (4) ◽

pp. 614 ◽

Cited By ~ 1

Author(s):

Nobuto Nakanishi ◽

Rie Tsutsumi ◽

Kanako Hara ◽

Masafumi Matsuo ◽

Hiroshi Sakaue ◽

...

Keyword(s):

Intensive Care Unit ◽

Intensive Care ◽

Muscle Atrophy ◽

Critically Ill Patients ◽

Chronic Illnesses ◽

Rehabilitation Management ◽

Muscle Dystrophy ◽

Thin Filaments ◽

Thick Filaments ◽

Post Intensive Care Syndrome

Titin is a giant protein that functions as a molecular spring in sarcomeres. Titin interconnects the contraction of actin-containing thin filaments and myosin-containing thick filaments. Titin breaks down to form urinary titin N-fragments, which are measurable in urine. Urinary titin N-fragment was originally reported to be a useful biomarker in the diagnosis of muscle dystrophy. Recently, the urinary titin N-fragment has been increasingly gaining attention as a novel biomarker of muscle atrophy and intensive care unit-acquired weakness in critically ill patients, in whom titin loss is a possible pathophysiology. Furthermore, several studies have reported that the urinary titin N-fragment also reflected muscle atrophy and weakness in patients with chronic illnesses. It may be used to predict the risk of post-intensive care syndrome or to monitor patients’ condition after hospital discharge for better nutritional and rehabilitation management. We provide several tips on the use of this promising biomarker in post-intensive care syndrome.

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The effect of heavy chain phosphorylation and solution conditions on the assembly of Acanthamoeba myosin-II.

The Journal of Cell Biology ◽

10.1083/jcb.109.4.1529 ◽

1989 ◽

Vol 109 (4) ◽

pp. 1529-1535 ◽

Cited By ~ 27

Author(s):

J H Sinard ◽

T D Pollard

Keyword(s):

Light Scattering ◽

Ionic Strength ◽

Myosin Heavy Chain ◽

Heavy Chain ◽

Critical Concentration ◽

Myosin Ii ◽

Acidic Ph ◽

Thick Filaments ◽

Low Ionic Strength

At low ionic strength, Acanthamoeba myosin-II polymerizes into bipolar minifilaments, consisting of eight molecules, that scatter about three times as much light as monomers. With this light scattering assay, we show that the critical concentration for assembly in 50-mM KCl is less than 5 nM. Phosphorylation of the myosin heavy chain over the range of 0.7 to 3.7 P per molecule has no effect on its KCl dependent assembly properties: the structure of the filaments, the extent of assembly, and the critical concentration for assembly are the same. Sucrose at a concentration above a few percent inhibits polymerization. Millimolar concentrations of MgCl2 induce the lateral aggregation of fully formed minifilaments into thick filaments. Compared with dephosphorylated minifilaments, minifilaments of phosphorylated myosin have a lower tendency to aggregate laterally and require higher concentrations of MgCl2 for maximal light scattering. Acidic pH also induces lateral aggregation, whereas basic pH leads to depolymerization of the myosin-II minifilaments. Under polymerizing conditions, millimolar concentrations of ATP only slightly decrease the light scattering of either phosphorylated or dephosphorylated myosin-II. Barring further modulation of assembly by unknown proteins, both phosphorylated and dephosphorylated myosin-II are expected to be in the form of minifilaments under the ionic conditions existing within Acanthamoeba.

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