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PubMed1984

Connectin filaments in stretched skinned fibers of frog skeletal muscle.

Indirect immunofluorescence microscopy of highly stretched skinned frog semi-tendinous muscle fibers revealed that connectin, an elastic protein of muscle, is located in the gap between actin and myosin filaments and also in the region of myosin filaments except in their centers. Electron microscopic observations showed that there were easily recognizable filaments extending from the myosin filaments to the I band region and to Z lines in the myofibrils treated with antiserum against connectin. In thin sections prepared with tannic acid, very thin filaments connected myosin filaments to actin filaments. These filaments were also observed in myofibrils extracted with a modified Hasselbach-Schneider solution (0.6 M KCl, 0.1 M phosphate buffer, pH 6.5, 2 mM ATP, 2 mM MgCl2, and 1 mM EGTA) and with 0.6 M Kl. SDS PAGE revealed that connectin (also called titin) remained in extracted myofibrils. We suggest that connectin filaments play an important role in the generation of tension upon passive stretch. A scheme of the cytoskeletal structure of myofibrils of vertebrate skeletal muscle is presented on the basis of our present information of connectin and intermediate filaments.

Animals↗

PubMed1989

Binding of connectin to myosin filaments.

Binding of native connectin (2,100 kDa fragment of alpha-connectin) to myosin filaments was investigated using a sedimentation technique and densitometric estimations of the separated proteins. In the presence of 60 mM KCl and 5 mM phosphate buffer, pH 7.0, as much as 1.5 mol of connectin was bound to 1 mol of myosin, suggesting that some 150 connectin filaments bound to a single myosin filament of approximately 0.5 micron in length. This value was much more than the ratio found in muscle (12:1). It appeared that C protein did not affect the binding of connectin to myosin filaments.

Animals↗

PubMed1994

Purification and characterization of the 1,200-kDa subfragment of connectin filaments produced by 0.1 mM calcium ions.

When myofibrils prepared from chicken leg muscle were treated with a solution containing 0.1 mM CaCl2 and 30 micrograms/ml of leupeptin, alpha-connectin, which exists as a longitudinal thin filament in a sarcomere, was split into beta-connectin and a 1,200-kDa subfragment. The native subfragment was successfully purified without using any denaturant: It was extracted with 1 M KI solution from the Ca-treated myofibrils and purified by TSKgel G6000PW column chromatography. About 10 mg of the subfragment was yielded from 100 g of starting muscle. Using immunofluorescence microscopy and immunoelectron microscopy, we show here that polyclonal antibodies against the 1,200-kDa subfragment bind to the Z-disk and the epitope, which is about 0.34 micron apart from the Z-disk at a sarcomere length of 2.6 microns; the 1,200-kDa subfragment constitutes the proximal region of connectin filaments. Purified alpha-actinin decorated alpha-connectin and the 1,200-kDa subfragment on nitrocellulose blots of myofibrillar proteins separated by SDS-PAGE. Therefore, we conclude that connectin filaments are anchored to the Z-disk by the binding of the 1,200-kDa subfragment to alpha-actinin.

Actinin↗

PubMed1981

Connectin, an elastic protein of muscle. Effects of proteolytic enzymes in situ.

The effects of various proteolytic enzymes on the high molecular weight protein (connectin) present in a direct sodium dodecyl sulfate extract of myofibrils from chicken breast muscle were studied in detail. To keep the high molecular weight proteins intact, myofibrils had to be prepared in the presence of EGTA. Trypsin, chymotrypsin, papain, and nagarse readily hydrolyzed connectin (doublet band of titin) and the band 3 protein (N2-line protein). Pepsin did not attack connectin, but digested the band 3 protein and myosin. Calcium-activated neutral proteinase hydrolyzed the band 3 protein, leaving connectin intact. On the other hand, serine protease digested connectin but not the band 3 protein.

Animals↗

PubMed1983

Preparation of native connectin from chicken breast muscle.

Connectin, an elastic protein of striated muscle, was isolated in a native state from chicken breast muscle. Myofibrils were well washed with 5 mM NaHCO3 and extracted with 0.1 M sodium phosphate buffer, pH 5.6. After washing with water, myofibrils were extracted with 0.1 M phosphate buffer, pH 6.6. The filtrate contained native connectin. The yield of connectin was as high as 200 mg, starting from 50 g of muscle. Since connectin had been isolated only in a denatured state before, the new preparation method enables investigation of possible interactions of connectin with other myofibrillar structural proteins.

Amino Acids↗

PubMed1984

Proteolytic fragments of connectin cause aggregation of myosin filaments but not of actin filaments.

Proteolytic fragments of 400 kD isolated from chymotrypsin-treated connectin, a muscle elastic protein, still retained the ability to cause aggregation of myosin filaments but lost the actin-bundling action. Tryptic digests of connectin showed similar effects. However, when connectin was hydrolyzed by pepsin to peptides smaller than approximately 40 kD, no such action was seen for both myosin and actin filaments. It is suggested that the actin bundling action of connectin filaments is due to topological restrictions. A modified reproducible procedure for the preparation of native connectin from chicken breast muscle is described in detail.

Actins↗

PubMed1986

Sodium dodecyl sulfate gel electrophoresis studies of connectin-like high molecular weight proteins of various types of vertebrate and invertebrate muscles.

Using an SDS gel electrophoresis method, connectin, very high molecular weight (approximately 10(6) dalton) protein, was detected in an SDS extract of whole tissues of various types of muscles of vertebrates and invertebrates. Connectin bands were clearly recognized in all the types of striated muscles (skeletal and cardiac) of the vertebrates examined: rabbit, chicken, turtle, snake, newt, frog, and fish. This was also the case with skeletal muscle of prochordate, Amphioxus. In invertebrates, the situation was much complicated. Connectin-like protein bands were detected in C. elegans (nematode), but not in earthworm (annelid). Smaller sizes of proteins (approximately 10(6)) were faintly found in molluscan adductor muscles. In arthropods, connectin-like proteins were clearly detected in some muscles (e.g., claw muscles of crab and crayfish; leg muscles of several insects) but not at all in other muscles (e.g., tail muscles of crayfish and shrimp; thoracic muscles of some insects). These peculiar observations might be related to the presence of such specific elastic proteins as projectin in honeybee flight muscle. The present study has revealed that connectin is an elastic protein of vertebrate striated muscle, skeletal and cardiac muscles.

Animals↗

PubMed2002

Profiles of connectin (titin) in atrophied soleus muscle induced by unloading of rats.

Responses of the properties of connectin molecules in the slow-twitch soleus (Sol) and fast-twitch extensor digitorum longus muscles of rats to 3 days of unloading with or without 3-day reloading were investigated. The wet weight (relative to body wt) of Sol, not of extensor digitorum longus, in the unloaded group was significantly less than in the age-matched control (P < 0.05). Immunoelectron microscopic analyses showed that a monoclonal antibody against connectin (SM1) bound to the I-band region close to the edge of the A band at resting length and moved reversibly away from the Z line as the muscle fibers were stretched. In Sol, the displacement of the SM1-bound dense spots in response to stretching decreased after hindlimb suspension. There were no changes in the molecular weights and the percent distributions of alpha- and beta-connectin in both muscles after hindlimb suspension. A significant increment of percent beta-connectin in Sol was observed after 3 days of reloading after hindlimb suspension (P < 0.05). It is suggested that the elasticity of connectin filaments in the I-band region of the atrophied Sol fibers was reduced relative to that of the control fibers. The lack of the elasticity in atrophied muscle fibers may cause a decrease in contractile function.

Animals↗

PubMed1996

Molecular cloning of a partial cDNA clone encoding the C terminal region of chicken breast muscle connectin.

The cDNA sequence encoding the C terminal region of chicken skeletal muscle connectin was described. Its predicted amino acid sequence had 1,021 amino acids comprising six motif Ils (Immunoglobulin C2 domain) and five interdomains. The sequence showed 70-75% homology with that of human cardiac connectin, but 168 amino acids including one motif II were missing in chicken skeletal muscle connectin. The C terminal sequence of chicken skeletal muscle connectin reported by the previous work (Maruyama et al., 1994) was erroneous due to the accidental ligation of the cDNA clone encoding a N terminal region of connectin with a partial porin cDNA clone.

Amino Acid Sequence↗

PubMed1979

Post-mortem changes in skeletal muscle connectin.

Changes in connectin and elasticity of skeletal muscle were determined during post-mortem ageing. The amount of connectin decreased with increasing time of post-mortem storage whereas the rate of the decrease depended on the source of muscles. The loss in elasticity of muscle coincided well with the decrease in connectin contents. Electron microscopically, a network structure between the Z discs vanished when the amount of connectin fell to zero. We have concluded that the continuous net structure of connectin is responsible for about 30% of the total elasticity of living skeletal muscle and its degradtaion is responsible for post-mortem tenderization of meat.

Animals↗

PubMed1993

Distribution of connectin (titin), nebulin and alpha-actinin at myotendinous junctions of chicken pectoralis muscles: an immunofluorescence and immunoelectron microscopic study.

The distribution of connectin (titin), nebulin and alpha-actinin in the areas of myotendinous junctions of chicken pectoralis muscles was examined by immunocytochemical methods. Staining with antibodies against connectin (4C9, SM1 and P1200) and nebulin formed 'doublets' flanking nonterminal Z-bands; near the end of muscle fibres 'singlets' were seen within the terminal sarcomere on the side adjacent to the terminal Z-bands. The apical regions of muscle processes, where no myosin filaments are present although actin filaments exist, were reactive with anti-nebulin but not with anti-connectin. Antibodies against pectoralis (skeletal muscle type) alpha-actinin stained non terminal Z-bands and that against gizzard (smooth muscle type) the sarcolemma. Terminal Z-bands were unreactive with both of these antibodies. These findings indicate that, although terminal and nonterminal Z-bands differ in their molecular composition, connectin and nebulin filaments appear to link myosin and actin filaments, respectively, to both Z-band types.

Actinin↗

PubMed2005

Possible functions of p94 in connectin-mediated signaling pathways in skeletal muscle cells.

Calpains are intracellular Ca2+ -requiring 'modulator proteases', which modulate cellular functions by limited and specific proteolysis. p94/calpain3, a skeletal-muscle specific calpain, has been one of the representative calpain species which indicates physiological importance of calpain proteolytic system; a defect of proteolytic activity of p94 causes limb girdle muscular dystrophy type2A (LGMD2A, also called 'calpainopathy'). Immunohistochemical studies on myofibrils showed that p94 localizes at the Z- and N2-line regions of sarcomeres. It was also identified by the yeast two hybrid studies that p94 binds to the N2A and M-line regions of connectin. Furthermore, genetic studies indicate that p94 is indispensable for skeletal muscles, although its precise functions are still unclear. Interestingly, connectin provides sarcomere not only with elasticity but also with binding sites to various multi-functional proteins such as muscle ankyrin repeat proteins (MARPs), muscle RING finger proteins (MURFs), titin-capping protein (T-cap/telethonin), sarcomeric-alpha-actinin, p94 etc. Binding sites for these proteins are not randomly placed along connectin but rather accumulated in the Z-, N2-, and/or M-line regions, indicating the existence of 'signal complexes' unique to each regions. The concept of these complexes are strongly supported by the facts that mutations of connectin or its binding proteins in these regions severely perturb muscle functions, as in the case of LGMD2A caused by mutations in the p94 gene. Therefore, it is hypothesized that the 'signal complexes' in the Z-, N2-, and M-lines modulate muscle cell homeostasis by transducing signals of external stimulations/stresses to trigger appropriate response at various different cellular events such as protein modification and gene expressions. In this article, we performed detailed immunohistochemical analyses of p94 on isolated single myofibers. Together with recent findings about p94, it is suggested that sarcomeric localization of p94, especially its M-line localization, is affected by the combination of cellular contexts such as contractile status of myofibrils, fiber type compositions, sarcomeric maturation, and the composition of the 'signal complexes' in each region.

Actinin↗

PubMed1994

Connectin, an elastic protein of striated muscle.

Connectin, also called titin, a giant elastic protein of striated muscle (approximately 3000 kDa) mainly consists of fibronectin type III and immunoglobulin C2 globular domains, the beta-sheets of which are parallel to the main axis of the molecule. One connectin molecule runs through the I band and binds onto the myosin filament up to the M line starting from the Z line. It positions the myosin filament at the center of a sarcomere. Connectin is also responsible for resting tension generation. Biodiversity of the connectin family exists in invertebrate muscle.

Animals↗

PubMed1985

Connectin filaments link thick filaments and Z lines in frog skeletal muscle as revealed by immunoelectron microscopy.

In an earlier study connectin, an elastic protein of striated muscle, was found to be associated with "gap filaments" originating from the thick filaments in the myofibril, but it was not clear whether it extends to Z lines or not (Maruyama, K., H. Sawada, S. Kimura, K. Ohashi, H. Higuchi, and Y. Umazume, 1984, J. Cell Biol., 99:1391-1397). In the present immunoelectron microscopic study using polyclonal antibodies against native connectin, we have concluded that the connectin structures are directly linked to Z lines from the thick (myosin) filaments in myofibrils of skinned fibers of frog skeletal muscle. There were five distinct antibody-binding stripes in each half of the A band and two stripes in the A-I junction region. Deposits of antibodies were recognized in I bands and Z lines. We suggest that connectin filaments run alongside the thick filaments, starting from a region approximately 0.15 micron from the center of the A band.

Animals↗

PubMed1991

Isolation and characterization of 1,200 kDa peptide of alpha-connectin.

When rabbit skeletal muscle myofibrils were kept for 12 h at 4 degrees C, alpha-connectin was partially degraded and 1,200 kDa peptide was newly formed [Takahashi, K. & Takai, H. (1988) Abst. 80th Jpn. Soc. Zootech. Sci. p-102]. The latter was isolated together with remaining alpha-connectin. Ultracentrifugation of the mixture at low ionic strength resulted in sedimentation of alpha-connectin, leaving the 1,200 kDa peptide in the supernatant. Physicochemical properties of the isolated 1,200 kDa peptide were investigated: UV absorption spectra, circular dichroism spectra, amino acid composition, and molecular size and shape. Polyclonal antibodies against the 1,200 kDa peptide [PcAb(1200)] bound the Z line and I-band. The position of the stripe in the I band near the N1 line due to the binding of PcAb(1200) moved both away from the Z lines and from the A band as sarcomeres were elongated. Therefore, it is considered that the 1,200 kDa portion of alpha-connectin is elastic.

Actins↗

PubMed1994

A partial connectin cDNA encoding a novel type of RSP motifs isolated from chicken embryonic skeletal muscle.

A partial cDNA encoding 811 amino acids of connectin (titin), a giant elastic protein of muscle (3,000 kDa), was cloned from a chicken embryonic skeletal muscle cDNA library using antibodies to muscle connectin. The encoded product was the C terminal segment of connectin. The predicted sequences consisted of 5 type II motifs (immunoglobulin C2 type) separated by 5 interdomain insertions. One interdomain insertion had significant homology (RSP) to KSP repeats found in human cardiac C-terminal connectin and another had a high sequence homology to porin (67.7%; 31 amino acids).

Amino Acid Sequence↗

PubMed1992

Connectin, a target of homeotic gene control in Drosophila.

The homeotic genes of Drosophila encode transcription factors that specify morphological differences between segments. To identify the genes that they control, we developed a chromatin immunopurification approach designed to isolate in vivo binding sites for the products of the homeotic gene Ultrabithorax. Here, we report the analysis of one immunopurified binding site. This 110 bp fragment maps within a regulatory region of a gene under homeotic control, connectin. A 4 kb DNA fragment, including the immunopurified binding site, is sufficient to reproduce the appropriate homeotic control within a subset of the full tissue distribution of connectin. Analysis of the role of the 110 bp binding site indicates that it mediates transcriptional controls by Ultrabithorax and other homeotic genes. This is the first report of a functional in vivo binding site isolated using the chromatin immunopurification method. We also show that the protein product of the connectin gene is predicted to be a cell-surface molecule containing leucine-rich repeats. The protein, connectin, can mediate cell-cell adhesion thus suggesting a direct link between homeotic gene function and processes of cell-cell recognition.

Amino Acid Sequence↗

PubMed2000

Developmental relationship of myosin binding proteins (myomesin, connectin and C-protein) to myosin in chicken somites as studied by immunofluorescence microscopy.

The developmental relationship of myosin binding proteins (myomesin, connectin and C-protein) to myosin was studied in chicken cervical somites by immunofluorescence microscopy. Muscle and non-muscle myosins initially appeared as slender rods at the same sites, and then, fused to form non-striated fibrils. As muscle myosin formed striated structures (A bands), non-muscle myosin disappeared from this structure. Myomesin (reactive with monoclonal antibodies MyB4 and MyBB78) and connectin (carboxy terminal region, reactive with monoclonal antibody T51) were seen as dots in the center of these myosin rods. These proteins then formed characteristic mature striations on non-striated fibrils of myosin. Earlier alignment of these myosin binding proteins rather than myosin indicates that the correct assembly of these proteins seems to be related to the formation of initial myosin rods as well as subsequent linear and periodic alignment of myosin molecules to form early A bands. Connectin spots reactive with 9D10 were scattered around myosin rods/myomesin dots/connectin T51 dots. These spots may represent radiating connectin filaments from these rods/dots to link myosin rods to the I-Z-I structures of myofibrils to be incorporated. Since the slow isoform of C-protein formed its characteristic bands ("doublets") prior to H zone formation within A bands by myosin, this isoform may help to precisely align myosin filaments within the A band region. The presence of the slow, then the slow and the cardiac, and finally the co-existence of the slow and the fast isoforms of C-protein may interfere with the incorporation and co-polymerization of non-adult isoforms into myofibrils.

Animals↗