The organization of titin filaments in the half-sarcomere revealed by monoclonal antibodies in immunoelectron microscopy: a map of ten nonrepetitive epitopes starting at the Z line extends close to the M line.

Abstract

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...

Distinct roles for telethonin N‐versus C‐terminus in sarcomere assembly and maintenance

“…Titin is the largest known protein, spanning half the sarcomere from the Z-disc to the M-line (Maruyama et al, 1977;Wang et al, 1979;Fü rst et al, 1988). In mature striated muscle, it provides passive muscle elasticity (Maruyama, 1976;Maruyama et al, 1977;Mitsui et al, 1987), and has been proposed to act as a stretch sensor (Knöll et al, 2002;Miller et al, 2004;Linke, 2008).…”

“…In mature striated muscle, it provides passive muscle elasticity (Maruyama, 1976;Maruyama et al, 1977;Mitsui et al, 1987), and has been proposed to act as a stretch sensor (Knöll et al, 2002;Miller et al, 2004;Linke, 2008). During sarcomere assembly, titin is hypothesized to act as a molecular ruler and organizational scaffold (Fü rst et al, 1988;Maruyama, 1976;Maruyama et al, 1985, Trinick, 1994. Therefore, titin must be rigidly and precisely anchored within the sarcomere, and this is accomplished in part by a small 19-kDa Z-disc protein called telethonin, also known as T-cap (Valle et al, 1997;Gregorio et al, 1998;Zou et al, 2003Zou et al, , 2006Pinotsis et al, 2006).…”

Gigantic Business

“…The titin molecule extends from the Z-disk (NH 2 terminus) to the M-band (COOH terminus) of the half sarcomere. 12 Multiple alternative splicing events, particularly in the I-band region of titin (≈230 exons), result in 3 main isoform variants: N2B (≈3 000 kDa) and N2BA (≈3200-3800 kDa) in the heart and N2A in skeletal muscles ( Figure 2). 13 Rare isoforms of titin include the full-length variants Novex-1 and Novex-2, and the truncated Novex-3 (625 kDa).…”

The pattern of MyoD and contractile protein localization in primary epaxial myotome reflects the dynamic progression of nascent myoblast differentiation

“…Second, only desmin was consistently present in 6-hr myoblasts while the presence of titin and myosin, as well as five other contractile proteins, was variable at this time point suggestive of a stochastic process in the activation of a cohort contractile protein expression. In fully differentiated muscle, titin protein molecules span a complete semi-sarcomere from Z-disc (N terminal) to M-line (C-terminal), a distance of approximately 1 m (Wang and Greaser, 1985;Maruyama, 1986;Furst et al, 1988). The human titin gene is large (294 kb) and encodes an exceptionally long polypeptide (38,138 amino acids).…”