Abstract. mAbs specific for titin or nebulin were characterized by immunoblotting and fluorescence microscopy. Immunoelectron microscopy on relaxed chicken breast muscle revealed unique transverse striping patterns. Each of the 10 distinct titin antibodies provided a pair of delicate decoration lines per sarcomere. The position of these pairs was centrally symmetric to the M line and was antibody dependent. The results provided a linear epitope map, which starts at the Z line (antibody T20), covers five distinct positions along the I band (T21, T12, T4, T1, Tll), the A-I junction (T3), and three distinct positions within the A band (T10, T22, T23). The epitope of T23 locates 0.2 txm before the M line. In immunoblots, the two antibodies decorating at or just before the Z line (T20, T21) specifically recognized the insoluble titin TI component but did not recognize TII, a proteolytic derivative. All other titin antibodies recognized TI and TII. Thus titin molecules appear as polar structures lacking over large regions repetitive epitopes. One physical end seems related to Z line anchorage, while the other may bind close to the M line. Titin epitopes influenced by the contractional state of the sarcomere locate between the N1 line and the A-I junction (T4, T1, Tll). We discuss the results in relation to titin molecules having half-sarcomere lengths. The three nebulin antibodies so far characterized again give rise to distinct pairs of stripes. These locate close to the N2 line.T HE pioneering work of the laboratories of Maruyama and Wang has suggested that sarcomeric structure relies not only on the well-known thick and thin filaments with their various associated proteins but also involves an elastic component (for recent reviews see 19,35). While the nature of the elastic filaments is still poorly understood, it seems clear that they are built from very high molecular weight polypeptides (20,22,38). One major component of the system is titin. Its polypeptide molecular weight is variously given as 1 or 2.8 million (21,31,32,39). Titin is usually present as a doublet on low porosity gels and only the second species TII can be purified under native conditions (10,11,31,40). TII is thought to arise by limited proteolysis from the nonextractable TI species (19,35). Although electron micrographs of purified TII indicate morphological heterogeneity, they clearly document very thin and rather long threads. While the exact biophysical properties are still in dispute, length measurements range from ~0.6 to 1.2 Ixm (21,31,40). In agreement with the proposal that it plays a pivotal role in sarcomere integrity, titin is found both in skeletal and cardiac muscle, although possibly not as identical molecules (7,9,17). Much less is known about nebulin from skeletal muscle. Its molecular weight is ~0. 5-0.8 million (21, 35, 38). As it has not been isolated in native form, its function and shape are not known. In addition, there are reports that nebulin is not expressed in cardiac muscle (9,17).There is distinct disagreement about...

Abstract. The M band of vertebrate cross-striated myofibrils has remained an enigmatic structure. In addition to myosin thick filaments, two major structural proteins, myomesin and M-protein, have been localized to the M band. Also, titin is expected to be anchored in this structure. To begin to understand the molecular layout of these three proteins, a panel of 16 polyclonal and monoclonal antibodies directed against unique epitopes of defined sequence was assembled, and immunoelectron microscopy was used to locate the position of the epitopes at the sarcomere level. The results allow the localization and orientation of defined domains of titin, myomesin, and M-protein at high resolution. The 250-kD carboxy-terminal region of titin clearly enters the M band with the kinase domain situated ~52 nm from the central Ml-line. The positions of three additional epitopes are compatible with the view that the titin molecule reaches ~60 nm into the opposite sarcomere half. Myomesin also seems to bridge the central Ml-line and is oriented parallel to the long axis of the myofibril. The neighboring molecules are oriented in an antiparallel and staggered fashion. The amino-terminal portion of the protein, known to contain a myosin binding site, seems to adopt a specific three-dimensional arrangement. While myomesin is present in both slow and fast fibers, M-protein is restricted to fast fibers. It appears to be organized in a fundamentally different manner: the central portion of the polypeptide is around the Ml-line, while the terminal epitopes seem to be arranged along thick filaments. This orientation fits the conspicuously stronger M1-lines in fast twitch fibers. Obvious implications of this model are discussed.T HE stable and ordered arrangement of thick and thin filaments in sarcomeric muscle myofibrils arises from a complex cytoskeletal framework (for review see Small et al., 1992). Its major structures, the M bands and Z discs, seem to be interconnected by the giant protein titin (also called connectin). In the M band, the thick, hexagonal filament lattice appears to be stabilized by additional components. Some aspects of M band substructure have been deduced from negatively stained longitudinal cryosections (SjOstr6m and Squire, 1977). Thus, the M band appears as a zone of higher density, about 75 nm wide, which depending on fiber type and animal species consists of three to five major cross striations. These striations are thought to reflect bridging structures, called

Assembly of muscle sarcomeres is a complex dynamic process and involves a large number of proteins. A growing number of these have regulatory functions and are transiently present in the myofibril. We show here that the novel tubulin-associated RING/B-box protein MURF2 associates transiently with microtubules, myosin and titin during sarcomere assembly. During sarcomere assembly, MURF2 first associates with microtubules at the exclusion of tyrosinated tubulin. Then, MURF2-labelled microtubules associate transiently with sarcomeric myosin and later with A-band titin when non-striated myofibrils differentiate into mature sarcomeres. Finally, MURF2 labelled microtubules disappear from the sarcomere after the incorporation of myosin filaments and the elongation of titin. This suggests that the incorporation of myosin into nascent sarcomeres and the elongation of titin require an active, microtubule-dependent transport process and that MURF2-associated microtubules play a role in the alignment and extension of nascent sarcomeres. MURF2 is expressed in at least four isoforms, of which a 27 kDa isoform is cardiac specific. A C-terminal isoform is generated by alternative reading frame use, a novelty in muscle proteins. In mature cardiac sarcomeres, endogenous MURF2 can associate with the M-band, and is translocated to the nucleus. MURF2 can therefore act as a transient adaptor between microtubules, titin and nascent myosin filaments, as well as being involved in signalling from the sarcomere to the nucleus.

No abstract

Filamin, also called actin binding protein-280, is a dimeric protein that cross-links actin filaments in the cortical cytoplasm. In addition to this ubiquitously expressed isoform (FLN1), a second isoform (ABP-L/␥-filamin) was recently identified that is highly expressed in mammalian striated muscles. A monoclonal antibody was developed, that enabled us to identify filamin as a Z-disc protein in mammalian striated muscles by immunocytochemistry and immunoelectron microscopy. In addition, filamin was identified as a component of intercalated discs in mammalian cardiac muscle and of myotendinous junctions in skeletal muscle. Northern and Western blots showed that both, ABP-L/␥-filamin mRNA and protein, are absent from proliferating cultured human skeletal muscle cells. This muscle specific filamin isoform is, however, up-regulated immediately after the induction of differentiation. In cultured myotubes, ABP-L/␥-filamin localises in Z-discs already at the first stages of Z-disc formation, suggesting that ABP-L/␥-filamin might play a role in Z-disc assembly.