Architecture of the sarcomere matrix of skeletal muscle: immunoelectron microscopic evidence that suggests a set of parallel inextensible nebulin filaments anchored at the Z line.

TLDR

Nebulin is a giant, 600–800 kDa myofibrillar protein that constitutes a cytoskeletal matrix component alongside actin and myosin in skeletal muscle sarcomeres. A monospecific antibody was employed to localize nebulin by immunoelectron microscopy in mechanically split rabbit psoas muscle fibers. Immunoblotting and gel analysis showed skeletal nebulin is distinct from cardiac and smooth muscle forms and exists in at least two size variants, while immunoelectron microscopy revealed six pairs of fixed, non‑stretching transverse densities 0.1–1.0 µm from the Z line, supporting a model in which nebulin forms inextensible filaments anchored at the Z line in parallel with titin, suggesting both proteins act as structural templates and length regulators for actin and myosin.

Abstract

Nebulin, a giant myofibrillar protein (600-800 kD) that is abundant (3%) in the sarcomere of a wide range of skeletal muscles, has been proposed as a component of a cytoskeletal matrix that coexists with actin and myosin filaments within the sarcomere. Immunoblot analysis indicates that although polypeptides of similar size are present in cardiac and smooth muscles at low abundance, those proteins show no immunological cross-reactivity with skeletal muscle nebulin. Gel analysis reveals that nebulins in various skeletal muscles of rabbit belong to at least two classes of size variants. A monospecific antibody has been used to localize nebulin by immunoelectron microscopy in a mechanically split rabbit psoas muscle fiber preparation. Labeled split fibers exhibit six pairs of stripes of antibody-imparted transverse densities spaced at 0.1-1.0 micron from the Z line within each sarcomere. These epitopes maintain a fixed distance to the Z line irrespective of sarcomere length and do not exhibit the characteristic elastic stretch-response of titin epitopes within the I band domain. It is proposed that nebulin constitutes a set of inextensible filaments attached at one end to the Z line and that nebulin filaments are in parallel, and not in series, with titin filaments. Thus the skeletal muscle sarcomere may have two sets of nonactomyosin filaments: a set of I segment-linked nebulin filaments and a set of A segment-linked titin filaments. This four-filament sarcomere model raises the possibility that nebulin and titin might act as organizing templates and length-determining factors for actin and myosin respectively.