NEBULIN IS A FULL-LENGTH TEMPLATE OF ACTIN-FILAMENTS IN THE SKELETAL-MUSCLE SARCOMERE - AN IMMUNOELECTRON MICROSCOPIC STUDY OF ITS ORIENTATION AND SPAN WITH SITE-SPECIFIC MONOCLONAL-ANTIBODIES

Nebulin, a giant myofibrillar protein with size variants from 700 to 9 00 kDa in various skeletal muscles, has been proposed to constitute a set of inextensible filaments anchored at the Z-line and coextensive w ith actin filaments. To elucidate the architectural organization of th is fourth set of myofilaments in the skeletal muscle sarcomere, we per formed immunoelectron microscopic localization of epitope profiles. of a number of site-specific monoclonal antibodies against cloned human nebulin fragments of known sequence loci. Monoclonal antibody N113, wh ich is directed to fragment ND8 at approximately 300 residues away fro m the C-terminus, labelled the edges of Z-lines in both human quadrice ps muscle and rabbit psoas muscle. Monoclonal antibody N101, which is directed to fragment NB5 near the N-terminal side, is localized to a s ingle locus at 0.89 mu m from the Z-line in human quadriceps muscle an d 0.80 mu m hom the Z-line in rabbit psoas muscle. Additionally, monoc lonal antibody N109, which is directed to fragment NA3 on the carboxy side of the adjacent fragment NB5, is localized at 0.76 mu m away from the Z-line in rabbit psoas muscle. This one-to-one correspondence bet ween epitope loci and sequence loci demonstrates that a single nebulin polypeptide spans the length of the thin filament with its C-terminus anchored at the Z-line. The epitope spacings of site-specific antibod ies are consistent with the notion that the nebulin filament is unifor m in mass density along its length. We conclude that the thin filament , as defined morphologically by electron microscopy, is a composite fi lament of the conventional actin thin filament (actin/tropomyosin/trop onin) and coextensible nebulin polypeptides which act as full-length m olecular templates that regulate or stabilize colaterally the actin fi lament in the skeletal muscle sarcomere.