Varying the Number of Heads in Phosphorylated Smooth Muscle Myosin Filaments Provides Evidence for Attachment Limited Kinetics of in vitro Actin-Sliding Velocities

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.

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

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2001892117 ◽

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.

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

The Journal of Cell Biology ◽

10.1083/jcb.200107037 ◽

2002 ◽

Vol 156 (1) ◽

pp. 101-112 ◽

Cited By ~ 32

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.

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Polylysine binding to unphosphorylated smooth muscle myosin enhances formation and stabilizes myosin filaments in vitro

Acta Physiologica Scandinavica ◽

10.1046/j.1365-201x.2002.00950.x ◽

2002 ◽

Vol 174 (4) ◽

pp. 337-346

Author(s):

P. T. SZYMANSKI ◽

D. G. FERGUSON ◽

R. J. PAUL

Keyword(s):

Smooth Muscle ◽

Smooth Muscle Myosin ◽

Myosin Filaments ◽

Muscle Myosin

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Vectorial activation of smooth muscle myosin filaments and its modulation by telokin

Canadian Journal of Physiology and Pharmacology ◽

10.1139/y05-053 ◽

2005 ◽

Vol 83 (10) ◽

pp. 899-912 ◽

Cited By ~ 1

Author(s):

Apolinary Sobieszek

Keyword(s):

Smooth Muscle ◽

Light Chain ◽

Smooth Muscle Myosin ◽

Activation Mechanism ◽

Myosin Filament ◽

Activation Process ◽

Diffusional Process ◽

Myosin Filaments ◽

Muscle Myosin

Smooth muscle myosin copurifies with myosin light chain kinase (MLCK) and calmodulin (CaM) as well as with variable amounts of myosin phosphatase. Therefore, myosin filaments formed in vitro also contain relatively high levels of these enzymes. Thus these filaments may be considered to be native-like because they are similar to those expected to exist in vivo. These endogenous enzymes are present at high concentrations relative to myosin, sufficient for rapid phosphorylation and dephosphorylation of the filaments at rates comparable to those observed for contraction and relaxation in intact muscle strips. The phosphorylation by MLCK/CaM complex appears to exhibit some directionality and is not governed by a random diffusional process. For the mixtures of myosin filaments with and without the endogenous MLCK/CaM complex, the complex preferentially phosphorylates its own parent filament at a higher rate than the neighboring filaments. This selective or vectorial-like activation is lost or absent when myosin filaments are dissolved at high ionic strength. Similar vectorial-like activation is exhibited by the reconstituted filament suspensions, but the soluble systems composed of isolated regulatory light chain or soluble myosin head subfragments exhibit normal diffusional kinetic behavior. At physiological concentrations, kinase related protein (telokin) effectively modulates the activation process by reducing the phosphorylation rate of the filaments without affecting the overall phosphorylation level. This results from telokin-induced liberation of the active MLCK/CaM complex from the filaments, so that the latter can also activate the neighboring filaments via a slower diffusional process. When this complex is bound at insufficient levels, this actually results in acceleration of the initial phosphorylation rates. In short, I suggest that in smooth muscle, telokin plays a chaperone role for myosin and its filaments.Key words: smooth muscle, regulation, myosin filament, phosphorylation, activation mechanism, myosin kinase, phosphatase, telokin.

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Assembly of smooth muscle myosin into side-polar filaments.

The Journal of Cell Biology ◽

10.1083/jcb.75.3.990 ◽

1977 ◽

Vol 75 (3) ◽

pp. 990-996 ◽

Cited By ~ 105

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.

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Filamentous smooth muscle myosin is regulated by phosphorylation.

The Journal of Cell Biology ◽

10.1083/jcb.109.6.2887 ◽

1989 ◽

Vol 109 (6) ◽

pp. 2887-2894 ◽

Cited By ~ 72

Author(s):

K M Trybus

Keyword(s):

Smooth Muscle ◽

Steady State ◽

Smooth Muscle Myosin ◽

Heavy Meromyosin ◽

Phosphate Release ◽

Product Release ◽

Single Turnover ◽

Myosin Filaments ◽

Muscle Myosin ◽

Low Rate

The enzymatic activity of filamentous dephosphorylated smooth muscle myosin has been difficult to determine because the polymer disassembles to the folded conformation in the presence of MgATP. Monoclonal antirod antibodies were used here to "fix" dephosphorylated myosin in the filamentous state. The steady-state actin-activated ATPase of phosphorylated filaments was 30-100-fold higher than that of antibody-stabilized dephosphorylated filaments, suggesting that phosphorylation can activate ATPase activity independent of changes in assembly. The degree of regulation may exceed 100-fold, because steady-state measurements slightly overestimate the rate of product release from dephosphorylated filaments. Single-turnover experiments in the absence of actin showed that although dephosphorylated folded myosin released products at the low rate of 0.0005 s-1 (Cross, R. A., K. E. Cross, A. Sobieszek. 1986. EMBO [Eur. Mol. Biol. Organ.] J. 5:2637-2641) the rate of product release from dephosphorylated filaments was only 3-12-fold higher, depending on the ionic strength. The addition of actin did not increase this rate to any appreciable extent. Dephosphorylated filaments and dephosphorylated heavy meromyosin (Sellers, J. R. 1985. J. Biol. Chem. 260:15815-15819) thus have similar low rates of phosphate release both in the presence and absence of actin. These results show that light chain phosphorylation alone, without invoking other mechanisms, is an effective switch for regulating the activity of smooth muscle myosin filaments.

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