scholarly journals Assembly of smooth muscle myosin into side-polar filaments.

1977 ◽  
Vol 75 (3) ◽  
pp. 990-996 ◽  
Author(s):  
R Craig ◽  
J Megerman
Keyword(s):  
Smooth Muscle ◽  
Opposite Polarity ◽  
Skeletal Muscle Myosin ◽  
Myosin Filaments ◽  
Skeletal Myosin ◽  
Cross Bridges ◽  
Bipolar Filament ◽  
Muscle Myosin

The in vitro assembly of myosin purified from calf aorta muscle has been studied by electron microscopy. Two types of filament are formed: short bipolar filament similar to those formed from skeletal muscle myosin, and longer "side-polar" filaments having cross bridges with a single polarity along the entire length of one side and the opposite polarity along the other side. Unlike the case with skeletal myosin filaments, antiparallel interactions between myosin molecules occur along the whole length of side-polar filaments. The side-polar structure may be related to the in vivo form of myosin in vertebrate smooth muscle.

scholarly journals Light chain phosphorylation regulates the movement of smooth muscle myosin on actin filaments.

1985 ◽  
Vol 101 (5) ◽  
pp. 1897-1902 ◽  
Author(s):  
J R Sellers ◽  
J A Spudich ◽  
M P Sheetz
Keyword(s):  
Smooth Muscle ◽  
Actin Filaments ◽  
Smooth Muscles ◽  
Smooth Muscle Myosin ◽  
Vitro Assay ◽  
Skeletal Muscle Myosin ◽  
Myosin Filaments ◽  
Muscle Myosin ◽  
Rate Of Movement

In smooth muscles there is no organized sarcomere structure wherein the relative movement of myosin filaments and actin filaments has been documented during contraction. Using the recently developed in vitro assay for myosin-coated bead movement (Sheetz, M.P., and J.A. Spudich, 1983, Nature (Lond.)., 303:31-35), we were able to quantitate the rate of movement of both phosphorylated and unphosphorylated smooth muscle myosin on ordered actin filaments derived from the giant alga, Nitella. We found that movement of turkey gizzard smooth muscle myosin on actin filaments depended upon the phosphorylation of the 20-kD myosin light chains. About 95% of the beads coated with phosphorylated myosin moved at velocities between 0.15 and 0.4 micron/s, depending upon the preparation. With unphosphorylated myosin, only 3% of the beads moved and then at a velocity of only approximately 0.01-0.04 micron/s. The effects of phosphorylation were fully reversible after dephosphorylation with a phosphatase prepared from smooth muscle. Analysis of the velocity of movement as a function of phosphorylation level indicated that phosphorylation of both heads of a myosin molecule was required for movement and that unphosphorylated myosin appears to decrease the rate of movement of phosphorylated myosin. Mixing of phosphorylated smooth muscle myosin with skeletal muscle myosin which moves at 2 microns/s resulted in a decreased rate of bead movement, suggesting that the more slowly cycling smooth muscle myosin is primarily determining the velocity of movement in such mixtures.

scholarly journals Highly selective inhibition of myosin motors provides the basis of potential therapeutic application

2016 ◽  
Vol 113 (47) ◽  
pp. E7448-E7455 ◽  
Author(s):  
Serena Sirigu ◽  
James J. Hartman ◽  
Vicente José Planelles-Herrero ◽  
Virginie Ropars ◽  
Sheila Clancy ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Muscle Relaxation ◽  
Selective Inhibition ◽  
Pharmacological Agents ◽  
Skeletal Muscle Myosin ◽  
Starting Point ◽  
Myosin Motors ◽  
Muscle Myosin

Direct inhibition of smooth muscle myosin (SMM) is a potential means to treat hypercontractile smooth muscle diseases. The selective inhibitor CK-2018571 prevents strong binding to actin and promotes muscle relaxation in vitro and in vivo. The crystal structure of the SMM/drug complex reveals that CK-2018571 binds to a novel allosteric pocket that opens up during the “recovery stroke” transition necessary to reprime the motor. Trapped in an intermediate of this fast transition, SMM is inhibited with high selectivity compared with skeletal muscle myosin (IC50 = 9 nM and 11,300 nM, respectively), although all of the binding site residues are identical in these motors. This structure provides a starting point from which to design highly specific myosin modulators to treat several human diseases. It further illustrates the potential of targeting transition intermediates of molecular machines to develop exquisitely selective pharmacological agents.

scholarly journals Electron microscopy of synthetic myosin filaments. Evidence for cross-bridge. Flexibility and copolymer formation.

1975 ◽  
Vol 67 (1) ◽  
pp. 93-104 ◽  
Author(s):  
T D Pollard
Keyword(s):  
Skeletal Muscle ◽  
Smooth Muscle ◽  
Structural Features ◽  
Length Frequency ◽  
Single Population ◽  
Skeletal Muscle Myosin ◽  
Distinctive Features ◽  
Myosin Filaments ◽  
The Mean ◽  
Muscle Myosin

Electron micrographs of negatively stained synthetic myosin filaments reveal that surface projections, believed to be the heads of the constituent myosin molecules, can exist in two configurations. Some filaments have the projections disposed close to the filament backbone. Other filaments have all of their projections widely spread, tethered to the backbone by slender threads. Filaments formed from the myosins of skeletal muscle, smooth muscle, and platelets each have distinctive features, particularly their lengths. Soluble mixtures of skeletal muscle myosin with either smooth muscle myosin or platelet myosin were dialyzed against 0.1 M KC1 at pH 7 to determine whether the simultaneous presence of two types of myosin would influence the properties of the filaments formed. In every case, a single population of filaments formed from the mixtures. The resulting filaments are thought to be copolymers of the two types of myosin, for several reasons: (a) their length-frequency distribution is unimodal and differs from that predicted for a simple mixture of two types of myosin filaments; (b) their mean length is intermediate between the mean lengths of the filaments formed separately from the two myosins in the mixture; (c) each of the filaments has structural features characteristic of both of the myosins in the mixture; and (d) their size and shape are determined by the proportion of the two myosins in the mixture.

scholarly journals ATP-dependent movement of myosin in vitro: characterization of a quantitative assay.

1984 ◽  
Vol 99 (5) ◽  
pp. 1867-1871 ◽  
Author(s):  
M P Sheetz ◽  
R Chasan ◽  
J A Spudich
Keyword(s):  
Skeletal Muscle ◽  
Actin Filaments ◽  
Quantitative Assay ◽  
Vitro Assay ◽  
Skeletal Muscle Myosin ◽  
In Vitro Characterization ◽  
Actin Cables ◽  
Muscle Myosin

Sheetz and Spudich (1983, Nature (Lond.), 303:31-35) showed that ATP-dependent movement of myosin along actin filaments can be measured in vitro using myosin-coated beads and oriented actin cables from Nitella. To establish this in vitro movement as a quantitative assay and to understand better the basis for the movement, we have defined the factors that affect the myosin-bead velocity. Beads coated with skeletal muscle myosin move at a rate of 2-6 micron/s, depending on the myosin preparation. This velocity is independent of myosin concentration on the bead surface for concentrations above a critical value (approximately 20 micrograms myosin/2.5 X 10(9) beads of 1 micron in diameter). Movement is optimal between pH 6.8 and 7.5, at KCl concentrations less than 70 mM, at ATP concentrations greater than 0.1 mM, and at Mg2+ concentrations between 2 and 6 mM. From the temperature dependence of bead velocity, we calculate activation energies of 90 kJ/mol below 22 degrees C and 40 kJ/mol above 22 degrees C. Different myosin species move at their own characteristic velocities, and these velocities are proportional to their actin-activated ATPase activities. Further, the velocities of beads coated with smooth or skeletal muscle myosin correlate well with the known in vivo rates of myosin movement along actin filaments in these muscles. This in vitro assay, therefore, provides a rapid, reproducible method for quantitating the ATP-dependent movement of myosin molecules on actin.

scholarly journals Velocities of unloaded muscle filaments are not limited by drag forces imposed by myosin cross-bridges

2015 ◽  
Vol 112 (36) ◽  
pp. 11235-11240 ◽  
Author(s):  
Richard K. Brizendine ◽  
Diego B. Alcala ◽  
Michael S. Carter ◽  
Brian D. Haldeman ◽  
Kevin C. Facemyer ◽  
...  
Keyword(s):  
Duty Ratio ◽  
Step Size ◽  
Drag Forces ◽  
Contraction Speed ◽  
Myosin Filaments ◽  
Adp Release ◽  
Muscle Types ◽  
Cross Bridges ◽  
Muscle Myosin

It is not known which kinetic step in the acto-myosin ATPase cycle limits contraction speed in unloaded muscles (V0). Huxley’s 1957 model [Huxley AF (1957) Prog Biophys Biophys Chem 7:255–318] predicts that V0 is limited by the rate that myosin detaches from actin. However, this does not explain why, as observed by Bárány [Bárány M (1967) J Gen Physiol 50(6, Suppl):197–218], V0 is linearly correlated with the maximal actin-activated ATPase rate (vmax), which is limited by the rate that myosin attaches strongly to actin. We have observed smooth muscle myosin filaments of different length and head number (N) moving over surface-attached F-actin in vitro. Fitting filament velocities (V) vs. N to a detachment-limited model using the myosin step size d = 8 nm gave an ADP release rate 8.5-fold faster and ton (myosin’s attached time) and r (duty ratio) ∼10-fold lower than previously reported. In contrast, these data were accurately fit to an attachment-limited model, V = N·v·d, over the range of N found in all muscle types. At nonphysiologically high N, V = L/ton rather than d/ton, where L is related to the length of myosin’s subfragment 2. The attachment-limited model also fit well to the [ATP] dependence of V for myosin-rod cofilaments at three fixed N. Previously published V0 vs. vmax values for 24 different muscles were accurately fit to the attachment-limited model using widely accepted values for r and N, giving d = 11.1 nm. Therefore, in contrast with Huxley’s model, we conclude that V0 is limited by the actin–myosin attachment rate.

Distribution of phenotypically disparate myocyte subpopulations in airway smooth muscle

2005 ◽  
Vol 83 (1) ◽  
pp. 104-116 ◽  
Author(s):  
Andrew J Halayko ◽  
Gerald L Stelmack ◽  
Akira Yamasaki ◽  
Karol McNeill ◽  
Helmut Unruh ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Smooth Muscle ◽  
Airway Smooth Muscle ◽  
Lung Development ◽  
Similar Phenotype ◽  
Low Levels ◽  
Phenotype Heterogeneity ◽  
Muscle Myosin

Phenotype and functional heterogeneity of airway smooth muscle (ASM) cells in vitro is well known, but there is limited understanding of these features in vivo. We tested whether ASM is composed of myocyte subsets differing in contractile phenotype marker expression. We used flow cytometry to compare smooth muscle myosin heavy chain (smMHC) and smooth muscle-α-actin (sm-α-actin) abundance in myocytes dispersed from canine trachealis. Based on immunofluorescent intensity and light scatter characteristics (forward and 90° side scatter), 2 subgroups were identified and isolated. Immunoblotting confirmed smMHC and sm-α-actin were 10- and 5-fold greater, respectively, in large, elongate myocytes that comprised ~60% of total cells. Immunohistochemistry revealed similar phenotype heterogeneity in human bronchial smooth muscle. Canine tracheal myocyte subpopulations isolated by flow cytometry were used to seed primary subcultures. Proliferation of subcultures established with myocytes exhibiting low levels of smMHC and sm-α-actin was ~2× faster than subcultures established with ASM cells with a high marker protein content. These studies demonstrate broad phenotypic heterogeneity of myocytes in normal ASM tissue that is maintained in cell culture, as demonstrated by divergent proliferative capacity. The distinct roles of these subgroups could be a key determinant of normal and pathological lung development and biology.Key words: flow cytometry, phenotype, heterogeneity, asthma, differentiation.

scholarly journals Muscle myosins form folded monomers, dimers, and tetramers during filament polymerization in vitro

2020 ◽  
Vol 117 (27) ◽  
pp. 15666-15672
Author(s):  
Xiong Liu ◽  
Shi Shu ◽  
Edward D. Korn
Keyword(s):  
Electron Microscopy ◽  
Smooth Muscle ◽  
Muscle Contraction ◽  
Actin Filaments ◽  
Critical Concentration ◽  
Smooth Muscle Myosin ◽  
Myosin Filaments ◽  
Muscle Myosin

Muscle contraction depends on the cyclical interaction of myosin and actin filaments. Therefore, it is important to understand the mechanisms of polymerization and depolymerization of muscle myosins. Muscle myosin 2 monomers exist in two states: one with a folded tail that interacts with the heads (10S) and one with an unfolded tail (6S). It has been thought that only unfolded monomers assemble into bipolar and side-polar (smooth muscle myosin) filaments. We now show by electron microscopy that, after 4 s of polymerization in vitro in both the presence (smooth muscle myosin) and absence of ATP, skeletal, cardiac, and smooth muscle myosins form tail-folded monomers without tail–head interaction, tail-folded antiparallel dimers, tail-folded antiparallel tetramers, unfolded bipolar tetramers, and small filaments. After 4 h, the myosins form thick bipolar and, for smooth muscle myosin, side-polar filaments. Nonphosphorylated smooth muscle myosin polymerizes in the presence of ATP but with a higher critical concentration than in the absence of ATP and forms only bipolar filaments with bare zones. Partial depolymerization in vitro of nonphosphorylated smooth muscle myosin filaments by the addition of MgATP is the reverse of polymerization.

scholarly journals Smitin, a novel smooth muscle titin–like protein, interacts with myosin filaments in vivo and in vitro

2002 ◽  
Vol 156 (1) ◽  
pp. 101-112 ◽  
Author(s):  
Kyoungtae Kim ◽  
Thomas C.S. Keller
Keyword(s):  
Smooth Muscle ◽  
Ionic Strength ◽  
Smooth Muscle Cells ◽  
Muscle Cells ◽  
Smooth Muscle Myosin ◽  
Globular Domain ◽  
Contractile Apparatus ◽  
Myosin Filaments ◽  
Muscle Myosin

Smooth muscle cells use an actin–myosin II-based contractile apparatus to produce force for a variety of physiological functions, including blood pressure regulation and gut peristalsis. The organization of the smooth muscle contractile apparatus resembles that of striated skeletal and cardiac muscle, but remains much more poorly understood. We have found that avian vascular and visceral smooth muscles contain a novel, megadalton protein, smitin, that is similar to striated muscle titin in molecular morphology, localization in a contractile apparatus, and ability to interact with myosin filaments. Smitin, like titin, is a long fibrous molecule with a globular domain on one end. Specific reactivities of an anti-smitin polyclonal antibody and an anti-titin monoclonal antibody suggest that smitin and titin are distinct proteins rather than differentially spliced isoforms encoded by the same gene. Smitin immunofluorescently colocalizes with myosin in chicken gizzard smooth muscle, and interacts with two configurations of smooth muscle myosin filaments in vitro. In physiological ionic strength conditions, smitin and smooth muscle myosin coassemble into irregular aggregates containing large sidepolar myosin filaments. In low ionic strength conditions, smitin and smooth muscle myosin form highly ordered structures containing linear and polygonal end-to-end and side-by-side arrays of small bipolar myosin filaments. We have used immunogold localization and sucrose density gradient cosedimentation analyses to confirm association of smitin with both the sidepolar and bipolar smooth muscle myosin filaments. These findings suggest that the titin-like protein smitin may play a central role in organizing myosin filaments in the contractile apparatus and perhaps in other structures in smooth muscle cells.

scholarly journals Affinity for MgADP and force of unbinding from actin of myosin purified from tonic and phasic smooth muscle

2008 ◽  
Vol 295 (3) ◽  
pp. C653-C660 ◽  
Author(s):  
Renaud Léguillette ◽  
Nedjma B. Zitouni ◽  
Karuthapillai Govindaraju ◽  
Laura M. Fong ◽  
Anne-Marie Lauzon
Keyword(s):  
Smooth Muscle ◽  
Alternative Hypothesis ◽  
In Vitro Motility Assay ◽  
In Vitro Motility ◽  
Tonic Muscle ◽  
Latch State ◽  
Muscle Myosin ◽  
Phasic Smooth Muscle

Smooth muscle is unique in its ability to maintain force at low MgATP consumption. This property, called the latch state, is more prominent in tonic than phasic smooth muscle. Studies performed at the muscle strip level have suggested that myosin from tonic muscle has a greater affinity for MgADP and therefore remains attached to actin longer than myosin from phasic muscle, allowing for cross-bridge dephosphorylation and latch-bridge formation. An alternative hypothesis is that after dephosphorylation, myosin reattaches to actin and maintains force. We investigated these fundamental properties of smooth muscle at the molecular level. We used an in vitro motility assay to measure actin filament velocity (νmax) when propelled by myosin purified from phasic or tonic muscle at increasing [MgADP]. Myosin was 25% thiophosphorylated and 75% unphosphorylated to approximate in vivo conditions. The slope of νmax versus [MgADP] was significantly greater for tonic (−0.51 ± 0.04) than phasic muscle myosin (−0.15 ± 0.04), demonstrating the greater MgADP affinity of myosin from tonic muscle. We then used a laser trap assay to measure the unbinding force from actin of populations of unphosphorylated tonic and phasic muscle myosin. Both myosin types attached to actin, and their unbinding force (0.092 ± 0.022 pN for phasic muscle and 0.084 ± 0.017 pN for tonic muscle) was not statistically different. We conclude that the greater affinity for MgADP of tonic muscle myosin and the reattachment of dephosphorylated myosin to actin may both contribute to the latch state.

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