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 of connectin and was compared to the incorporation of -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 -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.     

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.     

Binding of actin filaments to connectin

The binding of actin filaments to connectin, a muscle elastic protein, was investigated by means of turbidity and sedimentation measurements and electron microscopy. In the presence of less than 0.12 M KCl at pH 7.0, actin filaments bound to connectin. Long actin filaments formed bundles. Short actin filaments also aggregated into irregular bundles or a meshwork, and were frequently attached perpendicularly to long bundles. The binding of F-actin to connectin was saturated at an equal weight ratio (molar ratio, 50 : 1), as determined by a cosedimentation assay. Larger amounts of sonicated short actin filaments appeared to bind to connectin than intact F-actin. Myosin S1-decorated actin filaments did not bind to connectin. The addition of S1 to connectin-induced actin bundles resulted in partial disaggregation. Thus, connectin does not appear to interfere with actin-myosin interactions, since myosin S1 binds to actin more strongly than connectin.     

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.     

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.     

An historical perspective of the discovery of titin filaments –Part 2

In 2017, a Special Issue of Biophysical Reviews was devoted to “Titin and Its Binding Partners. The issue contained a review: “An historical perspective of the discovery of titin filaments” by dos Remedios and Gilmour that was intended to be a history of the discovery of the giant protein titin, previously named connectin. The review took readers back to the earliest discovery of the so-called third filament component of skeletal and cardiac muscle sarcomeres and ended in 1969. Recently, my colleague Shin’ichi Ishiwata gently reminded me of two papers published in 1990 and 1993 that were unwittingly omitted from the original historical perspective. In the first paper (J Cell Biol 110:53–62, 1990), Funatsu et al. examined the elastic filaments in skeletal muscle using a combination of light and electron microscopy, but they also measured resting as well as passive stiffness mechanical measurements to establish that connectin (titin) is responsible for both stiffness and fiber tension. In the second paper (J Cell Biol 120:711–724, 1993), Funatsu et al. used permeabilised cardiac muscle myocytes (from rabbit papillary muscles) and focussed on filament ultrastructure using either freeze-substitution or deep-etched replica methods to visualise connectin/titin filaments in fibers with and without actin and myosin filaments.     

Titin organisation and the 3D architecture of the vertebrate-striated muscle I-band

The giant muscle protein titin (connectin) is known to serve as a cytoskeletal element in muscle sarcomeres. It elastically restrains lengthening sarcomeres, it aids the integrity and central positioning of the A-band in the sarcomere and it may act as a template upon which some sarcomeric components are laid down during myogenesis. A puzzle has been how titin molecules, arranged systematically within the hexagonal A-band lattice of myosin filaments, can redistribute through the I-band to their anchoring sites in the tetragonal Z-band lattice. Recent work by Liversage and colleagues has suggested that there are six titin molecules per half myosin filament. Since there are two actin filaments per half myosin filament in a half sarcomere, this means that there are three titin molecules interacting with each Z-band unit cell containing one actin filament in the same sarcomere and one of opposite polarity from the next sarcomere. Liversage et al. suggested that the three titins might be distributed with two on an actin filament of one polarity and one on the filament of opposite polarity. Here, we build on this suggestion and discuss the transition of titin from the A-band to the Z-band. We show that there are good structural and mechanical reasons why titin might be organised as Liversage et al., suggested and we discuss the possible relationships between A-band arrangements in successive sarcomeres along a myofibril.     

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

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.     

The N terminus of myosin-binding protein C extends toward actin filaments in intact cardiac muscle

Myosin and actin filaments are highly organized within muscle sarcomeres. Myosin-binding protein C (MyBP-C) is a flexible, rod-like protein located within the C-zone of the sarcomere. The C-terminal domain of MyBP-C is tethered to the myosin filament backbone, and the N-terminal domains are postulated to interact with actin and/or the myosin head to modulate filament sliding. To define where the N-terminal domains of MyBP-C are localized in the sarcomere of active and relaxed mouse myocardium, the relative positions of the N terminus of MyBP-C and actin were imaged in fixed muscle samples using super-resolution fluorescence microscopy. The resolution of the imaging was enhanced by particle averaging. The images demonstrate that the position of the N terminus of MyBP-C is biased toward the actin filaments in both active and relaxed muscle preparations. Comparison of the experimental images with images generated in silico, accounting for known binding partner interactions, suggests that the N-terminal domains of MyBP-C may bind to actin and possibly the myosin head but only when the myosin head is in the proximity of an actin filament. These physiologically relevant images help define the molecular mechanism by which the N-terminal domains of MyBP-C may search for, and capture, molecular binding partners to tune cardiac contractility.     

Immunohistochemistry of chaetognath body wall muscles

Abstract. A light and electron immunohistochemical study was carried out on the body wall muscles of the chaetognath Sagitta friderici for the presence of a variety of contractile proteins (myosin, paramyosin, actin), regulatory proteins (tropomyosin, troponin), and structural proteins (α‐actinin, desmin, vimentin). The primary muscle (～80% of body wall volume) showed the characteristic structure of transversely striated muscles, and was comparable to that of insect asynchronous flight muscles. In addition, the body wall had a secondary muscle with a peculiar structure, displaying two sarcomere types (S1 and S2), which alternated along the myofibrils. S1 sarcomeres were similar to those in the slow striated fibers of many invertebrates. In contrast, S2 sarcomeres did not show a regular sarcomeric pattern, but instead exhibited parallel arrays of 2 filament types. The thickest filaments (～10–15 nm) were arranged to form lamellar structures, surrounded by the thinnest filaments (～6 nm). Immunoreactions to desmin and vimentin were negative in both muscle types. The primary muscle exhibited the classical distribution of muscle proteins: actin, tropomyosin, and troponin were detected along the thin filaments, whereas myosin and paramyosin were localized along the thick filaments; immunolabeling of α‐actinin was found at Z‐bands. Immunoreactions in the S1 sarcomeres of the secondary muscle were very similar to those found in the primary muscle. Interestingly, the S2 sarcomeres of this muscle were labeled with actin and tropomyosin antibodies, and presented no immunore‐actions to both myosin and paramyosin. α‐Actinin in the secondary muscle was only detected at the Z‐lines that separate S1 from S2. These findings suggest that S2 are not true sarcomeres. Although they contain actin and tropomyosin in their thinnest filaments, their thickest filaments do not show myosin or paramyosin, as the striated muscle thick myofilaments do. These peculiar S2 thick filaments might be an uncommon type of intermediate filament, which were labeled neither with desmin or vimentin antibodies.     

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.     

Evidence for Superthin Filaments

We have examined a variety of invertebrate and vertebrate musclesin the electron microscope and have seen evidence of a third,very thin (25 Ä) filament in all of them. This filamenthas been seen in H zones, A bands, and I bands, as well as bridgingthe gap between actin and myosin filaments in greatly stretchedmuscle. It, thus, seems likely that, if a third filament exists,it is elastic and extends throughout the sarcomere from Z discto Z disc, unlike the hypothetical actin-actin(s) filament ofHanson and Huxley (1955), or the actinmyosin gap filaments ofSjostrand (1962) and Carlsen, et al. (1965). These filaments therefore provide an answer to some of the presentparadoxes of muscle ultrastructure.     

Architecture of the sarcomere matrix of skeletal muscle: immunoelectron microscopic evidence that suggests a set of parallel inextensible nebulin filaments anchored at the Z line

Nebulin, a giant myofibrillar protein (600-800 kD) that is abundant (3%) in the sarcomere of a wide range of skeletal muscles, has been proposed as a component of a cytoskeletal matrix that coexists with actin and myosin filaments within the sarcomere. Immunoblot analysis indicates that although polypeptides of similar size are present in cardiac and smooth muscles at low abundance, those proteins show no immunological cross-reactivity with skeletal muscle nebulin. Gel analysis reveals that nebulins in various skeletal muscles of rabbit belong to at least two classes of size variants. A monospecific antibody has been used to localize nebulin by immunoelectron microscopy in a mechanically split rabbit psoas muscle fiber preparation. Labeled split fibers exhibit six pairs of stripes of antibody-imparted transverse densities spaced at 0.1-1.0 micron from the Z line within each sarcomere. These epitopes maintain a fixed distance to the Z line irrespective of sarcomere length and do not exhibit the characteristic elastic stretch-response of titin epitopes within the I band domain. It is proposed that nebulin constitutes a set of inextensible filaments attached at one end to the Z line and that nebulin filaments are in parallel, and not in series, with titin filaments. Thus the skeletal muscle sarcomere may have two sets of nonactomyosin filaments: a set of I segment-linked nebulin filaments and a set of A segment-linked titin filaments. This four-filament sarcomere model raises the possibility that nebulin and titin might act as organizing templates and length- determining factors for actin and myosin respectively.     

Interactions of muscle beta-connectin with myosin, actin, and actomyosin at low ionic strengths

The interaction of the muscle elastic protein connectin with myosin and actin filaments was investigated by turbidimetry, viscosity, flow birefringence measurements, and electron microscopic observations. In KCl concentrations lower than 0.15 M at pH 7.0 at 25 degrees C, both myosin and actin filaments were aggregated by connectin. Myosin filaments were entangled with each other in the presence of connectin. Actin filaments were assembled into bundles under the influence of connectin just as under that of alpha-actinin. The physiological significance of the interactions of connectin with myosin and actin filaments is discussed in relation to the localization of connectin in myofibrils. The Mg2+-activated ATPase activity of actomyosin was appreciably enhanced by connectin in the presence of KCl concentrations lower than 0.1 M. The extent of activation by connectin was smaller than by alpha-actinin. The enhancement of the ATPase activity may be due to acceleration of the onset of superprecipitation of actomyosin.     

Stepwise length changes in single invertebrate thick filaments

Previous experiments on thick filaments of the anterior byssus retractor muscle of Mytilus and the telson-levator muscle of Limulus polyphemus have shown large, reversible length changes up to 23% and 66% of initial length, respectively, within the physiological tension range. Using nanofabricated cantilevers and newly developed high-resolution detection methods, we investigated the dynamics of isolated Mytilus anterior byssus retractor muscle thick filaments. Single thick filaments were suspended between the tips of two microbeams oriented perpendicular to the filament axis: a deflectable cantilever and a stationary beam. Axial stress was applied by translating the base of the deflectable nanolever away from the stationary beam, which bent the nanolever. Tips of flexible nanolevers and stationary beam were imaged onto a photodiode array to track their positions. Filament shortening and lengthening traces, obtained immediately after the motor had imposed stress on the filament, showed steps and pauses. Step sizes were 2.7 nm and integer multiples thereof. Steps of this same size paradigm have been seen both during contraction of single sarcomeres and during active interaction between single isolated actin and myosin filaments, raising the question whether all of these phenomena might be related.     

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.     

Reduction of density and anisotropic distribution of intermediate filaments occur during avian skeletal myogenesis

Chicken skeletal muscle taken from embryos in ovo was examined by thin-section electron microscopy. Measurements of filament diameters reveal three nonoverlapping groups of filaments: thin (actin myofibrillar) filaments with mean diameters of 5.3 +/- 0.6 nm (S.D.), thick (myosin myofibrillar) filaments with mean diameters of 15 +/- 1.4 nm, and intermediate filaments with mean diameters of 9.3 +/- 0.9 nm. During muscle development these diameters do not change. By counting the number of filaments observed in the sarcoplasm at different stages, we find that the spatial density of intermediate filaments decreases during avian myogenesis in ovo, from 91 intermediate filaments/micron 2 at 6 days to 43 intermediate filaments/micron 2 at 17 days in ovo. Initially randomly arranged, some intermediate filaments become associated with Z discs, sarcoplasmic reticulum, nuclear membrane, and the sarcolemma between 6 and 10 days in ovo. These associated intermediate filaments course both parallel and transverse to myofibrils, forming lateral connections between myofibrillar Z discs and longitudinal connections from Z disc to Z disc within myofibrils. Intermediate filaments also appear to connect Z discs with the nuclear membrane. The intermediate filament associations persist through day 17 of development, after which the presence of cytoskeletal filaments is obscured by the densely packed myofibrils and membranes. Intermediate filament distribution becomes anisotropic during development. A greater proportion of intermediate filaments in the immediate perimyofibrillar area are oriented parallel to myofibrils than in other areas, so that the majority of the intermediate filaments nearest the myofibrils course parallel to them. The longitudinal intramyofibrillar intermediate filaments persist throughout development, as shown by their existence in KI-extracted adult myofibrils.     

The structure of thick filaments on longitudinal sections of rabbit psoas muscle

By means of electron microscopy the longitudinal sections of chemically skinned fibres of rigorised rabbit psoas muscle have been examined at pH of rigorising solutions equal to 6, 7, 8 (I = 0.125) and ionic strengths equal to 0.04, 0.125, 0.34 (pH 7.0). It has been revealed that at pH 6.0 the bands of minor proteins localization in A-disks were seen very distinctly, while at pH 7.0 and I = 0.125 these bands can be revealed only by means of antibody labelling technique. At the ionic strength of 0.34 (pH 7.0) the periodicity of 14.3 nm in thick filaments was clearly observed, which was determined by packing of the myosin rods into the filament shaft and of the myosin heads (cross-bridges) on the filament surface. The number of cross-bridge rows in the filament equals 102. A new scheme of myosin cross-bridge distribution in thick filaments of rabbit psoas muscle has been suggested according to which two rows of cross-bridges at each end of a thick filament are absent. The filament length equals 1.64 +/- 0.01 micron. It has been shown that the length of thick filament as well as the structural organization of their end regions in rabbit psoas muscle and frog sartorius one are different.     

Fractal dimension analysis and mathematical morphology of structural changes in actin filaments imaged by electron microscopy

In this work, we examined structural changes of actin filaments interacting with myosin visualized by quick freeze deep-etch replica electron microscopy (EM) by using a new method of image processing/analysis based on mathematical morphology.In order to quantify the degree of structural changes, two characteristic patterns were extracted from the EM images. One is the winding pattern of the filament shape (WP) reflecting flexibility of the filament, and the other is the surface pattern of the filament (SP) reflecting intra-molecular domain-mobility of actin monomers constituting the filament. EM images were processed by morphological filtering followed by box-counting to calculate the fractal dimensions for WP (D_WP) and SP (D_SP). The result indicates that D_WP was larger than D_SP irrespective of the state of the filament (myosin-free or bound) and that both parameters for myosin-bound filaments were significantly larger than those for myosin-free filaments. Overall, this work provides the first quantitative insight into how conformational disorder of actin monomers is correlated with the myosin-induced increase in flexibility of actin filaments along their length as suggested by earlier studies with different techniques. Our method is yet to be improved in details, but promising as a powerful tool for studying the structural change of protein molecules and their assemblies, which can potentially be applied to a wide range of biological and biomedical images.     

Nonuniform distribution of myosin light chains within the thick filaments of lobster slow muscle: Immunocytochemical study.

The in situ distribution of the alpha and beta myosin light chains was investigated at the subsarcomeric and subfilament levels in individual fibers of the superficial flexor muscle (SFM) of the lobster, Homarus americanus. Polyclonal antibodies were produced against the two classes of myosin light chains and used for subsequent immunolocalization on thin sections of sarcomeres and on isolated filaments from both the medial and lateral fiber bundles of the SFM. The beta myosin light chains were uniformly distributed within the crossbridge region of sarcomeres of both medial and lateral bundles. The alpha myosin light chains were uniformly distributed within the crossbridge region of sarcomeres from the medial bundle, but were nonuniformly distributed over the crossbridge region of lateral bundle sarcomeres. In the latter, the number of alpha myosin light chains was highest toward the center of the thick filaments, diminishing towards the ends. Similar distributions of alpha light chains were found in isolated myosin filaments. These data demonstrate that heterogeneity in protein composition extends to the level of the myosin filament and suggest that the myosin filament substructure in lobster may be different than that found in vertebrate skeletal muscle.