Distinct families of Z-line targeting modules in the COOH-terminal region of nebulin

To learn how nebulin functions in the assembly and maintenance of I-Z-I bands, MYC- and GFP- tagged nebulin fragments were expressed in primary cultured skeletal myotubes. Their sites of incorporation were visualized by double staining with anti-MYC, antibodies to myofibrillar proteins, and FITC- or rhodamine phalloidin. Contrary to expectations based on in vitro binding studies, none of the nebulin fragments expressed in maturing myotubes were incorporated selectively into I-band approximately 1.0-micrometer F-alpha-actin-containing thin filaments. Four of the MYC/COOH-terminal nebulin fragments were incorporated exclusively into periodic approximately 0.1-micrometer Z-bands. Whereas both anti-MYC and Rho-phalloidin stained intra-Z-band F-alpha-actin oligomers, only the latter stained the pointed ends of the polarized approximately 1.0-micrometer thin filaments. Z-band incorporation was independent of the nebulin COOH-terminal Ser or SH3 domains. In vitro cosedimentation studies also demonstrated that nebulin SH3 fragments did not bind to F-alpha-actin or alpha-actinin. The remaining six fragments were not incorporated into Z-bands, but were incorporated (a) diffusely throughout the sarcoplasm and into (b) fibrils/patches of varying lengths and widths nested among normal striated myofibrils. Over time, presumably in response to the mediation of muscle-specific homeostatic controls, many of the ectopic MYC-positive structures were resorbed. None of the tagged nebulin fragments behaved as dominant negatives; they neither blocked the assembly nor induced the disassembly of mature striated myofibrils. Moreover, they were not cytotoxic in myotubes, as they were in the fibroblasts and presumptive myoblasts in the same cultures.