In developing muscle, relatively little is known about the synthesis and 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 assembly was examined by immunofluorescence microscopy. The distribution of nebulin was compared to other myofibrillar and cytoskeletal proteins (myosin, titin, actin, desmin, tubulin). At the onset of differentiation, we observed that nebulin is first seen in a diffuse distribution throughout the cytoplasm. At this time, muscle specific myosin and titin are also distributed in this manner. Myosin and titin become associated with the nascent myofibrils prior to the addition of nebulin. The mature striated pattern of myosin and titin also preceded the development of striations with nebulin. After nebulin becomes organized into a striated pattern, actin filaments separate across the A-band and form thin filaments of uniform length. These patterns of assembly suggest that nebulin is required for restricting the lengths of the thin filaments. We have employed the strategy of using ethyl methane sulfonate and taxol to perturb myofibril assembly to examine interactions critical 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 diminished as previously reported by others; however, nebulin was found associated with myofibrils in a mature striated distribution. In addition, our results indicate that the taxol treated cultures contain remnants of the Z-line. These results suggest that nebulin assembly into the myofibril requires interactions or anchorage at the Z-line and within the A-band.
|Number of pages||18|
|Journal||Cell Motility and the Cytoskeleton|
|Publication status||Published - 1996|
- protein ruler
ASJC Scopus subject areas
- Structural Biology
- Cell Biology