The processes involved in the assembly of complex, multimolecular biological structures are poorly understood. This project seeks to use the striated muscle myofibril as a model system in which to explore these mechanisms. Extremely large myofibrillar proteins are hypothesized to play a key role in myofibril assembly. Therefore, as a first step to studying the function of extremely large myofibrillar proteins using molecular genetic approaches, we are cloning, sequencing, and expressing portions of mouse nebulin, as well as N-RAP, a nebulin-related, muscle specific protein. In the past year, we used the effect of association with actin on their apparent solubility to assay the incorporation of nebulin fragments into preformed actin filaments, and to study the morphology of actin-nebulin complexes. Using this approach, we found that nebulin fragments 7a and 8c bound to actin filaments with high affinity. In addition, we found that the stoichiometry of binding is similar to that found in native myofibrils. Immunofluorescence and electron microscopy of these actin-nebulin complexes indicated that the nebulin fragments were reorganized from punctate aggregates to a filamentous form upon interaction with actin filaments. In initial experiments, the actin-nebulin cofilaments were indistinguishable from actin alone when viewed in the electron microscope after negative staining. To further elucidate its function, we have cloned and expressed the amino and carboxy termini of mouse nebulin. Analysis of the sequences obtained so far indicate a high degree of similarity between the terminal domains of mouse and human nebulin. Previously, we had discovered N-RAP, a novel, muscle specific protein with homology to nebulin. In the past year, we mapped the gene encoding N-RAP to mouse chromosome 19 using interspecific crosses, and to human chromosome 10 using radiation hybrid panels. Comparative analysis of the mouse and human genomes indicates that the N-RAP gene is located in regions of conserved synteny between the two species. The C-terminal half of N-RAP contains more than two complete 245 residue nebulin-like super repeats. The N-terminus contains the consensus sequence of a cysteine rich LIM domain, which may function in mediating protein-protein interactions. N-RAP is localized at the myotendon junction in skeletal muscle and at the intercalated disk in cardiac muscle, leading us to hypothesize that N-RAP may be involved in linking the ends of myofibrils to specialized protein complexes found beneath the sarcolemma. We further explored this hypothesis by surveying the ability of recombinant N-RAP domains to bind a variety of candidate proteins. Using solid phase binding assays, we observed preferential binding of actin filaments to the nebulin-like super repeats of N-RAP. In contrast, talin bound preferentially to the LIM domain containing region of N-RAP, while various regions of vinculin bound equally well to all the tested recombinant N-RAP fragments. In contrast, whole myosin and isolated myosin heads exhibited no binding affinity for N-RAP domains. The results support our original hypothesis that N-RAP may serve as an important mechanical link in transmitting tension from the myofibrils to the extracellular matrix. Future studies will explore the physiological functions of nebulin and N-RAP.
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