Organization of protein and mRNA for titin and other myofibril components during myofibrillogenesis in cultured chicken skeletal muscle

Abstract

Myofibril assembly requires the cell to join diverse components, correctly oriented to the rest of the cell. Titin, a huge elastic protein with a role in myogenesis, assembles during translation in vivo and may require spatially organized mRNA to allow assembly. By immunofluorescence, we examined titin and myosin protein organization early in skeletal muscle development in vitro; titin was the first organized, initially as spots, then as periodically spaced lines, and later as doublets. Titin mRNA organization during development was detected by fluorescent in situ hybridization. Only titin mRNA was seen in mononucleated myoblasts. Shortly after fusion, both titin protein and mRNA were diffuse. Titin mRNA remained diffuse when titin protein formed cables. Where titin protein formed linear arrays of spots, titin mRNA showed a colinear, continuous array. Thin mRNA remained in arrays colinear with young myofibrils until several slender myofibrils aligned laterally; then, titin mRNA formed periodic arrays. The titin probe encodes peptide sequence in the A band, where this region of titin mRNA is detected in the most organized cells. Nebulin undergoes a similar progression slightly later in development. This pattern, of narrowly spaced stripes, is too closely spaced to function in the soluble phase. Titin mRNA is the earliest mRNA to become so highly organized in muscle; it does so earlier and at a different location than do mRNAs for costamere proteins. These results, taken with earlier ones, suggest mRNA localization may be as key to somatic cell differentiation as it is to embryonic development.