Previously, we had discovered N-RAP, a novel, muscle specific protein with homology to nebulin. The C-terminal half of N-RAP contains more than two complete actin-binding super repeats similar to those found in nebulin. The N-terminus contains the consensus sequence of a cysteine rich LIM domain, which binds talin in vitro. Our work had localized N- RAP at the myotendon junction in skeletal muscle and at the intercalated disk in cardiac muscle, leading us to hypothesize that the N-RAP domains may be involved in linking the ends of myofibrils to specialized protein complexes found beneath the sarcolemma. In the past year, we used a variety of methods to test this hypothesis. Double immunofluorescent staining of cultured embryonic chick cardiomyocytes for N-RAP and either alpha-actinin, titin, or vinculin showed that myofibrillogenesis and cell contact formation are both associated with targeting of N-RAP to the longitudinal ends of cardiac myocytes. In addition, preliminary results from experiments in which defined regions of N-RAP were expressed in chick cardiomyocytes as fusions with green fluorescent protein (GFP) suggest that the N-RAP LIM domain is responsible for targeting the molecule to myofibril ends and cell contacts. Meanwhile, GFP-tagged N-RAP super repeats are incorporated into sarcomeric actin filaments. We found that, at the ultrastructural level, N-RAP localization is complementary to that of nebulin in skeletal muscle. Even though both proteins contain actin-binding super repeats, nebulin is exclusively found in the sarcomeres, while N-RAP is confined to the terminal bundles of actin filaments at the myotendon junction. These results are consistent with our model of N-RAP as a modular link between the terminal actin filaments of myofibrils and the protein complexes located beneath the cell membrane, with the N-RAP LIM domain targeting the molecule to the ends of the muscle cell. Furthermore, we found that N-RAP binds muscle LIM protein (MLP) in vitro and that the targeting of N-RAP to the intercalated disks in mice lacking MLP is imprecise. The results suggest that N-RAP targeting to the cardiac intercalated disk is influenced by its interaction with MLP. Double immunogold labeling of mouse cardiac muscle reveals that vinculin is located immediately adjacent to the fascia adherentes region of the intercalated disk membrane, while N-RAP extends ~100 nm further towards the interior of the cell. Furthermore, N-RAP remains tightly bound to myofibrils and fascia adherentes during biochemical purification, consistent with it being a key constituent in the mechanical link between these two structures. - muscle, heart, myofibril, nebulin, N-RAP
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