Repetitive titin epitopes with a 42 nm spacing coincide in relative position with known A band striations also identified by major myosin-associated proteins. An immunoelectron-microscopical study on myofibrils

DIETER O. FtJRST 0 RtJDIGER NAVE 0 MARY OSBORN 0 KLAUS WEBER 0 0 Max Planck Institute for Biophysical Chemistry , D-3400 Goettingen, FRG Repetitive titin epitopes with a 42 nm spacing coincide in relative position with known A band striations also identified by major myosin-associated Author for correspondence Summary A direct titin-thick filament interaction in certain regions of the A band is suggested by results using four new monoclonal antibodies specific for titin in immunoelectron microscopy. Antibodies T30, T31 and T32 identify quasi-repeats in the titin molecule characterized by a 42-43 nm repeat spacing. These stripes seem to coincide with striations established by others on negatively stained cryosections of the A band. Antibodies T30 and T32 recognize epitopes matching five or two of the seven striations per half sacromere known to harbor both the myosin-associated C-protein and an 86K (K = 103 Mr) protein. Antibody T31 labels two stripes in the P zone, which correspond to the two positions where decoration is seen with 86K protein, but not with C-protein. The single titin epitope defined by antibody T33 is located 55 nm prior to the center of the M band. This position seems to coincide with the M7 striation defined by others on negatively stained A bands. - Sarcomeric muscles have a highly ordered structure. This involves not only the well-known thick and thin filaments with their associated proteins, but also an elastic component, which is thought to be present as a third filament system (for recent reviews, see Maruyama, 1986; Wang, 1985). Given the relative abundance of titin in isolated myofibrils of skeletal and heart muscle, this giant protein is likely to be a major component of the third filament system. Although nebulin, another high molecular weight protein of skeletal muscle myofibrils, has also been proposed as a component of elastic filaments, recent experiments in several laboratories show that it is not present in cardiac muscle (Hu et al. 1986; Locker & Wild, 1986; Hoffman et al. 1987; Fiirst et al. 1988; Wang & Wright, 1988) and so is unlikely to be a general elastic component of all sarcomeric muscles. The T33 epitope position proves that the titin molecule, which is known to be anchored at the Z line, also penetrates into the complex architecture of the M band. The titin epitopes described here enable us to begin to correlate known ultrastructural aspects of the interior part of the A band with the disposition of the titin molecule in the sarcomere. They raise the question of whether there is a regular interaction pattern between titin and the thick filaments. Because of the limit of resolution of immunoelectron microscopy, it remains to be seen whether the repetitive titin epitopes of the C and P zones coincide exactly with the position of the myosin-associated proteins or lie on a similar lattice, out of register with these proteins. In low-porosity polyacrylamide gels, dodecyl sulfatesolubilized myofibrils show a typical titin doublet, designated TI and T I L Only T i l , thought to be a proteolytic derivative of the larger TI species (Maruyama, 1986; Wang, 1985), can be extracted under native conditions (Kimura & Maruyama, 1983; Trinick et al. 1984; Wang et al. 1984). Although electron micrographs of purified T i l show marked heterogeneity, the molecules appear as very thin and rather long strings (Maruyama et al. 1984; Trinick et al. 1984; Wang et al. 1984). Length estimates for titin molecules range from ~0-6 to l-2^m and so a single titin molecule could span the distance from the M band to either the N2 line (Wang, 1985) or the Z line (Maruyama, 1986). Strong experimental support for titin binding to the Z line comes from immunoelectron micrographs using 10 distinct titin monoclonal antibodies, each defining a unique epitope along the half sarcomere. The polar epitope map starts at the Z line and ends 0-2/im before the center of the M band (Fiirst et al. 1988). Antibodies decorating at or close to the Z line are specific for the larger T I component, whereas all other antibodies react with both components of the titin doublet. Thus the titin filament seems attached via the Tl-specific end to the Z. line structure. Since only epitopes located between the Ni line and the A/1 junction show stretch dependency in location, the putative elastic part of the titin filaments may be restricted to this region. Here we report on a second group of titin monoclonal antibodies, which begin to define possible interaction patterns of titin with the A band. In immunoelectron microscopy, antibodies T30, T31 and T32 define multiple quasi-repeats of the titin molecule spaced 42-43 nm apart. These occur in the region of the A band close to the M band. The positions of these new repetitive titin epitopes seem to coincide with seven of the nine stripes previously reported for some myosin-associated proteins, i.e. C-protein and 86K (K=103Afr) protein (Craig & Offer, 1976; Bahler et al. 1985a,6). We discuss some structural implications of this observation. Materials and methods Monoclonal antibodies The new monoclonals T30 to T33 arose in a fusion of PAI cells with spleen cells of a Balb/c mouse previously immunized with purified T i l (cf. Fiirst etal. 1988). Because, initially, immunofluorescence microscopy pointed to A band decoration, these antibodies were not considered important, as several A bandspecific epitopes had already been documented by immunoelectron microscopy (Fiirst et al. 1988). Later these hybridomas were made monoclonal by subcloning via limiting dilution and further characterized by immunofluorescence microscopy, Western blotting and immunoelectron microscopy. It became clear that they represented a new group. T30-T33 were studied as hybridoma supernatants. In addition, T30 and T33 were available as antibodies purified from ascites fluids elicited in Balb/c mice primed with pristane. Rabbit antibodies to Cprotein and to 86K protein from chicken breast muscle have been described (Bahler et al. 1985a,b). They were generously provided by Drs H. Eppenberger and T. Wallimann, ETH, Zurich, Switzerland. Titin antibody T23 was previously characterized by immunoelectron microscopy (Fiirst et al. 1988). Gel electrophoresis and immunoblotting Linear polyacrylamide gradient gels (2% to 10% acrylamide and 0 5 % crosslinker) were used without a stacking gel in the Laemmli (1970) buffer system as described (Fiirst et al. 1988). Polypeptides were electrophoretically transferred to nitrocellulose. Antibody decoration was revealed with peroxidase-labeled rabbit anti-mouse antibodies (Dakopatts, Copenhagen, Denmark) followed by peroxidase substrates. Immunofluorescence microscopy on frozen sections and isolated myofibrils was as described (Furst el al. 1988). Affinity-purified sheep anti-mouse antibodies were coupled to CNBr-activated Sepharose 4B (Pharmacia). They were used to absorb murine antibody T33 from overgrown supernatant. The Sepharos (...truncated)