Tropomodulin is associated with the free (pointed) ends of the thin filaments in rat skeletal muscle.

The length and spatial organization of thin filaments in skeletal muscle sarcomeres are precisely maintained and are essential for efficient muscle contraction. While the major structural components of skeletal muscle sarcomeres have been well characterized, the mechanisms that regulate thin filament length and spatial organization are not well understood. Tropomodulin is a new, 40.6-kD tropomyosin-binding protein from the human erythrocyte membrane skeleton that binds to one end of erythrocyte tropomyosin and blocks head-to-tail association of tropomyosin molecules along actin filaments. Here we show that rat psoas skeletal muscle contains tropomodulin based on immunoreactivity, identical apparent mobility on SDS gels, and ability to bind muscle tropomyosin. Results from immunofluorescence labeling of isolated myofibrils at resting and stretched lengths using anti-erythrocyte tropomodulin antibodies indicate that tropomodulin is localized at or near the free (pointed) ends of the thin filaments; this localization is not dependent on the presence of myosin thick filaments. Immunoblotting of supernatants and pellets obtained after extraction of myosin from myofibrils also indicates that tropomodulin remains associated with the thin filaments. 1.2-1.6 copies of muscle tropomodulin are present per thin filament in myofibrils, supporting the possibility that one or two tropomodulin molecules may be associated with the two terminal tropomyosin molecules at the pointed end of each thin filament. Although a number of proteins are associated with the barbed ends of the thin filaments at the Z disc, tropomodulin is the first protein to be specifically located at or near the pointed ends of the thin filaments. We propose that tropomodulin may cap the tropomyosin polymers at the pointed end of the thin filament and play a role in regulating thin filament length.

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Reduced thin filament length in nebulin-knockout skeletal muscle alters isometric contractile properties

AJP Cell Physiology ◽

10.1152/ajpcell.00503.2008 ◽

2009 ◽

Vol 296 (5) ◽

pp. C1123-C1132 ◽

Cited By ~ 43

Author(s):

David S. Gokhin ◽

Marie-Louise Bang ◽

Jianlin Zhang ◽

Ju Chen ◽

Richard L. Lieber

Keyword(s):

Skeletal Muscle ◽

Thin Filament ◽

Expression Patterns ◽

Nemaline Myopathy ◽

Myosin Heavy Chain Isoforms ◽

Filament Length ◽

Force Transmission ◽

Thin Filaments ◽

Tension Properties ◽

Tension Curves

Nebulin (NEB) is a large, rod-like protein believed to dictate actin thin filament length in skeletal muscle. NEB gene defects are associated with congenital nemaline myopathy. The functional role of NEB was investigated in gastrocnemius muscles from neonatal wild-type (WT) and NEB knockout (NEB-KO) mice, whose thin filaments have uniformly shorter lengths compared with WT mice. Isometric stress production in NEB-KO skeletal muscle was reduced by 27% compared with WT skeletal muscle on postnatal day 1 and by 92% on postnatal day 7, consistent with functionally severe myopathy. NEB-KO muscle was also more susceptible to a decline in stress production during a bout of 10 cyclic isometric tetani. Length-tension properties in NEB-KO muscle were altered in a manner consistent with reduced thin filament length, with length-tension curves from NEB-KO muscle demonstrating a 7.4% narrower functional range and an optimal length reduced by 0.13 muscle lengths. Expression patterns of myosin heavy chain isoforms and total myosin content did not account for the functional differences between WT and NEB-KO muscle. These data indicate that NEB is essential for active stress production, maintenance of functional integrity during cyclic activation, and length-tension properties consistent with a role in specifying normal thin filament length. Continued analysis of NEB's functional properties will strengthen the understanding of force transmission and thin filament length regulation in skeletal muscle and may provide insights into the molecular processes that give rise to nemaline myopathy.

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Thin filaments are not of uniform length in rat skeletal muscle.

The Journal of Cell Biology ◽

10.1083/jcb.96.1.100 ◽

1983 ◽

Vol 96 (1) ◽

pp. 100-103 ◽

Cited By ~ 15

Author(s):

L Traeger ◽

M A Goldstein

Keyword(s):

Skeletal Muscle ◽

Thin Filament ◽

Fast Muscle ◽

Filament Length ◽

Rat Skeletal Muscle ◽

Adult Rats ◽

Thin Filaments ◽

Filament Assembly ◽

Old Rats ◽

Two Stages

The variation in thin filament length was investigated in slow and fast muscle from adult and neonatal rats. Soleus (slow) muscle from adult, 3-, 7-, and 9-d-old rats, and extensor digitorum longus (EDL; fast) muscle from adult rats were serially cross-sectioned. The number of thin filaments per 0.06 microns2 (TF#) was counted for individual myofibrils followed from the H zone of one sarcomere, through the I-Z-I region, to the H zone of an adjacent sarcomere TF# was pooled by distance from the Z band or AI junction. In both adult muscles, thin filament length varied from 0.18 to 1.20 microns, with approximately 25% of the thin filaments less than 0.7 microns in length. In 7- and 9-d soleus, thin filament length ranged from 0.18 to 1.08 microns; except for the longest (0.18 to 1.20 microns) filaments, the distribution of thin filament lengths was similar to that in adult muscle. In 3-d soleus, thin filament length was more uniform, with less than 5% of the filaments shorter than 0.7 microns. In all neonatal muscles, there were approximately 15% fewer thin filaments per unit area as compared to adult muscles. We conclude: (a) In rat skeletal muscle, thin filaments are not of uniform length, ranging in length from 0.18 to 1.20 microns. (b) There may be two stages of thin filament assembly in neonatal muscle: between 3 and 7 d when short thin filaments may be preferentially or synthesized or inserted near the Z-band, and between 9 d and adult when thin filaments of all lengths may be synthesized or inserted into the myofibril.

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New Insights into the Structural Roles of Nebulin in Skeletal Muscle

Journal of Biomedicine and Biotechnology ◽

10.1155/2010/968139 ◽

2010 ◽

Vol 2010 ◽

pp. 1-6 ◽

Cited By ~ 15

Author(s):

Coen A. C. Ottenheijm ◽

Henk Granzier

Keyword(s):

Skeletal Muscle ◽

Thin Filament ◽

Force Production ◽

Nemaline Myopathy ◽

Structure And Function ◽

Filament Length ◽

Thin Filaments ◽

Muscle Structure ◽

And Function

One important feature of muscle structure and function that has remained relatively obscure is the mechanism that regulates thin filament length. Filament length is an important aspect of muscle function as force production is proportional to the amount of overlap between thick and thin filaments. Recent advances, due in part to the generation of nebulin KO models, reveal that nebulin plays an important role in the regulation of thin filament length. Another structural feature of skeletal muscle that is not well understood is the mechanism involved in maintaining the regular lateral alignment of adjacent sarcomeres, that is, myofibrillar connectivity. Recent studies indicate that nebulin is part of a protein complex that mechanically links adjacent myofibrils. Thus, novel structural roles of nebulin in skeletal muscle involve the regulation of thin filament length and maintaining myofibrillar connectivity. When these functions of nebulin are absent, muscle weakness ensues, as is the case in patients with nemaline myopathy with mutations in nebulin. Here we review these new insights in the role of nebulin in skeletal muscle structure.

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Titin isoform size is not correlated with thin filament length in rat skeletal muscle

Frontiers in Physiology ◽

10.3389/fphys.2014.00035 ◽

2014 ◽

Vol 5 ◽

Cited By ~ 10

Author(s):

Marion L. Greaser ◽

Jonathan M. Pleitner

Keyword(s):

Skeletal Muscle ◽

Thin Filament ◽

Filament Length ◽

Rat Skeletal Muscle

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Tropomodulin isoforms regulate thin filament pointed-end capping and skeletal muscle physiology

The Journal of General Physiology ◽

10.1085/jgp1355oia4 ◽

2010 ◽

Vol 135 (5) ◽

pp. i4-i4

Author(s):

David S. Gokhin ◽

Raymond A. Lewis ◽

Caroline R. McKeown ◽

Roberta B. Nowak ◽

Nancy E. Kim ◽

...

Keyword(s):

Skeletal Muscle ◽

Thin Filament ◽

Muscle Physiology ◽

End Capping ◽

Pointed End

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Troponin and Titin Coordinately Regulate Length-dependent Activation in Skinned Porcine Ventricular Muscle

The Journal of General Physiology ◽

10.1085/jgp.200709895 ◽

2008 ◽

Vol 131 (3) ◽

pp. 275-283 ◽

Cited By ~ 38

Author(s):

Takako Terui ◽

Munguntsetseg Sodnomtseren ◽

Douchi Matsuba ◽

Jun Udaka ◽

Shin'ichi Ishiwata ◽

...

Keyword(s):

Skeletal Muscle ◽

Thin Filament ◽

Left Ventricular ◽

Ventricular Muscle ◽

Passive Force ◽

Active Force ◽

Fast Skeletal Muscle ◽

Thin Filaments ◽

Cross Bridges ◽

Length Dependent Activation

We investigated the molecular mechanism by which troponin (Tn) regulates the Frank-Starling mechanism of the heart. Quasi-complete reconstitution of thin filaments with rabbit fast skeletal Tn (sTn) attenuated length-dependent activation in skinned porcine left ventricular muscle, to a magnitude similar to that observed in rabbit fast skeletal muscle. The rate of force redevelopment increased upon sTn reconstitution at submaximal levels, coupled with an increase in Ca2+ sensitivity of force, suggesting the acceleration of cross-bridge formation and, accordingly, a reduction in the fraction of resting cross-bridges that can potentially produce additional active force. An increase in titin-based passive force, induced by manipulating the prehistory of stretch, enhanced length-dependent activation, in both control and sTn-reconstituted muscles. Furthermore, reconstitution of rabbit fast skeletal muscle with porcine left ventricular Tn enhanced length-dependent activation, accompanied by a decrease in Ca2+ sensitivity of force. These findings demonstrate that Tn plays an important role in the Frank-Starling mechanism of the heart via on–off switching of the thin filament state, in concert with titin-based regulation.

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The nebulin repeat protein Lasp regulates I-band architecture and filament spacing in myofibrils

The Journal of Cell Biology ◽

10.1083/jcb.201401094 ◽

2014 ◽

Vol 206 (4) ◽

pp. 559-572 ◽

Cited By ~ 27

Author(s):

Isabelle Fernandes ◽

Frieder Schöck

Keyword(s):

Thin Filament ◽

Muscle Protein ◽

Nemaline Myopathy ◽

Single Amino Acid ◽

Actin Binding ◽

Filament Length ◽

Thin Filaments ◽

Single Amino Acid Change ◽

Filament Spacing

Mutations in nebulin, a giant muscle protein with 185 actin-binding nebulin repeats, are the major cause of nemaline myopathy in humans. Nebulin sets actin thin filament length in sarcomeres, potentially by stabilizing thin filaments in the I-band, where nebulin and thin filaments coalign. However, the precise role of nebulin in setting thin filament length and its other functions in regulating power output are unknown. Here, we show that Lasp, the only member of the nebulin family in Drosophila melanogaster, acts at two distinct sites in the sarcomere and controls thin filament length with just two nebulin repeats. We found that Lasp localizes to the Z-disc edges to control I-band architecture and also localizes at the A-band, where it interacts with both actin and myosin to set proper filament spacing. Furthermore, introducing a single amino acid change into the two nebulin repeats of Lasp demonstrated different roles for each domain and established Lasp as a suitable system for studying nebulin repeat function.

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Calcium ion-regulated thin filaments from vascular smooth muscle

Biochemical Journal ◽

10.1042/bj1850355 ◽

1980 ◽

Vol 185 (2) ◽

pp. 355-365 ◽

Cited By ~ 79

Author(s):

S B Marston ◽

R M Trevett ◽

M Walters

Keyword(s):

Skeletal Muscle ◽

Smooth Muscle ◽

Atpase Activity ◽

Thin Filament ◽

Troponin I ◽

Troponin C ◽

Myosin Atpase ◽

Protein G ◽

Thin Filaments ◽

Myosin Atpase Activity

Myosin and actin competition tests indicated the presence of both thin-filament and myosin-linked Ca2+-regulatory systems in pig aorta and turkey gizzard smooth-muscle actomyosin. A thin-filament preparation was obtained from pig aortas. The thin filaments had no significant ATPase activity [1.1 +/- 2.6 nmol/mg per min (mean +/- S.D.)], but they activated skeletal-muscle myosin ATPase up to 25-fold [500 nmol/mg of myosin per min (mean +/- S.D.)] in the presence of 10(-4) M free Ca2+. At 10(-8) M-Ca2+ the thin filaments activated myosin ATPase activity only one-third as much. Thin-filament activation of myosin ATPase activity increased markedly in the range 10(-6)-10(-5) M-Ca2+ and was half maximal at 2.7 × 10(-6) M (pCa2+ 5.6). The skeletal myosin-aorta-thin-filament mixture gave a biphasic ATPase-rate-versus-ATP-concentration curve at 10(-8) M-Ca2+ similar to the curve obtained with skeletal-muscle thin filaments. Thin filaments bound up to 9.5 mumol of Ca2+/g in the presence of MgATP2-. In the range 0.06-27 microM-Ca2+ binding was hyperbolic with an estimated binding constant of (0.56 +/- 0.07) x 10(6) M-1 (mean +/- S.D.) and maximum binding of 8.0 +/- 0.8 mumol/g (mean +/- S.D.). Significantly less Ca2+ bound in the absence of ATP. The thin filaments contained actin, tropomyosin and several other unidentified proteins. 6 M-Urea/polyacrylamide-gel electrophoresis at pH 8.3 showed proteins that behaved like troponin I and troponin C. This was confirmed by forming interspecific complexes between radioactive skeletal-muscle troponin I and troponin C and the aorta thin-filament proteins. The thin filaments contained at least 1.4 mumol of a troponin C-like protein/g and at least 1.1 mumol of a troponin I-like protein/g.

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