Terminal regions of mouse nebulin: Sequence analysis and complementary localization with N-RAP This article is a US Government work and, as such, is in the public domain in the United States of America.

Abstract

The regions of mouse nebulin extending from the ends of the super repeats to the C-terminus and N-terminus were cloned and sequenced. Comparison of the mouse sequence with the previously published human sequence shows that the terminal regions of nebulin are highly conserved. The four phosphorylation motifs and SH3 domain found at the C-terminus of mouse nebulin are identical to those found in human nebulin, with the exception of four conservative substitutions. The modules linking this C-terminal region to the super repeats have deletions relative to both fetal and adult human nebulins that correspond to integral numbers of modules, making the mouse C-terminal simple repeat region among the shortest observed to date. The N-terminal region and the C-terminal modules were expressed in Escherichia coli and used for antibody production. Immunofluorescent labeling of these regions of nebulin in isolated myofibrils demonstrates that they are located near the center of the sarcomere and near the Z-line, respectively. Immunogold labeling with antibodies raised against the N-terminal nebulin sequence localizes this region in the A-band near the tips of the thin filaments. Nebulin localization is complementary to that of N-RAP, another muscle-specific protein containing nebulin-like super repeats; nebulin is exclusively found in the sarcomeres, while N-RAP is confined to the terminal bundles of actin filaments at the myotendinous junction. Cell Motil. Cytoskeleton 3:211-222, 2000 Published 2000 Wiley-Liss, Inc.

Ultrastructural and Biochemical Localization of N-RAP at the Interface between Myofibrils and Intercalated Disks in the Mouse Heart

“…The previous identification of N-RAP at skeletal muscle myotendon junctions and cardiac intercalated disks suggested an association of N-RAP with the ends of myofibrils (1). Ultrastructural observations in both skeletal muscles (8) and the heart (Figure 2) support this; in both cases, immunoelectron microscopy showed that N-RAP is concentrated at the terminal actin filaments that link the myofibrils to the cell membranes. Interestingly, vinculin antibodies generally labeled close to the fascia adherens membrane, while the N-RAP antibody was detected close to the membrane as well as inside the terminal cell processes.…”

“…Myofibrils were stained for N-RAP in flow chambers as previously described (8). In brief, they were incubated for 30 min in blocking solution [5% goat serum in phosphatebuffered saline (PBS)], followed by a 1 h incubation with anti-N-RAP antibody diluted 1:1000 in blocking solution, and then a 1 h incubation with fluorescein-linked antibody from donkey against rabbit immunoglobulins (Amersham Corp., Arlington Heights, IL) diluted 1:20 in blocking solution.…”

“…The secondary antibodies (Jackson Immunoresearch, West Grove, PA) were each adsorbed against serum proteins from the nontarget species, to minimize cross-reactivity with the nontarget primary antibody. Standard blocking and washing steps were included in the labeling procedure, as described previously (8). Sections were viewed and photographed by use of a Jeol 1200EX transmission electron microscope, at an accelerating voltage of 100 kV.…”

“…In adult tissues, N-RAP is concentrated at the myotendinous junctions of skeletal muscle and at the intercalated disks of cardiac muscle (1). Using immunoelectron microscopy, we recently showed that N-RAP is specifically associated with the terminal bundles of actin filaments at the myotendinous junctions (8). In addition, N-RAP was found concentrated at the cell periphery along with the earliest detectable myofibril precursors in spreading cultures of embryonic chick cardiomyocytes (9).…”