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. TII, the extractable form of titin, was purified from myofibrils and separated by high resolution gel permeation chromatography into two fractions (TII^ and TIIa). Novel specimen orientation methods used before metal shadowing and EM result in striking pictures of the two forms. Molecules layered on mica become uniformly oriented when subjected to centrifugation. TIIa comprises a very homogeneous fraction. All molecules reveal a single globular head at one end on a long and very thin rod of uniform diameter. The lengths of the rods have a very narrow distribution (900 + 50 nm). TII^ molecules seem lateral oligomers of TIIs, attached to each other via the head regions. While dimers are the predominant species, trimers and some higher oligomers can also be discerned. Mild proteolysis destroys the heads and converts TII^ and TIIs into THB-like rods. Similar molecules also result from titin purified from myofibrils by certain established purification schemes. Headless titin molecules show in gel electrophoresis only the TII band, while head bearing molecules give rise to two additional polypeptides at 165 and 190 kD. Immunoelectron microscopy of myofibrils identifies both titinassociated proteins as M band constituents. We speculate that in the polar images of TII the globular head region corresponds to the M band end of the titin molecules. This hypothesis is supported by immunoelectron micrographs of TIIa molecules using titin antibodies of known epitope location in the half sarcomere. This proposal complements our previous immunoelectron microscopic data on myofibrils. They showed that epitopes present only on the nonextractable TI species locate to the Z line and its immediately adjacent region (Fiirst, D. O., M. Osborn, R. Nave, and K. Weber. 1988. J. Cell Biol. 106:1563-1572. Thus, the two distinct ends of the titin molecule attach to Z and M band material respectively.

Desmin protofilaments and the proteolytically derived a-helical rod domain have been characterized by highresolution gel permeation chromatography (GPC) using columns calibrated for the determination of viscosity radii. Additional characterization by chemical cross-linking and the determination of sedimentation values allowed the calculation of the molecular dimensions of the molecular species isolated. In dilute buffers GPC separated desmin rod preparations into two complexes: a dimer species (single coiled coil) with a length of 50 & 5 nm and a tetramer species (two coiled coils) with a length of 65 & 5 nm. Thus the two coiled coils in the tetramer are staggered by approximately 15 nm. The hydrodynamically derived lengths of the rod dimer and tetramer are supported by electron microscopy after metal shadowing. The hydrodynamic properties of desmin protofilaments follow that of the rod tetramer. The rod is organized from double-stranded coiled coils [4] in which the participating a-helical chains are aligned in parallel and in register [5, 61. Usually two coiled coils are found tightly associated in the tetrameric protofilament unit, which is the building block of the intermediate filaments [4, 7-101. Two main questions arise about the structure of the tetramer: are the two coiled coils of the tetramer arranged in parallel or antiparallel manner and are the coiled coils aligned in register or with a stagger? Protofilaments and the isolated rod, lacking the head and tail domains due to limited proteolysis, have been extensively studied by electron microscopy [lo-151. Molecules displaying the unit length of the coiled coil (about 40 -50 nm) as well as particles with the one and a half fold length (about 70 nm) have been recognized in these studies. Because a firm correlation of the observed length variants with the number of chains per particle was not reached, the mode of alignment remained unresolved.We have now re-investigated the shape of the tetramer by hydrodynamic identification of distinct oligomer species. We show that the chymotryptically derived rod domain of chicken desmin (residues 70-415) is present in dilute buffers as a mixture of dimers (single coiled coils) and tetramers (double coiled coils), which can be separated by gel permeation chromatography (GPC). The chromatographically derivedCorrespondence to

Nebulin is a high molecular weight polypeptide (mass 060.8 million) which accounts for 3% of the myofibrillar mass in skeletal muscle. Due to its resistance to extraction under native conditions, relatively little is known about the biochemistry of the molecule. Here we report in vitro binding of a-actinin (a major Z-line protein) to nebulin. After solubilization with sodium dodecylsulfate myofibrillar polypeptides separated by gel electrophoresis were blotted on nitrocellulose and probed with 12sI-labelled a-actinin. Nebulin is the only polypeptide decorated by a-actinin. This result gives biochemical support for the hypothesis, based on recent immunoelectron micrographs, that nebulin could form in skeletal muscle a fourth filament system, possibly extending to the Z-line. a