scholarly journals Fiber-type-specific expression of essential (alkali) myosin light chains in human skeletal muscles.

1996 ◽  
Vol 44 (10) ◽  
pp. 1141-1152 ◽  
Author(s):  
K Jostarndt ◽  
A Puntschart ◽  
H Hoppeler ◽  
R Billeter
Keyword(s):  
Light Chain ◽  
Skeletal Muscles ◽  
Myosin Light Chain ◽  
Fiber Type ◽  
Expression Patterns ◽  
Light Chains ◽  
Myosin Light Chains ◽  
Type I ◽  
Specific Expression ◽  
Human Skeletal Muscles

We studied the expression patterns of the essential (alkali) myosin light-chain isoforms in adult human skeletal muscles, using in situ hybridization and single-fiber protein analysis. In analogy to other species, we found that the fiber type-specific expression of essential myosin light chains is regulated via the availability of the respective mRNAs in a given fiber. In contrast to other species, the slow isoform 1sa was only expressed in the most oxidative Type I fibers (Subtype IA) in addition to 1sb. These fibers also contained high levels of carbonic anhydrase III. Within the fibers, the essential myosin light-chain mRNAs were located preferentially in the perinuclear regions and to a lesser extent in the intermyofibrillar spaces, a distribution that excludes cotranslational assembly of these light chains into the myofibrils as the main mechanism. In comparing leg and shoulder muscles, we found less distinct fiber typing in the expression patterns of the essential myosin light chains in the leg muscles than in muscles from the shoulder region.

scholarly journals Fast myosin light chain expression in chicken muscles studied by in situ hybridization.

1992 ◽  
Vol 40 (10) ◽  
pp. 1547-1557 ◽  
Author(s):  
R Billeter ◽  
M Messerli ◽  
E Wey ◽  
A Puntschart ◽  
K Jostarndt ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Fiber Type ◽  
Light Chains ◽  
Myosin Light Chains ◽  
Two Dimensional ◽  
Fast Myosin ◽  
Chicken Muscles

We have studied the fiber type-specific expression of the fast myosin light chain isoforms LC 1f, LC 2f, and LC 3f in adult chicken muscles using in situ hybridization and two-dimensional gel electrophoresis. Type II (fast) fibers contain all three fast myosin light chain mRNAs; Types I and III (slow) fibers lack them. The myosin light chain patterns of two-dimensional gels from microdissected single fibers match their mRNA signals in the in situ hybridizations. The results confirm and extend previous studies on the fiber type-specific distribution of myosin light chains in chicken muscles which used specific antibodies. The quantitative ratios between protein and mRNA content were not the same for all three fast myosin light chains, however. In bulk muscle samples, as well as in single fibers, there was proportionally less LC 3f accumulated for a given mRNA concentration than LC 1f or LC 2f. Moreover, the ratio between LC 3f mRNA and protein was different in samples from muscles, indicating that LC 3f is regulated somewhat differently than LC 1f and LC 2f. In contrast to other in situ hybridization studies on the fiber type-specific localization of muscle protein mRNAs, which reported the RNAs to be located preferentially at the periphery of the fibers, we found all three fast myosin light chain mRNAs quite evenly distributed within the fiber's cross-sections, and also in the few rare fibers which showed hybridization signals several-fold higher than their surrounding counterparts. This could indicate principal differences in the intracellular localization among the mRNAs coding for various myofibrillar protein families.

Myosin light chains in normal and pathological human skeletal muscles

1983 ◽  
Vol 6 (1) ◽  
pp. 40-47 ◽  
Author(s):  
Fran�oise Pons ◽  
Jocelyne Leger ◽  
Michel Georgesco ◽  
Fran�ois Bonnel ◽  
Jean J. Leger
Keyword(s):  
Skeletal Muscles ◽  
Light Chains ◽  
Myosin Light Chains ◽  
Human Skeletal Muscles

scholarly journals Embryonic chicken skeletal, cardiac, and smooth muscles express a common embryo-specific myosin light chain.

1985 ◽  
Vol 100 (6) ◽  
pp. 2025-2030 ◽  
Author(s):  
H Takano-Ohmuro ◽  
T Obinata ◽  
M Kawashima ◽  
T Masaki ◽  
T Tanaka
Keyword(s):  
Light Chain ◽  
Gel Electrophoresis ◽  
Myosin Light Chain ◽  
Smooth Muscles ◽  
Light Chains ◽  
Myosin Light Chains ◽  
Striated Muscles ◽  
Two Dimensional Gel Electrophoresis ◽  
Embryonic Chicken ◽  
Muscle Myosin

It has been demonstrated that embryonic chicken gizzard smooth muscle contains a unique embryonic myosin light chain of 23,000 mol wt, called L23 (Katoh, N., and S. Kubo, 1978, Biochem. Biophys. Acta, 535:401-411; Takano-Ohmuro, H., T. Obinata, T. Mikawa, and T. Masaki, 1983, J. Biochem. (Tokyo), 93:903-908). When we examined myosins in developing chicken ventricular and pectoralis muscles by two-dimensional gel electrophoresis, the myosin light chain (Le) that completely comigrates with L23 was detected in both striated muscles at early developmental stages. Two monoclonal antibodies, MT-53f and MT-185d, were applied to characterize the embryonic light chain Le of striated muscles. Both monoclonal antibodies were raised to fast skeletal muscle myosin light chains; the former antibody is specific to fast muscle myosin light chains 1 and 3, whereas the latter recognizes not only fast muscle myosin light chains but also the embryonic smooth muscle light chain L23. The immunoblots combined with both one- and two-dimensional gel electrophoresis showed that Le reacts with MT-185d but not with MT-53f. These results strongly indicate that Le is identical to L23 and that embryonic chicken skeletal, cardiac, and smooth muscles express a common embryo-specific myosin light chain.

scholarly journals Myosin light chain kinase-regulated endothelial cell contraction: the relationship between isometric tension, actin polymerization, and myosin phosphorylation.

1995 ◽  
Vol 130 (3) ◽  
pp. 613-627 ◽  
Author(s):  
Z M Goeckeler ◽  
R B Wysolmerski
Keyword(s):  
Endothelial Cell ◽  
Light Chain ◽  
Isometric Contraction ◽  
Myosin Light Chain Kinase ◽  
Myosin Light Chain ◽  
Myosin Ii ◽  
Isometric Tension ◽  
Stress Fibers ◽  
Light Chains ◽  
Myosin Light Chains

The phosphorylation of regulatory myosin light chains by the Ca2+/calmodulin-dependent enzyme myosin light chain kinase (MLCK) has been shown to be essential and sufficient for initiation of endothelial cell retraction in saponin permeabilized monolayers (Wysolmerski, R. B. and D. Lagunoff. 1990. Proc. Natl. Acad. Sci. USA. 87:16-20). We now report the effects of thrombin stimulation on human umbilical vein endothelial cell (HUVE) actin, myosin II and the functional correlate of the activated actomyosin based contractile system, isometric tension development. Using a newly designed isometric tension apparatus, we recorded quantitative changes in isometric tension from paired monolayers. Thrombin stimulation results in a rapid sustained isometric contraction that increases 2- to 2.5-fold within 5 min and remains elevated for at least 60 min. The phosphorylatable myosin light chains from HUVE were found to exist as two isoforms, differing in their molecular weights and isoelectric points. Resting isometric tension is associated with a basal phosphorylation of 0.54 mol PO4/mol myosin light chain. After thrombin treatment, phosphorylation rapidly increases to 1.61 mol PO4/mol myosin light chain within 60 s and remains elevated for the duration of the experiment. Myosin light chain phosphorylation precedes the development of isometric tension and maximal phosphorylation is maintained during the sustained phase of isometric contraction. Tryptic phosphopeptide maps from both control and thrombin-stimulated cultures resolve both monophosphorylated Ser-19 and diphosphorylated Ser-19/Thr-18 peptides indicative of MLCK activation. Changes in the polymerization of actin and association of myosin II correlate temporally with the phosphorylation of myosin II and development of isometric tension. Activation results in a 57% increase in F-actin content within 90 s and 90% of the soluble myosin II associates with the reorganizing F-actin. Furthermore, the disposition of actin and myosin II undergoes striking reorganization. F-actin initially forms a fine network of filaments that fills the cytoplasm and then reorganizes into prominent stress fibers. Myosin II rapidly forms discrete aggregates associated with the actin network and by 2.5 min assumes a distinct periodic distribution along the stress fibers.

Effect of muscle length on isometric stress and myosin light chain phosphorylation in gallbladder smooth muscle

1991 ◽  
Vol 260 (6) ◽  
pp. G920-G924 ◽  
Author(s):  
R. J. Washabau ◽  
M. B. Wang ◽  
C. L. Dorst ◽  
J. P. Ryan
Keyword(s):  
Smooth Muscle ◽  
Guinea Pig ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Muscle Length ◽  
Stress Sensitivity ◽  
Light Chains ◽  
Myosin Light Chains ◽  
Myosin Light Chain Phosphorylation ◽  
Myofilament Activation

These experiments were designed to characterize the effect of muscle length on isometric stress, sensitivity to stimulation, and phosphorylation of the 20,000-Da myosin light chains in guinea pig gallbladder smooth muscle. Basal, active, and total isometric stress were determined in acetylcholine- or K(+)-treated (10(-4) M ACh, 80 mM KCl) muscle strips at 0.6-1.3 times the optimal muscle length (Lo) for isometric stress development. The effect of muscle length on the sensitivity to ACh and K+ was determined in cumulative dose-response experiments (10(-8) to 10(-4) M ACh, 10-80 mM KCl) at 0.7, 1.0, and 1.3 Lo. The effect of muscle length on myosin light chain phosphorylation was determined in ACh- or K(+)-treated (10(-4) M ACh, 80 mM KCl) muscle strips at 0.7, 1.0, and 1.3 Lo. In gallbladder smooth muscle, 1) active isometric stresses at 0.7 and 1.3 Lo were less than active isometric stress at 1.0 Lo; 2) the sensitivity of developed stress was similar at 1.0 and 1.3 Lo but decreased at 0.7 Lo; 3) the decline in isometric stress and sensitivity at 0.7 Lo was associated with reduced levels of phosphorylated myosin light chain; and 4) the decline in isometric stress at 1.3 Lo was not associated with reduced amounts of phosphorylated myosin light chain. These results suggest that the decline in active stress and sensitivity at short muscle lengths (L less than Lo) in gallbladder smooth muscle is due, at least in part, to decreases in the activation of the myofilaments. The decline in active isometric stress at long muscle lengths (L greater than Lo) is not due to changes in myofilament activation.

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.

Dictyostelium discoideum myosin: isolation and characterization of cDNAs encoding the regulatory light chain

1989 ◽  
Vol 9 (7) ◽  
pp. 3073-3080
Author(s):  
S R Tafuri ◽  
A M Rushforth ◽  
E R Kuczmarski ◽  
R L Chisholm
Keyword(s):  
Amino Acid ◽  
Light Chain ◽  
Base Pair ◽  
Calcium Binding ◽  
Expression Patterns ◽  
Light Chains ◽  
Myosin Light Chains ◽  
Base Pairs ◽  
Isolation And Characterization ◽  
Skeletal Myosin

Phosphorylation of the regulatory light chains (RMLC) of nonmuscle myosin can increase the actin-activated ATPase activity and filament formation. Little is known about these regulatory mechanisms and how the RMLC are involved in ATP hydrolysis. To better characterize the nonmuscle RMLC, we isolated cDNAs encoding the Dictyostelium RMLC. Using an antibody specific for the RMLC, we screened a lambda gt11 expression library and obtained a 200-base-pair clone that encoded a portion of the RMLC. The remainder of the sequence was obtained from two clones identified by DNA hybridization, using the 200-base-pair cDNA. The composite RMLC cDNA was 645 nucleotides long. It contained 60 base pairs of 5' untranslated, 483 bases of coding, and 102 base pairs of 3' untranslated sequence. The amino acid sequence predicted an 18,300-dalton protein that shares 42% amino acid identity with Dictyostelium calmodulin and 30% identity with the chicken skeletal myosin RMLC. This sequence contained three regions that were similar to the E-F hand calcium-binding domains found in calmodulin, troponin C, and other myosin light chains. A sequence similar to the phosphorylation sequence found in chicken gizzard and skeletal myosin light chains was found at the amino terminus. Genomic Southern blot analysis suggested that the Dictyostelium genome contains a single gene encoding the RMLC. Analysis of RMLC expression patterns during Dictyostelium development indicated that accumulation of this mRNA increases just before aggregation and again during culmination. This pattern is similar to that obtained for the Dictyostelium essential myosin light chain and suggests that expression of the two light chains is coordinated during development.

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