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PubMed1998

Deterioration of connectin/titin and nebulin filaments by an excess of protease inhibitors.

We studied the effect of protease inhibitors at a high concentration on connectin and nebulin filaments in myofibrils. Calpastatin domain I at 0.1 mM bound to connectin and nebulin filaments, and deteriorated their physico-chemical properties; the calcium-binding ability of connectin and nebulin filaments was suppressed, the susceptibility of both filaments to trypsin was markedly decreased, and the resting tension of mechanically skinned fibers was increased by 2.5 times that of the control at a sarcomere length of 3.6 microns. This indicates that the connectin filaments were made more rigid. The same phenomenon was observed from the treatment of skinned fibers with 1 mM leupeptin whose resting tension was increased to 2 times the control value. Microscopically, both protease inhibitors induced dense aggregation and disappearance of the regular striation of myofibrils due to their non-specific binding to many myofibrillar proteins. The use of excess calpastatin domain I and leupeptin should therefore be avoided in physiological and biochemical studies on connectin and nebulin filaments, as well as on myofibrils.

Animals↗
PubMed1997

Binding of the N-terminal 63 kDa portion of connectin/titin to alpha-actinin as revealed by the yeast two-hybrid system.

Connectin/titin is a 3000 kDa protein which links the myosin filament to the Z-line in vertebrate striated muscle sarcomeres. To search for the Z-line proteins to which connectin binds, the yeast two-hybrid system was applied using cDNA coding the N-terminal 63 kDa fragment of connectin. Two clones coding the C-terminal half region of alpha-actinin (amino acids, 343-897 and 446-897) were obtained. Enzyme-linked immunosorbent assay clearly demonstrated the interactions of alpha-actinin and the N-terminal 63 kDa fragment of connectin in vitro. Thus it is concluded that the N-terminal 63 kDa portion of connectin binds to alpha-actinin in the Z-line of myofibrillar sarcomeres.

Actinin↗
PubMed2006

Suppressed disassembly of autolyzing p94/CAPN3 by N2A connectin/titin in a genetic reporter system.

p94/calpain 3 is a skeletal muscle-specific member of the Ca(2+)-regulated cytosolic cysteine protease family, the calpains. Defective p94 protease activity originating from gene mutations causes a muscular dystrophy called calpainopathy, indicating the indispensability of p94 for muscle survival. Because of the existence of the p94-specific regions IS1 and IS2, p94 undergoes very rapid and exhaustive autolysis. To elucidate the physiological relevance of this unique activity, the autolytic profiles of p94 and the effect of the p94 binding protein, connectin/titin, on this process were investigated. In vitro analysis of p94 autolysis showed that autolysis in IS1 proceeds without immediate disassembly into fragments and that the newly identified cryptic autolytic site in IS2 is critical for disassembling autolyzed fragments. As a genetic system to assay p94 autolysis semiquantitatively, p94 was expressed in yeast as a hybrid protein between the DNA binding and activation domains of the yeast transcriptional activator Gal4. Transcriptional activation by the Gal4-p94:WT hybrid protein is precluded by p94 autolysis. Complete or partial loss of autolytic activity by C129S active site mutation, limb girdle muscular dystrophy type 2A pathogenic missense mutations, or PCR-based random mutagenesis could be detected by semiquantitative restoration of Gal4-dependent beta-galactosidase gene expression. Using this system, the N2A connectin fragment that binds to p94 was shown to suppress p94 autolytic disassembly. The proximity of the IS2 autolytic and connectin-binding sites in p94 suggested that N2A connectin suppresses IS2 autolysis. These data indicate the importance of p94-connectin interaction in the control of p94 functions by regulating autolytic decay of p94.

Animals↗
PubMed1999

Organization of connectin/titin filaments in sarcomeres of differentiating chicken skeletal muscle cells.

Very long, elastic connectin/titin molecules position the myosin filaments at the center of a sarcomere by linking them to the Z line. The behavior of the connectin filaments during sarcomere formation in differentiating chicken skeletal muscle cells was observed under a fluorescent microscope using the antibodies to the N terminal (located in the Z line), C terminal (M line), and C zone (myosin filament) regions ofconnectin and was compared to the incorporation of alpha-actinin and myosin into forming sarcomeres. In early stages of differentiating muscle cells, the N terminal region of connectin was incorporated into a stress fiber-like structure (SFLS) together with alpha-actinin to form dots, whereas the C terminal region was diffusely distributed in the cytoplasm. When both the C and N terminal regions formed striations in young myofibrils, the epitope to the C zone of A-band region, that is the center between the A-I junction and the M-line, initially was diffuse in appearance and later formed definite striations. It appears that it took some time for the N and C terminal regions of connectin to form a regular organization in a sarcomere. Thus the two ends of the connectin filaments were first fixed followed by the specific binding of the middle portion onto the myosin filament during sarcomere formation.

Animals↗
PubMed1989

Myofibril assembly is linked with vinculin, alpha-actinin, and cell-substrate contacts in embryonic cardiac myocytes in vitro.

The relationship of nascent myofibrils with the accumulation of adhesion plaque proteins and the formation of focal cell contacts was studied in embryonic chick cardiac myocytes in vitro. The cultures were double-stained with various combinations of the specific antiactin drug phalloidin and antibodies against vinculin, alpha-actinin, connectin (titin), myosin heavy chain, fibronectin, and desmin and examined under fluorescence and interference reflection microscopy. In the areas of myofibril assembly, vinculin and alpha-actinin plaques were formed at the ventral sarcolemmae. These areas overlapped with the sites of cell-to-substrate focal contacts and extracellular fibronectin. Because the myofibrils always ran in a straight line between these sites, polarized lines appeared to be generated within the cells in response to their physical (e.g., stress) and/or biochemical environment (e.g., adhesion plaque proteins). The possible presence of other factors cannot be ruled out for the proper alignment of myofibrils. As soon as myofibrils came to span between these adhesion sites, they exhibited typically mature cross-striated characteristics. Thus, the formation of these inferred lines has some relation to, or is in fact necessary for, the maturation of myofibrils, in addition to the directional arrangement of sarcomeric proteins. Additionally, synthesis and distribution of myosin and connectin were tightly linked during early developmental (premyofibril and myofibril) stages. The spatial deployment of desmin was not coupled with vinculin. Thus, connectin and desmin do not appear to form the initial scaffold of sarcomeres.

Actinin↗
PubMed1995

Trypanosoma cruzi: monoclonal antibody to cytoskeleton recognizes giant proteins of the flagellar attachment zone.

Cytoskeletal preparations of Trypanosoma cruzi trypomastigotes and epimastigotes contain a protein recognized by a monoclonal antibody (2G4) which is connected to the flagellar attachment zone of both stages of the parasite. Western blot analysis revealed that the antibody was able to recognize protein bands of molecular masses higher than 700 kDa up to 2500 kDa. These giant proteins do not seem to share sequences with beta-connectin since an anti-beta-connectin antibody did not recognize the T. cruzi proteins nor did the 2G4 monoclonal antibody recognize authentic beta-connectin. Immunofluorescence and immunogold electron microscopy provided evidence that this protein is located inside the cell body of the parasite, closely related to a corset of four microtubules known as subpellicular microtubule quartet. Immunogold labeling shows that the protein accompanies the flagellar attachment zone as long as the flagellum adheres to the cell body. It is proposed that these microtubule-associated proteins recognized by the 2G4 monoclonal antibody exist only in trypanosomatid forms having a junctional complex between the flagellum and the cell body and may act as transmembrane elements connecting the subpellicular microtubular quartet with the flagellum at the desmosome region.

Animals↗
PubMed1996

A 11.5-kb 5'-terminal cDNA sequence of chicken breast muscle connectin/titin reveals its Z line binding region.

A partial 5'-region cDNA (11.5 kb) of chicken breast muscle connectin/titin encoding 3752 amino acids was sequenced. The predicted amino acid sequence contains 31 immunoglobulin C2 motifs and 10 interdomains. The sequence suggests a skeletal muscle type of connectin isoforms. Immunoelectron microscopic studies using antisera raised against several products of the cDNA fragments expressed in E. coli revealed that a region of some 800 amino acids from the N terminus of connectin is involved in its binding to the Z line in a sarcomere.

Animals↗
PubMed1992

Spatial relationship of nebulin relative to other myofibrillar proteins during myogenesis in embryonic chick skeletal muscle cells in vitro.

The developmental expression of nebulin was studied in embryonic chick skeletal muscle cells in vitro by means of immunofluorescence microscopy. Initially nebulin appeared homogeneously or in a punctate form in the cytoplasm, and then it was assembled into I-Z-I-like complexes containing actin and alpha-actinin but not myosin and connectin (titin). Striated patterns of nebulin ('singlets') in myofibrils appeared simultaneously with those of alpha-actinin (Z-bands), myosin (A-bands) and connectin ('doublets'), but earlier than those of actin. After actin striations were formed as myofibrils matured, each nebulin band started to exhibit 'droplets'. The delayed development of nebulin compared to the I-Z-I brush formation and the myofibril maturation seems to indicate that this giant myofibrillar protein is unnecessary for both the initial (formation of I-Z-I-like structures) and the subsequent (regular alignment of myofibrils) phases of myofibrillogenesis.

Animals↗
PubMed1999

Genetic variability of foetal bovine myoblasts in primary culture.

Selected strains of adult bovines and those which either have high muscle growth capacity or are double-muscled present particular characteristics of muscle fibres and collagen at slaughter that favour meat tenderness. For double-muscled bovines, it has been shown that these characteristics originated during foetal life. However, no studies have been done to determine the origin of muscle growth superiority in bovine with high muscle growth capacity compared to those with a low muscle growth capacity. Therefore, the objective of this study was to examine the proliferation and differentiation phases of myoblasts in primary culture taken from high and low muscle growth capacity foetuses at 110 days post-conception. These cultures were analysed on 1, 2, 3, 4, 6, 8, 10 days of culture. The proliferation phase was monitored by appropriate marker antibodies. The differentiation was studied by immunocytochemistry with specific antibodies for foetal, I, II (IIa and IIb), I and IIb, I and IIa myosin heavy chains (MHCs) and connectin respectively, and by immunoblotting with desmin antibody. A higher proliferation, a lower fusion and a delayed differentiation of the 'late markers' namely MHCs fast (IIa and IIb) and connectin were shown in high muscle growth capacity foetuses compared to low capacity ones. The results indicate that the muscle growth superiority of high muscle growth capacity bovines seems, therefore, to have a similar foetal origin to that of double-muscled ones.

Animals↗
PubMed1997

Zeugmatin is part of the Z-band targeting region of titin.

Originally, zeugmatin was identified as a 600-800 kD muscle specific protein in Z-bands of cardiac and skeletal muscles by Maher et al. (1985). In this presentation we review our work on myofibrillogenesis and present evidence that zeugmatin is actually part of the Z-Band region of titin and that this region of titin plays an important role in the assembly of the Z-bands and myofibrils. Rhee et al. (1994) reported that during myofibrillogenesis, zeugmatin antibody localization is detected in fully formed Z-bands in the mature myofibrils, in the Z-bodies of the nascent myofibrils, but not in the Z-bodies of the premyofibrils. These observations lead to the suggestion that zeugmatin might be responsible for the fusion of the Z-bodies to form the solid Z-bands of the mature myofibrils (Rhee et al. 1994). As part of a study to test aspects of this model of myofibrillogenesis, we isolated a 1.8 kb cDNA from a chicken cardiac expression library using an anti-zeugmatin antibody (Turnacioglu et al., 1996). We found this chicken cDNA to be 60% identical at the amino acid level to a segment of the Z-band region of human cardiac titin (connectin) sequenced by Labeit and Kolmerer (1995). This homology along with Western blot analysis with purified titin, suggested that zeugmatin is in fact part of the N-terminal region of chicken titin. When expressed in non-muscle cells, Z1.1 product colocalized with the alpha-actinin in stress fiber dense bodies and focal adhesions. Cultures of non-muscle cells, skeletal myotubes and cardiomyocytes were also transfected with a fusion construct (Z1.1GFP) consisting of the Z1.1 kb cDNA linked to the cDNA for green fluorescent protein (GFP). The Z1.1 kb cDNA encodes only 362 of the approximately 2,000 amino acids which comprise the Z-band region of titin; nevertheless, the Z1.1GFP fusion protein targets in vivo to the alpha-actinin rich Z-bands of contracting myofibrils. A dominant negative phenotype was observed in living cells expressing high levels of this Z1.1GFP fusion protein with inhibition of myofibrillogenesis as well as the disassembly of preexisting myofibrils in these cells. These data indicate that the Z-band region of titin (connectin) plays an important role in organizing and maintaining the structure of the myofibril.

Animals↗
PubMed2006

Meat tenderizing effect of injecting encapsulated Ca2+ in liposome into rabbit before slaughter.

Encapsulated calcium in liposome (L-Ca) produced by using egg phosphatidyl choline in the laboratory was injected into rabbit to evaluate the effect of calcium injection on the ageing of meat. After injecting L-Ca into the blood vessels of rabbit to increase the Ca2+ concentration in the body for 24 h, the fragmentation rate of myofibrils was observed. The fragmentation rates in the loin from the control group and L-Ca injected group were 2.56% and 3.10% in 2 days, 12.27% and 16.18% in 6 days, and 33.56% and 49.60% in 10 days, respectively (p<0.05). SDS-PAGE patterns of connectin and nebulin show that the total degradation of connectin by the control group took longer than 2-3 days, while it was within 1 day for the L-Ca-injected group. The control group took 8-10 days for nebulin, while the L-Ca-injected group took 2-3 days for total degradation. These results indicate that, injecting L-Ca into rabbit was effective for reducing the ageing period of meat without resulting in any physical shock or contamination.

Abattoirs↗
PubMed1976

An elastic protein from the cortical layer of the sea-urchin egg.

An elastic protein was isolated from the cortical layer of the fertilized eggs of two species of sea-urchin. Young's modulus of the fiber of the sea-urchin protein was approximately 1-10(7) dyne/cm2. The amino acid compositions of the elastic protein from the two species of sea-urchin eggs were very similar to that of connectin, elastic protein from vertebrate skeletal muscle. The fluorescent antibody against muscle connectin stained the cortical layer of the sea-urchin egg. It appears that the elastic protein is responsible for the elasticity of the egg cortex.

Amino Acids↗
PubMed1999

Regulation of bovine satellite cell proliferation and differentiation by insulin and triiodothyronine.

Satellite cells activity contributes to postnatal muscle growth. Herein, we have studied the respective influence of insulin and triiodothyronine (T3) on the proliferation and differentiation of primary bovine satellite cells isolated from Semitendinosus muscle of Montbéliard steers. Under basal conditions, satellite cells proliferated until the fifth day of culture, began to fuse into myotubes and expressed differentiation markers such as connectin, myogenin, and myosin heavy chain (MHC) isoforms. Insulin behaved as an effective mitogen. Moreover, it promoted extensive myotube formation and enhanced differentiation as shown by an increase in the accumulation of differentiation markers. Maximal differentiation occurred with insulin physiological range concentrations. A delay in the stimulation of differentiation was registered with a high dose that promoted maximal proliferation. Conversely, T3 decreased cell proliferation in a dose-dependent manner. In addition, fusion and biochemical differentiation (accumulation of connectin, MyoD1, myogenin, and myosin heavy chain isoforms) were also enhanced. Bovine satellite cells seemed to respond differentially to insulin and T3 for proliferation. Interestingly, both hormones displayed a myogenic influence. Our observations suggest that both hormones could influence bovine satellite cells in vivo and contribute to the regulation of postnatal muscle growth.

Animals↗
PubMed2001

Sarcomeric visco-elasticity of chemically skinned skeletal muscle fibres of the rabbit at rest.

The giant muscle protein titin (connectin), contained in the gap filament that connect a thick filament to the Z-line in a sarcomere, is generally considered to be responsible for the passive force (tension) and visco-elasticity in resting striated muscle. However, whether it can account for all the features of the resting tension response remains unclear. In this paper, we examine the basic features of the 'sarcomeric visco-elasticity' in a single resting mammalian muscle fibre and attempt to account for various tension components on the basis of known structural features of a sarcomere. At sarcomere length of approximately 2.6 microm, the force response to a ramp stretch of 2-5% is complex but can be resolved into four functionally different components. The behaviour displayed by the components ranges from pure viscous type (directly proportional to stretch velocity, ranging from 0.1 to 30 lengths s(-1)) to predominantly elastic type (insensitive to stretch velocity at 1-2 s time scale); simulations show two components of visco-elasticity with characteristically different relaxation times. The velocity-sensitive components (only) are enhanced by filament lattice compression (dextran - 500 kD) and by increased medium viscosity (dextran - 12 kD); also, the relaxation time of visco-elasticity is longer with increased medium viscosity. Amplitude of all the components and the relaxation time of visco-elasticity are increased at longer sarcomere length (range approximately 2.5 - 3.0 microm). The study, and quantitative analyses, extend our previous work on intact muscle fibres and suggest that the velocity-sensitive tension components in intact sarcomere arise from interactions between sarcomeric filaments, filament segments and inter-filamentary medium; the two components of visco-elasticity arise from distinct regions of titin (connectin) molecules.

Animals↗
PubMed2003

The entire cDNA sequences of projectin isoforms of crayfish claw closer and flexor muscles and their localization.

Projectin is a giant protein related to twitchin and titin/connectin, that is found in arthropod striated muscle. The complete sequence of a 1 MDa projectin from Drosophila muscle was recently deduced from a thorough analysis of the genomic DNA (Southgate and Ayme-Southgate, 2001). Here we report the complete sequence for projectin from crayfish claw closer muscle (8625 residues; 962,634 Da). The N-terminal sequence contains 12 unique 19-residue repeats rich in glutamic acid (E) and lysine (K). This region, termed the EK region, is clearly distinguishable from the PEVK-like domain of Drosophila projectin. The sequence of crayfish flexor projectin differs from that of closer muscle projectin in that there is a 114-residue deletion and a 35-residue insertion in the N-terminal region. Immunofluorescence microscopy demonstrated that projectin is mainly localized within the sarcomeric A band in both closer and flexor muscles, although the N-terminal region was shown to extrude into the I band region. In the closer muscles, invertebrate connectin (D-titin) connects the Z line to the edge of the A band (Fukuzawa et al., 2001). We have shown that invertebrate connectin is also present in flexor muscle sarcomeres, although in very low abundance.

Amino Acid Motifs↗
PubMed1990

Elastic filaments in skeletal muscle revealed by selective removal of thin filaments with plasma gelsolin.

Muscle needs an elastic framework to maintain its mechanical stability. Removal of thin filaments in rabbit skeletal muscle with plasma gelsolin has revealed the essential features of elastic filaments. The selective removal of thin filaments was confirmed by staining with phalloidin-rhodamine for fluorescence microscopy, examination of arrowhead formation with myosin subfragment 1 by electron microscopy, and analysis by SDS-PAGE. Thin section electron microscopy revealed the elastic fine filaments (approximately 4 nm in diameter) connecting thick filaments and the Z line. After removal of thin filaments, both rigor stiffness and active tension generation were lost, but the resting tension remained. These observations indicate that the thin filament-free fibers maintain a framework composed of the serial connections of thick filaments, the elastic filaments, and the Z line, which gives passive elasticity to the contractile system of skeletal muscle. The resting tension that remained in the thin filament-free fibers was decreased by mild trypsin treatment. The only protein component that was digested in parallel with the decrease in the resting tension and the disappearance of the elastic filaments was alpha-connectin (also called titin 1), which was transformed from the alpha to the beta form (from titin 1 to 2, respectively). Thus, we conclude that the main protein component of the elastic filaments is alpha-connectin (titin 1).

Actin Cytoskeleton↗
PubMed1995

Determination of the protein components of native thin filaments isolated from natural actomyosin: nebulin and alpha-actinin are associated with actin filaments.

A new method was developed for extracting natural actomyosin with full length connectin (titin) and nebulin from rabbit skeletal muscle. To determine the protein components of native thin filaments, thin filaments were isolated from natural actomyosin by sedimentation in a H2O/D2O/sucrose density gradient. Both alpha-actinin and nebulin were cosedimented with actin filaments, while connectin was not. This shows that the native thin filaments contained alpha-actinin and nebulin. This indicates that the native thin filaments were more complicated than synthetic filaments reconstituted from actin, tropomyosin, and troponin. It is known that synthetic filaments are less Ca2+ sensitive than native thin filaments. This difference in Ca2+ sensitivity may be due to the differences in components and/or the different structures of native and synthetic filaments. The newly developed methods described here for extracting natural actomyosin and for isolating native thin filaments are useful for addressing these important problems related to the structure and function of native thin filaments.

Actinin↗
PubMed1996

A giant protein associated with the anterior pole of a trypanosomatid cell body skeleton.

A megadalton protein was found to be a cytoskeleton component of the promastigote forms of the flagellate Phytomonas serpens. This protein migrated on sodium dodecyl sulfate polyacrylamide gel electrophoresis as a doublet of polypeptides with a molecular mass similar to muscle beta-connectin (titin) 2500-3000 kDa. A polyclonal antibody raised against this protein reacts, by immunoblot analysis, with Phytomonas serpens and two others Phytomonas species. In addition, the Phytomonas serpens protein was immunoprecipitated after being metabolically labeled with [35S]methionine. This antibody did not cross-react with the cytoskeletal proteins of Trypanosoma cruzi, Crithidia luciliae thermophila, Crithidia fasciculata and Leptomonas samueli or with beta-connectin (titin). Indirect immunofluorescence microscopy analysis revealed a punctate fluorescence staining at the anterior region of the parasite's body skeleton. Moreover, immunogold electron microscopy of cytoskeletal preparations and of thin sections of whole cells indicates that the giant protein appears to cap the anterior end of the cell body microtubules at the level of the junctional complex. We suggest that this giant protein may serve as a linker between the cell body skeleton and the flagellum membrane.

Animals↗