Nspl1, a new Z-band-associated protein

Molecular characterization of a novel gene designated Neuroendocrine-Specif ic Protein-Like-1 (Nspl1) had revealed that this gene is expressed as two t ranscripts, a 1.2 kb transcript found predominantly in skeletal muscle and a 2.1 kb transcript expressed in the brain. The exceptionally high level of skeletal muscle expression prompted us to determine where the protein is l ocalized to skeletal muscle. In vitro studies were performed using two plas mid constructs that generate full-length Nspl1 muscle-specific protein fuse d to the green fluorescent protein (GFP). In one construct, the GFP cDNA wa s fused to the N-terminus of the Nspl1 cDNA while in the second construct, the GFP cDNA was fused to the C-terminus of the Nspl1 cDNA. Transfection of either plasmid into mononucleated myoblasts showed that the Nspl1-GFP chim eric protein was associated with intermediate filaments. This was confirmed by using an antibody to stain desmin and finding that GFP-Nspl1 colocalize s with desmin. Chick primary myoblasts were transfected with the chimeric c DNAs and allowed to differentiate into mature myotubes. Results from this a nalysis and the use of monoclonal antibody to stain alpha-actinin, further localized the Nspl1 protein to the Z-band of mature myotubes. Confocal micr oscopy of the myotubes containing Nspl1-GFP demonstrates that Nspl1 is dist ributed continuously throughout the Z-disks.