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PubMed1992

Localization and elasticity of connectin (titin) filaments in skinned frog muscle fibres subjected to partial depolymerization of thick filaments.

The localization and elasticity of connectin (titin) filaments in skinned fibres of frog skeletal muscle were examined for changes in the localization of connectin and in resting tension during partial depolymerization of thick filaments with a relaxing solution containing increased KCl concentrations. Immunoelectron microscopic studies revealed that deposites of antibodies against connectin at a sarcomere length of 3.0 microns remained at about 0.8 microns from the M-line, until the thick filament was depolymerized to the length of approximately 0.4 microns. On further depolymerization, the bound antibodies were found to move towards the Z-line and, on complete depolymerization, were observed to be within 0.3 microns of the Z-line; a marked decrease in resting tension accompanied this further depolymerization. These results suggest that connectin filament starts from the Z-line, extends to the M-line, and contributes to resting tension. After partial depolymerization of thick filaments, the distances between the anti-connectin deposits and the Z-line and between anti-connectin deposits and the M-line increased with sarcomere length, suggesting that connectin filaments are elastic along their entire length.

Actin Cytoskeleton↗

PubMed1990

Degradation of connectin (titin) in Fukuyama type congenital muscular dystrophy: immunochemical study with monoclonal antibodies.

Connectin (also called titin) is a myofibrillar elastic filament which links a thick filament to a neighbouring Z line in a sarcomere and thus contributes significantly to the elasticity of myofibrils. In a previous study, we demonstrated by Western blot analysis of the biopsied skeletal muscles using an anti-connectin monoclonal antibody that connectin was degraded extensively after 5 years of age in Duchenne muscular dystrophy (DMD), while it was degraded mildly in Becker muscular dystrophy and only minimally in myotonic dystrophy, limb girdle dystrophy, amyotrophic lateral sclerosis and Charcot-Marie-Tooth disease. In the present study, we investigated the degradation state of connectin in Fukuyama type congenital muscular dystrophy (FCMD) by a similar method using 2 distinct anti-connectin monoclonal antibodies. In FCMD, connectin degradation began much earlier than in DMD: Definite degradation was already observed in 5-8-month-old patients. It was presumed that connectin degradation would play an important role in the myofibrillar degeneration in the early stage of FCMD.

Antibodies, Monoclonal↗

PubMed1997

Connectin mediates adhesion in Drosophila.

The Drosophila cell-surface molecule connectin mediates cell-cell adhesion in vitro, and its expression pattern in vivo fits well with an adhesion role in the embryonic neuromuscular system. However, connectin mutants do not show dramatic neuromuscular defects, and ectopic expression studies so far have not supported an adhesion role. Here, we demonstrate that connectin mutants do have a phenotype; the normally connectin-positive pleural muscles fail to adhere closely together. An in vivo adhesion role is supported by misexpression studies, which result in excessive adhesion of normally connectin-negative muscles. Misexpression also causes defects in axon pathfinding. While a previous study interpreted similar defects as indicating a repulsion role for connectin, we argue that the phenotypes are consistent with connectin's adhesion role.

Animals↗

PubMed2002

Detection of giant myofibrillar proteins connectin and nebulin in fish meat by electrophoresis in 3-5 gradient sodium dodecyl sulfate polyacrylamide slab gels.

An improved method was investigated for sodium dodecyl sulfate polyacrylamide slab gel electrophoresis (SDS-PAGE) to facilitate the analysis of the giant myofibrillar proteins, connectin and nebulin, in fish meat by using jack mackerel (Trachurus japonicus) as the sample fish. It was established that separation of the alpha-connectin band from the beta-connectin band by SDS-PAGE could be achieved by using 3-5% gradient gels with glycerol to facilitate the formation of a gradient with polymerization at 35 degrees C. SDS-PAGE samples of white dorsal muscle from the jack mackerel were homogenized with a 2% SDS solution containing an inhibitor mixture (1 microg/mL of phenylmethanesulfonyl fluoride, 1 microg/mL of leupeptin, and 1 microg/mL of E-64) and heated at 50 degrees C for 20 min. Heating these samples at 100 degrees C for 2 min resulted in the disintegration of connectin but did not affect nebulin. A purified myofibril sample and a whole muscle sample showed similar changes in the overall rate of degradation of whole connectin and nebulin during the postmortem storage period, but it was clear that beta-connectin was cleaved from alpha-connectin during the preparation of myofibrils at the early stage postmortem. Storage of the SDS-PAGE samples at -85 degrees C was preferable to storage at -18 degrees C for a long period.

Animals↗

PubMed2001

Invertebrate connectin spans as much as 3.5 microm in the giant sarcomeres of crayfish claw muscle.

In crayfish claw closer muscle, the giant sarcomeres are 8.3 microm long at rest, four times longer than vertebrate striated muscle sarcomeres, and they are extensible up to 13 microm upon stretch. Invertebrate connectin (I-connectin) is an elastic protein which holds the A band at the center of the sarcomere. The entire sequence of crayfish I-connectin was predicted from cDNA sequences of 53 424 bp (17 352 residues; 1960 kDa). Crayfish I-connectin contains two novel 68- and 71-residue repeats, and also two PEVK domains and one kettin region. Kettin is a small isoform of I-connectin. Immunoblot tests using antibody to the 68-residue repeats revealed the presence of I-connectin also in long sarcomeres of insect leg muscle and barnacle ventral muscle. Immunofluorescence microscopy demonstrated that the two repeats, the long spacer and the two PEVK domains contribute to sarcomere extension. These regions rich in charged amino acids, occupying 63% of the crayfish I-connectin molecule, may allow a span of a 3.5 microm distance as a new class of composite spring.

Amino Acid Sequence↗

PubMed1989

Isolation of alpha-connectin, an elastic protein, from rabbit skeletal muscle.

alpha-Connectin (also called titin 1) has been isolated from rabbit back muscle. Myofibrils were well washed with 5 mM NaHCO3 and then extracted with 0.2 M sodium phosphate, pH 7.0. The extract was dialyzed against 0.1 M potassium phosphate, pH 7.0, to sediment myosin. The supernatant, adjusted to 0.18 M potassium phosphate, pH 7.0, and 4 M urea, was subjected to DEAE Toyopearl column chromatography. beta-Connectin was eluted in the flow-through fraction and alpha-connectin was eluted at around 0.1 M NaCl, when a 0 to 0.25 M NaCl gradient was applied. The separated alpha-connectin was dialyzed against 0.2 M potassium phosphate, pH 7.0. The resultant alpha-connectin showed the same mobility as that in an SDS extract of rabbit back muscle on SDS gel electrophoresis using 1.8% polyacrylamide gels. A monoclonal antibody against chicken breast muscle beta-connectin reacted with the alpha-connectin isolated from rabbit back muscle.

Animals↗

PubMed1984

Molecular size and shape of beta-connectin, an elastic protein of striated muscle.

Connectin is an elastic protein of vertebrate striated muscle, and consists of doublet components, alpha and beta (also called titins 1 and 2). In the present study, beta-connectin isolated in the native state was investigated in order to characterize its molecular size and shape. The molecular weight was approximately 2.1 X 10(6) (SDS gel electrophoresis) or 2.7 X 10(6) (sedimentation equilibrium). The sedimentation coefficient (SO20, w) was 17S in 0.1 M phosphate buffer, pH 7.0. The intrinsic viscosity measured in an Ostwald-type viscometer was 1.8 dl/g. However, the viscosity was greatly dependent on the velocity gradient, and at a very low velocity gradient of 0.0007 s-1, a solution of connectin (0.3 mg/ml) showed a viscosity value of 17,000 cp. Flow birefringence measurements suggested a length distribution ranging from 300 to 450 nm. Electron microscopic observations revealed that connectin is a long flexible filament and the peaks of frequency of length distribution were at 150, 300, 450, and 600 nm. It was tentatively assumed that the connectin molecule is 300-400 nm long and 34-38 nm wide. It is likely that beta-connectin is derived from alpha-connectin, which has an apparent molecular weight of 2.8 X 10(6).

Animals↗

PubMed1982

Studies on the structure of connectin in muscle.

Enzymic hydrolysis, followed by amino acid analysis, provided no evidence for the presence of epsilon-(gamma-glutamyl) lysine or other isopeptide crosslinks in connectin. Gel elecrrophoresis in the presence of sodium dodecyl sulphate did not reveal any difference in connectin between normal and lathyritic muscle, indicating that lysyl oxidase does not initiate cross-link formation in connectin. Although connectin may be covalently crosslinked by some unknown mechanism, the available evidence suggests that the subunit of MW approximately to 900 000 is synthesised as a single polypeptide chain. In developing fetal muscle, myosin heavy chains are apparent some weeks earlier than connectin. This, together with the known susceptibility of connectin to hydrolysis, suggests that connectin exists in an exposed environment rather than as a core to the thick filament.

Amino Acids↗

PubMed1985

Studies on exercise and an elastic protein "connectin" in hindlimb muscle of growing rat.

We examined the effect of physical training on the properties of an elastic protein, connectin, in the hindlimb muscles of 6 growing rats running on a treadmill, in comparison with its properties in 6 growing non-running rats. The experimental results are as follows. 1) The body weight of exercised (R-) and control (C-) rats gradually increased during housing for 31 days, from an average of 46.2 g to 275 g. The average amount of running in the 6 R-rats was 38,160 +/- 12,122 (S.D.) g X m/hr. 2) The SDS-insoluble connectin in R-rat muscle was lower in quantity than that in C-rat muscle, but the SDS-soluble connectin of R-rat muscle was greater in quantity. 3) Amino acid compositions of SDS-soluble connectin in R- and C-rats, calculated from SDS-insoluble connectin and soleus ghost, were significantly different. 4) The soleus ghost preparation consisted mainly of connectin as shown by SDS polyacrylamide gel electrophoresis and by immunohistological staining. 5) The maximum passive tension of the soleus ghost, calculated from the factors of T/phi and (dT/dx)/phi, was higher in R-rats than in C-rats. The above results suggest that physical training might alter the biosynthetic pattern of connectin to suit the developing muscle, just as physical training alters that of myosin isoenzymes.

Amino Acids↗

PubMed1994

Biodiversity of the localization of the epitopes to connectin antibodies in the sarcomeres of lamprey, electric ray, and horse mackerel skeletal muscles.

SDS gel electrophoresis showed the presence of connectin, approximately 3000 kDa, in skeletal muscles of fishes, lamprey, electric ray and horse mackerel. The antibodies to avian or mammalian skeletal muscle connectin, 3B9 and Pc1200, reacted with all the fish connectins. However, a monoclonal antibody SM1 recognized electric ray connectin but did not react with lamprey and horse mackerel connectins. Immunoelectron microscopy revealed that the epitope to Pc1200 was localized at the periphery of the Z line of all the fish muscle sarcomeres and that to SM1 was located at the N2 line in the I band of ray muscle. These two localizations are the same as those in rabbit, chicken and frog skeletal muscles. On the other hand, the positions of epitopes to 3B9 were variable in the three classes of fishes, although all of them were localized in the A band. Some of the epitope positions were common to those in chicken skeletal muscle. Thus the present work demonstrates biodiversity of connectin in fish skeletal muscles, distinct from avian and mammalian skeletal muscles.

Animals↗

PubMed1994

Ectopic expression of connectin reveals a repulsive function during growth cone guidance and synapse formation.

Connectin, a cell surface protein of the leucine-rich repeat family in Drosophila, is expressed on the surface of a subset of embryonic muscles (primarily lateral muscles), on the growth cones and axons of the motoneurons that innervate these muscles (primarily SNa motoneurons), and on several associated glial cells. When coupled with its ability to mediate homophilic cell adhesion, these results led to the suggestion that Connectin functions as an attractive signal for SNa pathfinding and targeting. In the present study, we ectopically expressed Connectin on ventral muscles normally innervated by SNb motoneurons. The SNb growth cones change both their morphology and their trajectory when they encounter ectopic Connectin-positive ventral muscles, displaying "bypass," "detour," and "stall" phenotypes. Moreover, SNb synapse formation is prevented by Connectin expression on ventral muscles. These results reveal a repulsive function for Connectin during motoneuron growth cone guidance and synapse formation.

Animals↗

PubMed1989

Elastic behavior of connectin filaments during thick filament movement in activated skeletal muscle.

Connectin (also called titin) is a huge, striated muscle protein that binds to thick filaments and links them to the Z-disc. Using an mAb that binds to connectin in the I-band region of the molecule, we studied the behavior of connectin in both relaxed and activated skinned rabbit psoas fibers by immunoelectron microscopy. In relaxed fibers, antibody binding is visualized as two extra striations per sarcomere arranged symmetrically about the M-line. These striations move away from both the nearest Z-disc and the thick filaments when the sarcomere is stretched, confirming the elastic behavior of connectin within the I-band of relaxed sarcomeres as previously observed by several investigators. When the fiber is activated, thick filaments in sarcomeres shorter than 2.8 microns tend to move from the center to the side of the sarcomere. This translocation of thick filaments within the sarcomere is accompanied by movement of the antibody label in the same direction. In that half-sarcomere in which the thick filaments move away from the Z-disc, the spacings between the Z-disc and the antibody and between the antibody and the thick filaments both increase. Conversely, on the side of the sarcomere in which the thick filaments move nearer to the Z-line, these spacings decrease. Regardless of whether I-band spacing is varied by stretch of a relaxed sarcomere or by active sliding of thick filaments within a sarcomere of constant length, the spacings between the Z-line and the antibody and between the antibody and the thick filaments increase with I-band length identically. These results indicate that the connectin filaments remain bound to the thick filaments in active fibers, and that the elastic properties of connectin are unaltered by calcium ions and cross-bridge activity.

Actin Cytoskeleton↗

PubMed1984

Connectin content and its post-mortem changes in fish muscle.

Connectin was isolated from fish dorsal myofibrils by an SDS-gel filtration method and estimated to account for approximately 13% of the total myofibrillar proteins. There was no significant difference in the amount of connectin among seven fish species but rabbit skeletal myofibrils contained a slightly higher content (16%) of connectin. The high molecular weight connectins from carp and rabbit both showed a doublet band, consisting of bands 1 and 2, on SDS-polyacrylamide gel electrophoresis using a large-pore gel. However, rabbit band 1 (a component of the connectin doublet) was found to migrate more slowly than carp band 1. During post-mortem ageing of the muscles, it was observed that the band 1 component rapidly disappeared with a concomitant increase in band 2 component and then the band 2 component was transformed slowly into faster migrating components. These results suggest that post-mortem ageing has qualitatively similar effects on the submolecular compositions of carp and rabbit connectins. However, the apparent rate of disappearance of the band 1 component was considerably higher in carp muscle than that in rabbit muscle.

Animals↗

PubMed1993

Connectin content in rabbit cardiac and skeletal muscle.

1. The contents of connectin in rabbit cardiac and skeletal muscle were estimated by densitometry of the bands in SDS gel electrophoresis. 2. The weight ratio of connectin to myosin was 1:9.5 in cardiac muscle and 1:4.2 in skeletal muscle. 3. Assuming that the molecular weights of myosin and skeletal muscle connectin were 5.25 x 10(5) and 4.2 x 10(6) respectively, and a myosin filament consists of 300 myosin molecules, it was calculated that there were approx. 4 connectin molecules per half of myosin filament in rabbit skeletal muscle. 4. In cardiac muscle, the molecular weight of connectin was assumed to be 3.6 x 10(6) and it was estimated that there were about 2 connectin filaments per half of myosin filament.

Animals↗

PubMed1977

Connectin, an elastic protein of muscle. Comparative Biochemistry.

Connectin, an elastic protein, was isolated from both skeletal and cardiac muscles of various species of vertebrates, and also from smooth muscles (gizzard) of the chicken. The amino acid compositions of these preparations were very similar. Connectin was also obtained from claw and tail muscles of the crayfish, but preparations from clam adductor muscles and insect thoracic muscles were heavily contaminated with collagen and resilin, respectively. Connectin-like protein was obtained from cell membranes of erythrocytes and fluorescent anti-connectin staining suggested that it is located on the cytoplasmic surface of the membrane. An attempt to isolate an elastic protein from insoluble residues of amoebae of the slime mold and those of bacterial cell body (Salmonella) was inconclusive. The present comparative bio-chemical study has shown that connectin or connectin-like protein is widely distributed in various types of muscles and in some nonmuscle cells.

Amino Acids↗

PubMed1992

Connectin: a homophilic cell adhesion molecule expressed on a subset of muscles and the motoneurons that innervate them in Drosophila.

Each abdominal hemisegment in the Drosophila embryo contains a stereotyped array of 30 muscles, each specifically innervated by one or a few motoneurons. We screened 11,000 enhancer trap lines, isolated several expressing beta-galactosidase in small subsets of muscle fibers prior to innervation, and identified two of these as inserts in connectin and Toll, members of the leucine-rich repeat gene family. Connectin contains a signal sequence, ten leucine-rich repeats, and a putative phosphatidylinositol membrane linkage; in S2 cells, connectin can mediate homophilic cell adhesion. Connectin is expressed on the surface of eight muscles, the motoneurons that innervate them, and several glial cells along the pathways leading to them. During synapse formation, the protein localizes to synaptic sites; afterward, it largely disappears. Thus, connectin is a novel cell adhesion molecule whose expression suggests a role in target recognition.

Amino Acid Sequence↗

PubMed1993

Characterization of beta-connectin (titin 2) from striated muscle by dynamic light scattering.

Connectin (titin) is a large filamentous protein (single peptide) with a molecular mass of approximately 3 MDa, contour length approximately 900 nm, and diameter approximately 4 nm, and resides in striated muscle. Connectin links the thick filaments to the Z-lines in a sarcomere and produces a passive elastic force when muscle fiber is stretched. The aim of this study is to elucidate some aspects of physical properties of isolated beta-connectin (titin 2), a proteolytic fragment of connectin, by means of dynamic light-scattering (DLS) spectroscopy. The analysis of DLS spectra for beta-connectin gave the translational diffusion coefficient of 3.60 x 10(-8) cm2/s at 10 degrees C (or the hydrodynamic radius of 44.1 nm), molecular mass little smaller than 3.0 MDa (for a literature value of sedimentation coefficient), the root-mean-square end-to-end distance of 163 nm (or the radius of gyration of 66.6 nm), and the Kuhn segment number of 30 and segment length of 30 nm (or the persistence length of 15 nm). These results permitted to estimate the flexural rigidity of 6.0 x 10(-20) dyn x cm2 for filament bending, and the elastic constant of 7 dyn/cm for extension of one persistence length. Based on a simple model, implications of the present results in muscle physiology are discussed.

Animals↗

PubMed2002

Single-molecule measurement of elasticity of serine-, glutamate- and lysine-rich repeats of invertebrate connectin reveals that its elasticity is caused entropically by random coil structure.

Invertebrate connectin (I-connectin) is a 1960 kDa elastic protein linking the Z line to the tip of the myosin filament in the giant sarcomere of crayfish claw closer muscle (Fukuzawa et al., 2001 EMBO J 20: 4826-4835). I-Connectin can be extended up to 3.5 microns upon stretch of giant sarcomeres. There are several extensible regions in I-connectin: two long PEVK regions, one unique sequence region and Ser-, Glu- and Lys-rich 68 residue-repeats called SEK repeats. In the present study, the force measurement of the single recombinant SEK polypeptide containing biotinylated BDTC and GST tags at the N and C termini, respectively, were performed by intermolecular force microscopy (IFM), a refined AFM system. The force vs. extension curves were well fit to the wormlike chain (WLC) model and the obtained persistence length of 0.37 +/- 0.01 nm (n = 11) indicates that the SEK region is a random coil along its full length. This is the first observation of an entropic elasticity of a fully random coil region that contributes to the physiological function of I-connectin.

Amino Acid Sequence↗