Koscak Maruyama

Regulatory and Cytoskeletal Proteins of Vertebrate Skeletal Muscle

Publisher Summary This chapter describes the structure and function of the major regulatory proteins, troponin and tropomyosin, and also of the main cytoskeletal protein, connectin (titin). The effective contractile machinery of vertebrate striated muscle represents an elaborate framework. The motion of myosin and actin filaments is controlled by regulatory proteins and their position is supported by cytoskeletal proteins. Approximately 65% of the total myofibrillar proteins are myosin and actin, the contractile proteins of muscle. There are a number of both regulatory and cyotoskeletal proteins. The troponin and tropomyosin are the best characterized proteins, together with actin and myosin, in the field of muscle biochemistry. They are involved in the Ca 2+ regulation of muscle contraction. On the other hand, connectin—an elastic protein—is a relative newcomer, and because of its huge molecular weight (more than 2 million), its physicochemical characterization has remained incomplete. Nevertheless, it may be appropriate to call attention to this protein, because new aspects of protein chemistry might be revealed from work on such a giant peptide as connectin.

Connectin, an Elastic Filamentous Protein of Striated Muscle

Publisher Summary This chapter reveals that connectin, also called titin, is a long, flexible filamentous protein of striated muscle that links thick myosin filaments to Z disks in a sarcomere. Connectin superthin filaments serve as an elastic component of striated muscle. Therefore, it is regarded as the fourth type of cytoskeletal structure following microtubules, intermediate filaments, and actin filaments. It has been established that a number of muscle structural proteins undergo characteristic changes in isoform expression during embryonic and neonatal development. The chapter presents the investigation of changes in connectin isoforms during embryonic and neonatal development of the chick using sodium dodecyl sulfate (SDS) gel electrophoresis checked by an immunoblot technique. Although the band was very faint, connectin-like high-molecular-weight protein was detected in breast muscles of 7-day incubated chick embryo. There are two ways to detect connectin in various types of tissues or organisms: detection of high-molecular-weight protein bands in 2–3% polyacrylamide gels in the presence of SDS preferably with confirmation by an immunoblot and also by immunofluorescence examinations of fixed cells.

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.

Binding of the N‐terminal 63 kDa portion of connectin/titin to α‐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 α‐actinin (amino acids, 343–897 and 446–897) were obtained. Enzyme‐linked immunosorbent assay clearly demonstrated the interactions of α‐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 α‐actinin in the Z‐line of myofibrillar sarcomeres.

Projectin is an invertebrate connectin (titin): Isolation from crayfish claw muscle and localization in crayfish claw muscle and insect flight muscle

SummaryA filamentous protein was isolated from crayfish claw muscle. This protein had physiochemical properties very similar to vertebrate skeletal muscle connectin (titin), although its apparent molecular mass (∼ 1200 kDa) was considerably lower than that of connectin (∼ 3000 kDa). Polyclonal as well as monoclonal antibodies against chicken skeletal muscle connectin reacted with the 1200 kDa protein from crayfish claw muscle. Conversely, polyclonal antibodies against crayfish 1200 kDa protein crossreacted with chicken connectin. Circular dichroic spectra indicated the abundance ofβ-sheet structure (∼ 60 %). Low-angle shadowed images showed filamentous structures (0.2 ∼ 0.5μm) by electron microscopy. Proteolysis of the 1200 kDa protein by α-chymotrypsin or V8 protease rapidly resulted in formation of 1000 kDa or 1100 and 800 kDa peptides. The amino acid composition was very similar to those of vertebrate connectins and of honeybee flight muscle projectin. Based on the molecular weight and amino acid composition, the 1200 kDa protein is regarded to be crayfish projectin.Immunofluorescence and immunoelectron microscopy revealed that crayfish projectin was localized in the A/I junction area and A-band except for its centre region in crayfish claw muscles. Polyclonal antibodies against crayfish claw muscle projectin reacted with 1200 kDa projectin of honeybee and beetle flight muscle. A monoclonal antibody against chicken skeletal muscle connectin also reacted with honeybee and beetle projectin. Immunoelectron microscopic observations revealed that anti-crayfish projectin antibodies bound the connecting filaments linking the Z-line and the thick filaments up to the M-line of honeybee muscle sarcomere. Anti-crayfish projectin antibodies bound the I-band region near the Z-line of beetle flight muscle.It is concluded that the 1200 kDa projectin from crayfish claw muscle is an invertebrate connectin (titin). Recent work with locust flight muscle mini-titin (Nave & Weber, 1990) is in good agreement with the present study, except that the isolated minititin estimated as 600 kDa appears to be a proteolytic product (∼ 1100 kDa) of the parent molecule (∼ 1200 kDa).

Generation of functional beta-actinin (CapZ) in an E. coli expression system.

Abstractβ-actinin (CapZ) is a heterodimeric actin-binding protein which caps the barbed end of actin filaments and nucleates actin-polymerization in a Ca2+-independent manner. In myofibrils it is localized in the Z-lines. As judged by these properties of β-actinin, it is conceivable that β-actinin is involved in the regulation of actin assembly, especially in the formation of I-Z-I complex during myofibrillogenesis. In this study, we devised a system to produce functional β-actinin in E. Coli.The cDNAs of βI′ and βII subunits of β-actinin were obtained by RT-PCR methods using the published sequence as references, and subcloned in a pET vector. When the proteins were produced with the cDNA of either βI′ or βII in E. coli, the proteins were insoluble and non-functional. However, when the cDNAs encoding the two subunits were cloned into a single vector and␣both proteins were expressed simultaneously, the proteins became soluble and purified as a functional heterodimer. The␣activity of the purified proteins was not distinguishable from that of β-actinin purified from skeletal muscle.

Behaviour of connectin (titin) and nebulin in skinned muscle fibres released after extreme stretch as revealed by immunoelectron microscopy

SummaryStretching of skinned fibres of frog skeletal muscle beyond the overlap of the thin and thick filaments followed by release to resting length results in disorganization of the thin filaments at the A-I junction of a sarcomere (Higuchiet al. (1988) J. Muscl. Res. Cell. Motility9, 491–8). Immunoelectron microscopic observations showed that the binding sites of antibodies against connectin (titin) returned to the original position after extreme stretch and release but those of anti-nebulin antibodies were largely disorganized. The binding sites of anti-connectin antibodies moved within an I band with the change in sarcomere length, but those of anti-nebulin antibodies did not. Nebulin remained in the I band at extreme stretch. Thus connectin filaments appear to be responsible for maintaining mechanical continuity of a sarcomere and appear to behave independently of thin filaments. It is suggested that nebulin is localized in the I band but not in the A band and is associated with thin filaments but not with the elastic structure of myofibrils.

Characterization and localization of α-connectin (titin 1): An elastic protein isolated from rabbit skeletal muscle

SummaryA simplified procedure to isolateα-connectin (titin 1, TI), a gigantic elastic protein, from rabbit skeletal muscle is described. A rapid column chromatography step to concentrateα-connectin is introduced. Separation ofα-connectin fromβ-connectin is introduced. Separation ofα-connectin fromβ-connectin (titin 2, TII) in the presence of 4 M urea at pH 7.0 did not cause any change in the secondary structure ofα-connectin as judged by circular dichroic spectra. Ultraviolet absorption spectra and the amino acid composition ofα-connectin (MW, approximately 3×106) were similar to those of its proteolytic product,β-connectin (MW, approximately 2×106). Circular dichroic spectra suggested that bothα- andβ-connectin consist of 60%β-sheet and 30%β-turn. It thus appears that the whole elastic filament of connectin has a foldedβ-strand structure. Proteolysis ofα-connectin by calpain resulted in formation ofβ-connectin and smaller peptides. Theα-connectin interacted with both myosin and actin filaments similarly toβ-connectin. Polyclonal antibodies raised against 1200 kDa peptides obtained from aged rabbit skeletal myofibrils reacted withα-connectin (titin 1, TI) but only weakly withβ-connectin (titin 2, TII) in rabbit skeletal muscle. Immunoelectron microscopy and indirect immunofluorescence microscopy revealed that the antibodies bound at the Z-line and at the epitope regions in the I-band near the binding site of a monoclonal antibody SMI whose position depends on sarcomere length. It thus appears thatβ-connectin extends from the edge of M-line to the above epitope region in the I-band.

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

SummaryThe developmental expression of nebulin was studied in embryonic chick skeletal muscle cellsin 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 andα-actinin but not myosin and connectin (titin). Striated patterns of nebulin (‘singlets’) in myofibrils appeared simultaneously with those ofα-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.

Assembly of connectin (titin) in relation to myosin and α-actinin in cultured cardiac myocytes

SummaryBy using polyclonal and monoclonal antibodies against connectin (titin) which stain the A-I junctional area and the A-band domain (polyclonal anti-connectin and monoclonal 4C9) and the I-band domain (monoclonal SM1), the developmental relationship of this elastic protein with sarcomeric proteins, especially myosin andα-actinin, was examined in embryonic chick cardiac myocytesin vitro under fluorescence microscopy. During premyofibril stages, I-Z-I proteins were detected first (α-actinin dots and diffuse actin [phalloidin and anti-troponin C] staining), and later in these areas connectin and myosin dots appeared with nearly identical distribution. Somewhat later, phalloidin-positive nonstriated fibrils were observed in a straight course. They were always reactive with antibodies against a-actinin and troponin C, but unreactive or only weakly reactive with anticonnectin and anti-myosin. Initially,α-actinin dots were aligned along these fibrils but did not form striations. As they aggregated to form Z-bands, connectin and myosin started to exhibit typical striations (‘doublets’ and A-bands, respectively). No difference in the staining pattern was observed with two kinds of monoclonal antibodies against different domains of connectin filaments (4C9 and SM1) at early phases. As myosin staining began to show clear A-bands, connectin epitopes became arranged in polarized positions. We conclude that primitive I-Z-I complexes appear prior to the assembly of connectin and myosin filaments and then connectin filaments, developing intimately and coordinately with myosin, become associated with theα-actinin lines. Thus it appears that the putative elastic protein connectin plays some role in integrating myosin filaments with the preexisting I-Z-I brushes. The occasional absence of connectin and A-bands between two Z-bands, beyond both of which clear sarcomeres have been formed, indicates that connectin is not a preformed scaffold of myofibrils on which sarcomeric proteins accumulate.