Myofibrillogenesis in striated muscles is a highly complex process that depends on the coordinated assembly and integration of a large number of contractile, cytoskeletal, and signaling proteins into regular arrays, the sarcomeres. It is also associated with the stereotypical assembly of the sarcoplasmic reticulum and the transverse tubules around each sarcomere. Three giant, muscle-specific proteins, titin (3-4 MDa), nebulin (600-800 kDa), and obscurin (approximately 720-900 kDa), have been proposed to play important roles in the assembly and stabilization of sarcomeres. There is a large amount of data showing that each of these molecules interacts with several to many different protein ligands, regulating their activity and localizing them to particular sites within or surrounding sarcomeres. Consistent with this, mutations in each of these proteins have been linked to skeletal and cardiac myopathies or to muscular dystrophies. The evidence that any of them plays a role as a "molecular template," "molecular blueprint," or "molecular ruler" is less definitive, however. Here we review the structure and function of titin, nebulin, and obscurin, with the literature supporting a role for them as scaffolding molecules and the contradictory evidence regarding their roles as molecular guides in sarcomerogenesis.
Obscurin is a large ( approximately 800-kDa), modular protein of striated muscle that concentrates around the M-bands and Z-disks of each sarcomere, where it is well positioned to sense contractile activity. Obscurin contains several signaling domains, including a rho-guanine nucleotide exchange factor (rhoGEF) domain and tandem pleckstrin homology domain, consistent with a role in rho signaling in muscle. We investigated the ability of obscurin's rhoGEF domain to interact with and activate small GTPases. Using a combination of in vitro and in vivo approaches, we found that the rhoGEF domain of obscurin binds selectively to rhoA, and that rhoA colocalizes with obscurin at the M-band in skeletal muscle. Other small GTPases, including rac1 and cdc42, neither associate with the rhoGEF domain of obscurin nor concentrate at the level of the M-bands. Furthermore, overexpression of the rhoGEF domain of obscurin in adult skeletal muscle selectively increases rhoA expression and activity in this tissue. Overexpression of obscurin's rhoGEF domain and its effects on rhoA alter the expression of rho kinase and citron kinase, both of which can be activated by rhoA in other tissues. Injuries to rodent hindlimb muscles caused by large-strain lengthening contractions increases rhoA activity and displaces it from the M-bands to Z-disks, similar to the effects of overexpression of obscurin's rhoGEF domain. Our results suggest that obscurin's rhoGEF domain signals at least in part by inducing rhoA expression and activation, and altering the expression of downstream kinases in vitro and in vivo.
We used four antibodies to regions of obscurin isoforms A and B, encoded by the obscurin gene, to investigate the location of these proteins in skeletal myofibers at resting and stretched lengths. Obscurin A (∼800 kDa) which was recognized by antibodies generated to the N‐terminal, Rho‐GEF, and the non‐modular C‐terminal domain that lacks the kinase‐like domains, localizes at the level of the M‐band. Obscurin B (∼900 kDa) which has the N‐terminal, Rho‐GEF, and the C‐terminal kinase‐like domains, localizes at the level of the A/I junction. Additional isoforms, which lack one or more of these epitopes, are present at the Z‐disk and Z/I junction.
Obscurin is a multidomain protein composed of adhesion and signaling domains that plays key roles in the organization of contractile and membrane structures in striated muscles. Overexpression of the second immunoglobulin domain of obscurin (Ig2) in developing myotubes inhibits the assembly of A- and M-bands, but not Z-disks or I-bands. This effect is mediated by the direct interaction of the Ig2 domain of obscurin with a novel isoform of myosin binding protein-C slow (MyBP-C slow), corresponding to variant-1. Variant-1 contains all the structural motifs present in the known forms of MyBP-C slow, but it has a unique COOH terminus. Quantitative reverse transcription-polymerase chain reaction indicated that MyBP-C slow variant-1 is expressed in skeletal muscles both during development and at maturity. Immunolabeling of skeletal myofibers with antibodies to the unique COOH terminus of variant-1 demonstrated that, unlike other forms of MyBP-C slow that reside in the C-zones of A-bands, variant-1 preferentially concentrates around M-bands, where it codistributes with obscurin. Overexpression of the Ig2 domain of obscurin or reduction of expression of obscurin inhibited the integration of variant-1 into forming M-bands in skeletal myotubes. Collectively, our experiments identify a new ligand of obscurin at the M-band, MyBP-C slow variant-1 and suggest that their interaction contributes to the assembly of M- and A-bands.
Obscurin is an approximately 800-kDa protein composed of structural and signaling domains that organizes contractile structures in striated muscle. We have studied the Rho-GEF domain of obscurin to understand its roles in morphogenesis and signaling. We used adenoviral overexpression of this domain, together with ultrastructural and immunofluorescence methods, to examine its effect on maturing myofibrils. We report that overexpression of the Rho-GEF domain specifically inhibits the incorporation of titin into developing Z-disks and disrupts the structure of the Z-disk and Z/I junction, and alters features of the A/I junction. The organization of other sarcomeric markers, including alpha-actinin, was not affected. We identified Ran binding protein 9 (RanBP9) as a novel ligand of the Rho-GEF domain and showed that binding is specific, with an apparent binding affinity of 1.9 microM. Overexpression of the binding region of RanBP9 also disrupted the incorporation of titin into developing Z-disks. Immunofluorescence localization during myofibrillogenesis indicated that the Rho-GEF domain assembles into sarcomeres before RanBP9, which first occurs in myonuclei and later in development translocates to the myoplasm, where it colocalizes with obscurin. Both the Rho-GEF domain and its binding region on RanBP9 bind directly to the N-terminal Ig domains of titin, which flank the Z-disk. Our results suggest that the Rho-GEF domain interacts with RanBP9 and that both can interact with the N-terminal region of titin to influence the formation of the Z-disk and A/I junction.
