scholarly journals Evidence for S2 flexibility by direct visualization of quantum dot–labeled myosin heads and rods within smooth muscle myosin filaments moving on actin in vitro

2021 ◽  
Vol 153 (3) ◽  
Author(s):  
Richard K. Brizendine ◽  
Murali Anuganti ◽  
Christine R. Cremo
Keyword(s):  
Coiled Coil ◽  
Geometric Constraints ◽  
The Other ◽  
Direct Visualization ◽  
Myosin Filaments ◽  
Different Color ◽  
Distance Working ◽  
Muscle Myosin ◽  
Kinetics Of

Myosins in muscle assemble into filaments by interactions between the C-terminal light meromyosin (LMM) subdomains of the coiled-coil rod domain. The two head domains are connected to LMM by the subfragment-2 (S2) subdomain of the rod. Our mixed kinetic model predicts that the flexibility and length of S2 that can be pulled away from the filament affects the maximum distance working heads can move a filament unimpeded by actin-attached heads. It also suggests that it should be possible to observe a head remain stationary relative to the filament backbone while bound to actin (dwell), followed immediately by a measurable jump upon detachment to regain the backbone trajectory. We tested these predictions by observing filaments moving along actin at varying ATP using TIRF microscopy. We simultaneously tracked two different color quantum dots (QDs), one attached to a regulatory light chain on the lever arm and the other attached to an LMM in the filament backbone. We identified events (dwells followed by jumps) by comparing the trajectories of the QDs. The average dwell times were consistent with known kinetics of the actomyosin system, and the distribution of the waiting time between observed events was consistent with a Poisson process and the expected ATPase rate. Geometric constraints suggest a maximum of ∼26 nm of S2 can be unzipped from the filament, presumably involving disruption in the coiled-coil S2, a result consistent with observations by others of S2 protruding from the filament in muscle. We propose that sufficient force is available from the working heads in the filament to overcome the stiffness imposed by filament-S2 interactions.

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

2015 ◽  
Vol 108 (2) ◽  
pp. 296a-297a
Author(s):  
Richard Brizendine ◽  
Diego Alcala ◽  
Brian Haldeman ◽  
Kevin Facemyer ◽  
Josh Baker ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Myosin ◽  
Myosin Filaments ◽  
Muscle Myosin ◽  
Kinetics Of

Métabolisme de l'acide indolyl-3-acétique dans les cultures de cellules d'Acer pseudoplatanus cultivés in vitro

1977 ◽  
Vol 55 (19) ◽  
pp. 2530-2534 ◽  
Author(s):  
F. Maillard ◽  
J.-P. Zrÿd
Keyword(s):  
Cell Wall ◽  
Acetic Acid ◽  
Culture Medium ◽  
Degradation Products ◽  
Cell Suspensions ◽  
The Other ◽  
Acer Pseudoplatanus ◽  
Kinetics Of

Incubation of cell suspensions of sycamore (Acer pseudoplatanus) with β-indoyl-3-acetic acid (IAA) first led to the formation of IAA-glycosides, then to that of IAA-aspartate. Great differences are observed between the kinetics of IAA transformed by two distinct strains: one, auxin dependent (S), the other, auxin independent (MB). Other degradation products are only found in the culture medium. The localization of IAA-degrading systems in the cell wall is postulated. The auxin requirement of the S strain is discussed.

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 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.

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 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.

A comparative study on the conditions of growth and sporulation of three strains of Clostridium petfringens type A

1996 ◽  
Vol 42 (3) ◽  
pp. 298-304 ◽  
Author(s):  
Mathilde Decaudin ◽  
Jean-Luc Tholozan
Keyword(s):  
Clostridium Perfringens ◽  
Slow Growth ◽  
Type A ◽  
High Volume ◽  
The Other ◽  
Additional Information ◽  
Mutant Strains ◽  
Dependent Strain ◽  
Kinetics Of

Different conditions of growth and sporulation of a strain of Clostridium perfringens type A (NCTC 8798) and two derived mutant strains, the lysozyme-germination dependent strain 8-6 and the revertant strain R3, have been determined. No sporulation was detected for the three strains in the Duncan and Strong (DS) medium; 100% sporulation was routinely obtained for the two mutant strains in the defined (D) medium. Factors promoting in vitro sporulation of C. perfringens type A were assayed: the volume of the culture, the type of preculture, and the addition of lysozyme in precultures. The paper also provides additional information on growth and sporulation of the mutant strains 8-6 and R3. Glucose concentrations up to 11 mM produced high percentages of sporulation. However, strain R3 still sporulated at 20% with 56 mM of glucose. A high volume of D medium led to slow growth kinetics and favoured sporulation. Faster kinetics of growth and the best percentage of sporulation were obtained with a young inoculum of the two mutant strains. On the other hand, the type of medium in the preculture (fluid thioglycollate (FTG) or basal carbonate yeast trypticase (BCYT)) did not influence the percentage of sporulation. However, while strain R3 was not affected by the addition of lysozyme in D medium, kinetics of growth were strongly influenced by this addition in strain 8-6, and the percentage of sporulation increased with a preculture in FTG medium and decreased when BCYT medium was used.Key words: Clostridium perfringens, medium, growth, sporulation.

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

2005 ◽  
Vol 83 (10) ◽  
pp. 899-912 ◽  
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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