ASSEMBLY OF NEBULIN INTO THE SARCOMERES OF AVIAN SKELETAL-MUSCLE

In developing muscle, relatively little is known about the synthesis a nd incorporation of the large actin binding protein, nebulin, into the sarcomere. To determine the temporal pattern of nebulin assembly into the myofibrils of differentiating skeletal muscle cells, myofibril as sembly was examined by immunofluorescence microscopy. The distribution of nebulin was compared to other myofibrillar and cytoskeletal protei ns (myosin, titin, actin, desmin, tubulin). At the onset of differenti ation, we observed that nebulin is first seen in a diffuse distributio n throughout the cytoplasm. At this time, muscle specific myosin and t itin are also distributed in this manner. Myosin and thin become assoc iated with the nascent myofibrils prior to the addition of nebulin. Th e mature striated pattern of myosin and thin also preceded the develop ment of striations with nebulin. After nebulin becomes organized into a striated pattern, actin filaments separate across the A-band and for m thin filaments of uniform length. These patterns of assembly suggest that nebulin is required for restricting the lengths of the thin fila ments. We have employed the strategy of using ethyl methane sulfonate and taxol to perturb myofibril assembly to examine interactions critic al for the addition of nebulin to the developing sarcomeres. The same temporal pattern of assembly seen in the normal cultures was observed in the ethyl methane sulfonate treated cultures, but at a much slower rate. In cultures treated with the microtubule stabilizing drug taxol, the amount of stress fibers and nascent I-bands was greatly diminishe d as previously reported by others; however, nebulin was found associa ted with myofibrils in a mature striated distribution. In addition, ou r results indicate that the taxol treated cultures contain remnants of the Z-line. These results suggest that nebulin assembly into the myof ibril requires interactions or anchorage at the Z-line and within the A-band. (C) 1996 Wiley-Liss, Inc.