Orientation and Motion of Myosin Light Chain and Troponin in Reconstituted Muscle Fibers as Detected by ESR with a New Bifunctional Spin Label

2003 ◽  
pp. 279-284 ◽  
Author(s):  
Toshiaki Arata ◽  
Motoyoshi Nakamura ◽  
Hidenobu Akahane ◽  
Tomoki Aihara ◽  
Shoji Ueki ◽  
...  
Keyword(s):  
Light Chain ◽  
Spin Label ◽  
Myosin Light Chain ◽  
Muscle Fibers

scholarly journals Fiber type- and position-dependent expression of a myosin light chain-CAT transgene detected with a novel histochemical stain for CAT.

1991 ◽  
Vol 115 (2) ◽  
pp. 423-434 ◽  
Author(s):  
M J Donoghue ◽  
J D Alvarez ◽  
J P Merlie ◽  
J R Sanes
Keyword(s):  
Light Chain ◽  
Transgene Expression ◽  
Myosin Light Chain ◽  
Fiber Type ◽  
Muscle Fibers ◽  
Single Type ◽  
Regulatory Sequences ◽  
Type I ◽  
Type Ii ◽  
Light Chain Gene

We recently generated and characterized transgenic mice in which regulatory sequences from a myosin light chain gene (MLC1f/3f) are linked to the chloramphenicol acetyltransferase (CAT) gene. Transgene expression in these mice is specific to skeletal muscle and graded along the rostrocaudal axis: adult muscles derived from successively more caudal somites express successively higher levels of CAT. To investigate the cellular basis of these patterns of expression, we developed and used a histochemical stain that allows detection of CAT in individual cells. Our main results are as follows: (a) Within muscles, CAT is detected only in muscle fibers and not in associated connective tissue, blood vessels, or nerves. Thus, the tissue specificity of transgene expression observed by biochemical assay reflects a cell-type specificity demonstrable histochemically. (b) Within individual muscles, CAT levels vary with fiber type. Like the endogenous MLC1f/3f gene, the transgene is expressed at higher levels in fast-twitch (type II) than in slow-twitch (type I) muscle fibers. In addition, CAT levels vary among type II fiber subtypes, in the order IIB greater than IIX greater than IIA. (c) Among muscles that are similar in fiber type composition, the average level of CAT per fiber varies with rostrocaudal position. This position-dependent variation in CAT level is apparent even when fibers of a single type are compared. From these results, we conclude that fiber type and position affect CAT expression independently. We therefore infer the existence of separate fiber type-specific and positionally graded transcriptional regulators that act together to determine levels of transgene expression.

Effect of Ca2+-independent myosin light chain kinase on different skinned smooth muscle fibers

1984 ◽  
Vol 401 (1) ◽  
pp. 107-109 ◽  
Author(s):  
M. Gagelmann ◽  
U. Mrwa ◽  
S. Bostrom ◽  
J. C. R�egg ◽  
D. Hartshorne
Keyword(s):  
Smooth Muscle ◽  
Light Chain ◽  
Myosin Light Chain Kinase ◽  
Myosin Light Chain ◽  
Muscle Fibers ◽  
Skinned Smooth Muscle

scholarly journals Myosin light chain 3f attenuates age-induced decline in contractile velocity in MHC type II single muscle fibers

Aging Cell ◽  
2011 ◽  
Vol 11 (2) ◽  
pp. 203-212 ◽  
Author(s):  
Jong-Hee Kim ◽  
Windy S. Torgerud ◽  
Kelsey H. H. Mosser ◽  
Hiroyuki Hirai ◽  
Shuichi Watanabe ◽  
...  
Keyword(s):  
Light Chain ◽  
Myosin Light Chain ◽  
Muscle Fibers ◽  
Single Muscle ◽  
Type Ii

scholarly journals Myosin Orientation in a Muscle Fiber Determined with High Angular Resolution Using Bifunctional Spin Labels

2019 ◽  
Author(s):  
Yahor Savich ◽  
Benjamin P. Binder ◽  
Andrew R. Thompson ◽  
David D. Thomas
Keyword(s):  
Muscle Fiber ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Angular Resolution ◽  
Muscle Fibers ◽  
Fiber Axis ◽  
Myosin Regulatory Light Chain ◽  
Paramagnetic Resonance ◽  
Lever Arm ◽  
Electron Paramagnetic

ABSTRACTWe have measured the orientation of the myosin light chain domain (lever arm) elements in demembranated muscle fibers by electron paramagnetic resonance (EPR), using a bifunctional spin label (BSL), with angular resolution of 4 degrees. Despite advances in X-ray crystallography and cryo-electron microscopy (cryo-EM), and fluorescence polarization, none of these techniques provide high-resolution structural information about the myosin light chain domain under ambient conditions in a muscle fiber. Two cysteines, 4 residues apart, were engineered on two α-helices in the myosin regulatory light chain (RLC), permitting stereoselective site-directed labeling with BSL. One labeled helix (helix E) is adjacent to the myosin lever arm, the other helix (helix B) is located farther apart from the motor domain beyond the “hinge” of the myosin. By exchanging BSL-labeled RLC onto oriented muscle fibers, we obtained EPR spectra that determined angular distributions of BSL with high resolution, which enabled the accurate determination of helix orientation of individual structural elements with respect to the muscle fiber axis. In the absence of ATP (rigor), each of the two labeled helices exhibited both ordered (σ ~ 9-11 degrees) and disordered (σ > 38 degrees) populations. We used these angles to determine the orientation of the myosin lever arm, concluding that the oriented population has lever arms that are perpendicular to the muscle fiber axis. This orientation is ~33 degrees different than predicted from a standard “lever arm down” model based on cryo-EM of actin decorated with isolated myosin heads, but it is compatible with fluorescence polarization and EM data obtained from muscle fibers. The addition of ATP, in the absence of Ca2+, shifted the orientation to a much more disordered distribution.SummaryWe used electron paramagnetic resonance to determine the orientation of elements within the myosin regulatory light chain in skinned skeletal muscle fibers. A bifunctional spin label provided sufficient resolution to detect an ordered population of lever arms perpendicular to actin.

scholarly journals Myosin lever arm orientation in muscle determined with high angular resolution using bifunctional spin labels

2019 ◽  
Vol 151 (8) ◽  
pp. 1007-1016 ◽  
Author(s):  
Yahor Savich ◽  
Benjamin P. Binder ◽  
Andrew R. Thompson ◽  
David D. Thomas
Keyword(s):  
High Resolution ◽  
Muscle Fiber ◽  
Light Chain ◽  
Fluorescence Polarization ◽  
Myosin Light Chain ◽  
Angular Resolution ◽  
Muscle Fibers ◽  
Fiber Axis ◽  
X Ray ◽  
Lever Arm

Despite advances in x-ray crystallography, cryo-electron microscopy (cryo-EM), and fluorescence polarization, none of these techniques provide high-resolution structural information about the myosin light chain domain (LCD; lever arm) under ambient conditions in vertebrate muscle. Here, we measure the orientation of LCD elements in demembranated muscle fibers by electron paramagnetic resonance (EPR) using a bifunctional spin label (BSL) with an angular resolution of 4°. To achieve stereoselective site-directed labeling with BSL, we engineered a pair of cysteines in the myosin regulatory light chain (RLC), either on helix E or helix B, which are roughly parallel or perpendicular to the myosin lever arm, respectively. By exchanging BSL-labeled RLC onto oriented muscle fibers, we obtain EPR spectra from which the angular distributions of BSL, and thus the lever arm, can be determined with high resolution relative to the muscle fiber axis. In the absence of ATP (rigor), each of the two labeled helices exhibits both ordered (σ ∼9–11°) and disordered (σ > 38°) populations. Using these angles to determine the orientation of the lever arm (LCD combined with converter subdomain), we observe that the oriented population corresponds to a lever arm that is perpendicular to the muscle fiber axis and that the addition of ATP in the absence of Ca2+ (inducing relaxation) shifts the orientation to a much more disordered orientational distribution. Although the detected orientation of the myosin light chain lever arm is ∼33° different than predicted from a standard “lever arm down” model based on cryo-EM of actin decorated with isolated myosin heads, it is compatible with, and thus augments and clarifies, fluorescence polarization, x-ray interference, and EM data obtained from muscle fibers. These results establish feasibility for high-resolution detection of myosin LCD rotation during muscle contraction.

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