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At least 109 records · Page 6Linked to original sources
PubMed1989

The organization of titin (connectin) and nebulin in the sarcomeres: an immunocytolocalization study.

Monospecific polyclonal antibodies against two exceptionally large proteins, titin (a-T) and nebulin (a-N) isolated from rabbit skeletal muscles, were raised in guinea pig. Using an immuno-pre-embedding method, we have localized at the ultrastructural level of resolution the reactivity sites in skinned muscle fibres. At resting length a-T and a-N antibodies recognize epitopes which only partially overlap. a-T antibodies decorate mostly the A band with at least four clearly distinguished lines of reaction and one line in the I band, all near the A/I limit; a-N antibodies bind to the same region, but with wider areas of reaction in both A and I bands. To study whether the localization of these reaction sites varies according to the sarcomere length, skinned rabbit psoas fibres were incubated at sarcomere lengths ranging from maximum shortening to overstretching. The results indicate that lines decorated by a-T move away from the Z disc when the sarcomere is lengthened. With respect to the M line, the behaviour was biphasic. When the sarcomere was stretched up to about 2.8 microns, the decorated lines maintain almost the same distance from the M line. When the sarcomere is stretched beyond 2.8 microns, all a-T epitopes move away from the M line and the molecule behaves elastically. At resting length the a-N decoration appears to be localized on three large adjacent bands at the I, A/I and A level. The a-N line of reaction at the edge of the A band moves away from the Z discs as the sarcomere lengthens, while a second line which seems to be localized at the tip of the thin filament moves away from M line when the sarcomere lengthens. In non-overlapping sarcomeres a-N antibodies decorate only the tip of the thin filaments. Our results indicate that titin forms a polar filament connecting the M line to the Z line. In short sarcomeres, the filament seems to have some connections with structures of the A band, since titin epitopes do not move during stretching. These connections are lost at longer sarcomere lengths. On the other hand, our results suggest that nebulin is probably not a constituent of the titin filament.

Animals↗
PubMed2001

Calcium binding to an elastic portion of connectin/titin filaments.

Alpha-connectin/titin-1 exists as an elastic filament that links a thick filament with the Z-disk, keeping thick filaments centered within the sarcomere during force generation. We have shown that the connectin filament has an affinity for calcium ions and its binding site(s) is restricted to the beta-connectin/titin-2 portion. We now report the localization and the characterization of calcium-binding sites on beta-connectin. Purified beta-connectin was digested by trypsin into 1700- and 400-kDa fragments. which were then subjected to fluorescence calcium-binding assays. The 400-kDa fragment possesses calcium-binding activity; the binding constant was 1.0 x 10(7) M(-1) and the molar ratio of bound calcium ions to the 400-kDa fragment reached a maximum of 12 at a free calcium ion concentration of approximately 1.0 microM. Antibodies against the 400-kDa fragment formed a sharp dense stripe at the boundary of the A and the I bands, indicating that the calcium-binding domain constitutes the N-terminal region of beta-connectin, that is, the elastic portion of connectin filaments. Furthermore, we estimated the N-terminal location of beta-connectin of various origins (n = 26). Myofibrils were treated with a solution containing 0.1 mM CaCl2 and 70 microM leupeptin to split connectin filaments into beta-connectin and a subfragment, and chain weights of these polypeptides were estimated according to their mobility in 2% polyacrylamide slab gels. The subfragment exhibited a similar chain weight of 1200+/-33 kDa (mean+/-SD), while alpha- and beta-connectins were variable in size according to their origin. These results suggest that the apparent length of the 1200-kDa subfragment portion is almost constant in all instances, about 0.34 microm at the slack condition, therefore that the C-terminus of the 1200-kDa subfragment, that is, the N-terminus of the calcium-binding domain, is at the N2 line region of parent filaments in situ. Because the secondary structure of the 400-kDa fragment was changed by the binding of calcium ions, connectin filaments could be expected to alter their elasticity during the contraction-relaxation cycle of skeletal muscle.

Animals↗
PubMed1993

Elastic filaments in situ in cardiac muscle: deep-etch replica analysis in combination with selective removal of actin and myosin filaments.

To clarify the full picture of the connectin (titin) filament network in situ, we selectively removed actin and myosin filaments from cardiac muscle fibers by gelsolin and potassium acetate treatment, respectively, and observed the residual elastic filament network by deep-etch replica electron microscopy. In the A bands, elastic filaments of uniform diameter (6-7 nm) projecting from the M line ran parallel, and extended into the I bands. At the junction line in the I bands, which may correspond to the N2 line in skeletal muscle, individual elastic filaments branched into two or more thinner strands, which repeatedly joined and branched to reach the Z line. Considering that cardiac muscle lacks nebulin, it is very likely that these elastic filaments were composed predominantly of connectin molecules; indeed, anti-connectin monoclonal antibody specifically stained these elastic filaments. Further, striations of approximately 4 nm, characteristic of isolated connectin molecules, were also observed in the elastic filaments. Taking recent analyses of the structure of isolated connectin molecules into consideration, we concluded that individual connectin molecules stretched between the M and Z lines and that each elastic filament consisted of laterally-associated connectin molecules. Close comparison of these images with the replica images of intact and S1-decorated sarcomeres led us to conclude that, in intact sarcomeres, the elastic filaments were laterally associated with myosin and actin filaments in the A and I bands, respectively. Interestingly, it was shown that the elastic property of connectin filaments was not restricted by their lateral association with actin filaments in intact sarcomeres. Finally, we have proposed a new structural model of the cardiac muscle sarcomere that includes connectin filaments.

Actin Cytoskeleton↗
PubMed2004

Identification of high molecular weight proteins in squid muscle by Western blotting analysis and postmortem rheological changes.

The high molecular weight protein connectin (also called titin) in Japanese common squid (Todarodes pacificus) mantle muscle was identified by western blotting analysis with 3B9, the mouse anti-chicken skeletal muscle connectin monoclonal antibody. Similarly to vertebrate samples, there exists connectin in invertebrate squid mantle muscle, and the amino acid sequences are assumed to resemble those present in the A band of vertebrate connectin, judging by the specificity of 3B9. Moreover, the connectin in squid muscle migrated in this study as a closely spaced doublet of alpha and beta (titins 1 and 2). Between 5 and 7 h post-mortem, the SDS PAGE patterns of the squid sample indicated a change of the doublet bands into a single beta-connectin band. Simultaneously, the rheological properties of the squid muscle changed substantially. This degradation of alpha-connectin into beta-connectin in the muscle can explain the critical change that occurs during the post-mortem tenderization of squid muscle.

Animals↗
PubMed1994

Electron microscopic filament lengths of connection and its fragments.

Connectin (titin) is an extraordinarily long filamentous protein of striated muscle. The particle lengths of alpha-connectin (titin 1) and its proteolytic products, beta-connectin (titin 2) and 1,200 kDa fragment, were measured with rotary-shadowed images of the filaments after orientation by centrifugation. It was observed that the 1,200 kDa fragment was frequently folded into a double strand, beta-connectin was partly folded, and alpha-connectin was easily split into beta-connectin and 1,200 kDa fragment. Taking these features into consideration, the average lengths of alpha- and beta-connectin and 1,200 kDa fragment were estimated to be approximately 1,250, 920, and 360 nm, respectively.

Animals↗
PubMed1990

Isolation, characterization, and immunochemical properties of a giant protein from sea urchin egg cytomatrix.

A giant protein of apparent molecular weight (Mr) 2000 kDa, as determined by SDS-PAGE, was isolated and partially purified, under denaturing conditions, from the detergent-resistant cytomatrix of unfertilized sea urchin egg. Immunoblot analysis and indirect immunofluorescence microscopy observations indicated that this high-molecular-weight protein cross-reacted with the immunospecific serum raised against chicken breast muscle beta-connectin. However, rotary-shadowing electron microscopy images of the protein revealed short threadlike structures which appear morphologically different from beta-connectin structure. Indirect immunofluorescence localization of the protein with anti-beta-connectin serum showed a distribution throughout the whole unfertilized egg cytomatrix. This immunofluorescence pattern seems to change upon egg fertilization, since at metaphase the fluorescence stain appears to be excluded from the mitotic apparatus region as revealed by the double immunolabeling with anti-beta-connectin serum and monoclonal anti-alpha-tubulin antibody. Moreover, when egg cortical fragments were double-labeled with anti-beta-connectin serum and rhodamin-conjugated phalloidin, it was observed that the microfilaments assembled after fertilization seem to be in close association with the protein at the cleavage furrow and other locations. The possible significance of this sea urchin egg connectin(titin)-like protein is discussed.

Animals↗
PubMed1997

Connectin/titin, giant elastic protein of muscle.

The structure and function of the giant elastic protein connectin/titin are described on the basis of recent investigations. The 3000 kDa protein links the Z line to the myosin filament in striated muscle sarcomeres. The NH2-terminal region of connectin filament is involved in the Z line binding, and the COOH-terminal region is bound onto the myosin filament with an overlap between the counter-connectin filaments at the M line. The PEVK region in the I band is shown to be mainly responsible for passive tension generation. The longitudinal continuity of myosin-, actin-free sarcomeres is explained by the linkage of freed connectin filaments extending from both sides of the Z lines in a sarcomere. The role of connectin in myofibrillar differentiation and the biodiversity of connectin-related proteins in the animal kingdom are briefly reviewed.

Animals↗