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

2014 ◽  
Vol 206 (4) ◽  
pp. 559-572 ◽  
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.

scholarly journals New Insights into the Structural Roles of Nebulin in Skeletal Muscle

2010 ◽  
Vol 2010 ◽  
pp. 1-6 ◽  
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.

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

2009 ◽  
Vol 296 (5) ◽  
pp. C1123-C1132 ◽  
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.

scholarly journals Experimentally Varying the Number of Super-Repeats in the Neb Gene of the Mouse - Assessing the Role of Nebulin in Thin Filament Length Regulation

2019 ◽  
Vol 116 (3) ◽  
pp. 551a
Author(s):  
Balazs Kiss ◽  
Paola Tonino ◽  
Justin Kolb ◽  
John E. Smith ◽  
Henk L. Granzier
Keyword(s):  
Thin Filament ◽  
Filament Length ◽  
Length Regulation

scholarly journals Skeletal muscle fiber atrophy: altered thin filament density changes slow fiber force and shortening velocity

2005 ◽  
Vol 288 (2) ◽  
pp. C360-C365 ◽  
Author(s):  
D. A. Riley ◽  
J. L. W. Bain ◽  
J. G. Romatowski ◽  
R. H. Fitts
Keyword(s):  
Thin Filament ◽  
Weight Bearing ◽  
Hindlimb Suspension ◽  
Shortening Velocity ◽  
Cross Sectional ◽  
Thin Filaments ◽  
Specific Tension ◽  
Fast Myosin ◽  
Working Length ◽  
Filament Spacing

Single skinned fibers from soleus and adductor longus (AL) muscles of weight-bearing control rats and rats after 14-day hindlimb suspension unloading (HSU) were studied physiologically and ultrastructurally to investigate how slow fibers increase shortening velocity ( V0) without fast myosin. We hypothesized that unloading and shortening of soleus during HSU reduces densities of thin filaments, generating wider myofilament separations that increase V0 and decrease specific tension (kN/m2). During HSU, plantarflexion shortened soleus working length 23%. AL length was unchanged. Both muscles atrophied as shown by reductions in fiber cross-sectional area. For AL, the 60% atrophy accounted fully for the 58% decrease in absolute tension (mN). In the soleus, the 67% decline in absolute tension resulted from 58% atrophy plus a 17% reduction in specific tension. Soleus fibers exhibited a 25% reduction in thin filaments, whereas there was no change in AL thin filament density. Loss of thin filaments is consistent with reduced cross bridge formation, explaining the fall in specific tension. V0 increased 27% in soleus but was unchanged in AL. The V0 of control and HSU fibers was inversely correlated ( R = −0.83) with thin filament density and directly correlated ( R = 0.78) with thick-to-thin filament spacing distance in a nonlinear fashion. These data indicate that reduction in thin filament density contributes to an increased V0 in slow fibers. Osmotically compacting myofilaments with 5% dextran returned density, spacing, and specific tension and slowed V0 to near-control levels and provided evidence for myofilament spacing modulating tension and V0.

scholarly journals The Caenorhabditis elegans muscle-affecting gene unc-87 encodes a novel thin filament-associated protein.

1994 ◽  
Vol 127 (1) ◽  
pp. 79-93 ◽  
Author(s):  
S Goetinck ◽  
R H Waterston
Keyword(s):  
Caenorhabditis Elegans ◽  
Gene Product ◽  
Thin Filament ◽  
Genomic Sequence ◽  
Muscle Protein ◽  
Thin Filaments ◽  
C Elegans ◽  
Neuronal Protein ◽  
And Function ◽  
Filament Protein

Mutations in the unc-87 gene of Caenorhabditis elegans affect the structure and function of bodywall muscle, resulting in variable paralysis. We cloned the unc-87 gene by taking advantage of a transposon-induced allele of unc-87 and the correspondence of the genetic and physical maps in C. elegans. A genomic clone was isolated that alleviates the mutant phenotype when introduced into unc-87 mutants. Sequence analysis of a corresponding cDNA clone predicts a 357-amino acid, 40-kD protein that is similar to portions of the vertebrate smooth muscle proteins calponin and SM22 alpha, the Drosophila muscle protein mp20, the deduced product of the C. elegans cDNA cm7g3, and the rat neuronal protein np25. Analysis of the genomic sequence and of various transcripts represented in a cDNA library suggest that unc-87 mRNAs are subject to alternative splicing. Immunohistochemistry of wildtype and mutant animals with antibodies to an unc-87 fusion protein indicates that the gene product is localized to the I-band of bodywall muscle. Studies of the UNC-87 protein in other muscle mutants suggest that the unc-87 gene product associates with thin filaments, in a manner that does not depend on the presence of the thin filament protein tropomyosin.

scholarly journals Cofilin loss in Drosophila contributes to myopathy through defective sarcomerogenesis and aggregate formation during muscle growth

2019 ◽  
Author(s):  
Mridula Balakrishnan ◽  
Shannon F. Yu ◽  
Samantha M. Chin ◽  
David B. Soffar ◽  
Stefanie E. Windner ◽  
...  
Keyword(s):  
Point Mutation ◽  
Muscle Function ◽  
Muscle Growth ◽  
Nemaline Myopathy ◽  
Aggregate Formation ◽  
Thin Filaments ◽  
Sarcomeric Protein ◽  
Thick Filaments

SUMMARYSarcomeres, the fundamental contractile units of muscles, are conserved structures composed of actin thin filaments and myosin thick filaments. How sarcomeres are formed and maintained is not well understood. Here, we show that knockdown of Drosophila Cofilin (DmCFL), an actin depolymerizing factor, leads to the progressive disruption of sarcomere structure and muscle function in vivo. Loss of DmCFL also results in the formation of sarcomeric protein aggregates and impairs sarcomere addition during growth. Strikingly, activation of the proteasome delayed muscle deterioration in our model. Further, we investigate how a point mutation in CFL2 that causes nemaline myopathy (NM) in humans, affects CFL function and leads to the muscle phenotypes observed in vivo. Our data provide significant insights to the role of CFLs during sarcomere formation as well as mechanistic implications for disease progression in NM patients.

scholarly journals Role of thin-filament regulatory proteins in relaxation of colonic smooth muscle contraction

2009 ◽  
Vol 297 (5) ◽  
pp. G958-G966 ◽  
Author(s):  
Sita Somara ◽  
Robert Gilmont ◽  
Khalil N. Bitar
Keyword(s):  
Smooth Muscle ◽  
Muscle Contraction ◽  
Thin Filament ◽  
Smooth Muscle Contraction ◽  
Regulatory Proteins ◽  
Actin Binding ◽  
Thin Filament Regulation ◽  
Actomyosin Interaction ◽  
Regulation Of Contraction

Coordinated regulation of smooth muscle contraction and relaxation is required for colonic motility. Contraction is associated with phosphorylation of myosin light chain (MLC20) and interaction of actin with myosin. Thin-filament regulation of actomyosin interaction is modulated by two actin-binding regulatory proteins: tropomyosin (TM) and caldesmon (CaD). TM and CaD are known to play crucial role in actomyosin interaction promoting contraction. Contraction is associated with phosphorylation of the small heat shock protein HSP27, concomitant with the phosphorylation of TM and CaD. Phosphorylation of HSP27 is attributed as being the prime modulator of thin-filament regulation of contraction. Preincubation of colonic smooth muscle cells (CSMC) with the relaxant neurotransmitter vasoactive intestinal peptide (VIP) showed inhibition in phosphorylation of HSP27 (ser78). Attenuation of HSP27 phosphorylation can result in modulation of thin-filament-mediated regulation of contraction leading to relaxation; thus the role of thin-filament regulatory proteins in a relaxation milieu was investigated. Preincubation of CSMC with VIP exhibited a decrease in phosphorylation of TM and CaD. Furthermore, CSMC preincubated with VIP showed a reduced association of TM with HSP27 and with phospho-HSP27 (ser78) whereas there was reduced dissociation of TM from CaD and from phospho-CaD. We thus propose that, in addition to alteration in phosphorylation of MLC20, relaxation is associated with alterations in thin-filament-mediated regulation that results in termination of contraction.

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

1993 ◽  
Vol 120 (2) ◽  
pp. 411-420 ◽  
Author(s):  
V M Fowler ◽  
M A Sussmann ◽  
P G Miller ◽  
B E Flucher ◽  
M P Daniels
Keyword(s):  
Skeletal Muscle ◽  
Thin Filament ◽  
Spatial Organization ◽  
Membrane Skeleton ◽  
Filament Length ◽  
Human Erythrocyte Membrane ◽  
Thin Filaments ◽  
Muscle Tropomyosin ◽  
Immunofluorescence Labeling ◽  
Pointed End

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.

scholarly journals The number of Z-repeats and super-repeats in nebulin greatly varies across vertebrates and scales with animal size

2020 ◽  
Vol 153 (3) ◽  
Author(s):  
Jochen Gohlke ◽  
Paola Tonino ◽  
Johan Lindqvist ◽  
John E. Smith ◽  
Henk Granzier
Keyword(s):  
Binding Site ◽  
Thin Filament ◽  
Muscle Protein ◽  
Isometric Force ◽  
Force Production ◽  
Actin Binding ◽  
Skeletal Muscle Protein ◽  
Shortening Velocity ◽  
Large Animals ◽  
Simple Repeats

Nebulin is a skeletal muscle protein that associates with the sarcomeric thin filaments and has functions in regulating the length of the thin filament and the structure of the Z-disk. Here we investigated the nebulin gene in 53 species of birds, fish, amphibians, reptiles, and mammals. In all species, nebulin has a similar domain composition that mostly consists of ∼30-residue modules (or simple repeats), each containing an actin-binding site. All species have a large region where simple repeats are organized into seven-module super-repeats, each containing a tropomyosin binding site. The number of super-repeats shows high interspecies variation, ranging from 21 (zebrafish, hummingbird) to 31 (camel, chimpanzee), and, importantly, scales with body size. The higher number of super-repeats in large animals was shown to increase thin filament length, which is expected to increase the sarcomere length for optimal force production, increase the energy efficiency of isometric force production, and lower the shortening velocity of muscle. It has been known since the work of A.V. Hill in 1950 that as species increase in size, the shortening velocity of their muscle is reduced, and the present work shows that nebulin contributes to the mechanistic basis. Finally, we analyzed the differentially spliced simple repeats in nebulin's C terminus, whose inclusion correlates with the width of the Z-disk. The number of Z-repeats greatly varies (from 5 to 18) and correlates with the number of super-repeats. We propose that the resulting increase in the width of the Z-disk in large animals increases the number of contacts between nebulin and structural Z-disk proteins when the Z-disk is stressed for long durations.

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