Titin and myosin, but not desmin, are linked during myofibrillogenesis in postmitotic mononucleated myoblasts.

Abstract

Abstract. Monoclonal antibodies specific for the muscle protein titin have been used in conjunction with muscle-specific antibodies against myofibrillar myosin heavy chains (MHCs) and desmin to study myogenesis in cultured cells. Desmin synthesis is initiated in replicating presumptive myoblasts, whereas the synthesis of titin and MHC is initiated simultaneously in their progeny, the postmitotic, mononucleated myoblasts. Both titin and MHC are briefly localized to nonstriated and thereafter to definitively striated myofibrils. At no stage during myofibrillogenesis is either protein observed as part of a sequence of mini-sarcomeres. Titin antibodies bind to the A-I junction, MHC antibodies to the A bands in nascent, maturing, and mature myofibrils. In contrast, desmin remains distributed as longitudinal filaments until well after the definitive myofibrils have aligned laterally. This tight temporal and topographical linkage between titin and myosin is also observed in postmitotic, mononucleated myoblasts and multinucleated myotubes when myoflbrillogenesis is perturbed with Colcemid or taxol. Colcemid induces elongating postmitotic mononucleated myoblasts and multinucleated myotubes to round up and form Colcemid myosacs. The myofibrils that emerge in these rounded cells are deployed in convoluted circles. The time required for their nonstriated myofibrils to transform into striated myofibrils is greatly protracted. Furthermore, as Colcemid induces immense desmin intermediate filament cables, the normal spatial relationships between emerging individual myofibrils is distorted. Despite these disturbances at all stages, the characteristic temporal and spatial relationship observed in normal myofibrils between titin and MHC is observed in myofibrils assembling in Colcemid-treated cells. Newly born postmitotic mononucleated myoblasts, or maturing myotubes, reared in taxol acquire a star-shaped configuration and are induced to assemble "pseudo-striated myofibrils" Pseudo-striated myofibrils consist of laterally aggregated 1.6-1xm long, thick filaments that interdigitate, not with thin filaments, but with long microtubules. These atypical myofibrils lack Z bands. Despite the absence of thin filaments and Z bands, titin localizes with its characteristic sarcomeric periodicity in pseudo-striated myofibrils. We conclude that the initiation and subsequent regulation of titin and myosin synthesis, and their spatial deployment within developing sarcomeres are tightly coupled events. These findings are discussed in terms of a model that proposes interaction between two relatively autonomous "organizing centers" in the assembly of each sarcomere.

Generation of noncentrosomal microtubule arrays

“…Notably, many MTs in the array become stabilized as the cells differentiate (Gundersen et al, 1989). The linear, noncentrosomal array of MTs in myotubes has been implicated in myofibrillogenesis as well as in establishing the elongated shape of the cell (Antin et al, 1981;Hill et al, 1986;Pizon et al, 2005;Tassin et al, 1985). Experiments using inhibitors of MTs have also implicated MTs in the mobility of acetylcholine receptors in primary cultures of chick embryonic muscle cells, suggesting a function for these arrays in the development of neuromuscular junctions (Connolly, 1984;Connolly and Oldfin, 1985).…”

Exon- and contraction-dependent functions of titin in sarcomere assembly

“…9). At the premyofibril stage, the Ttn N-terminus binds to the Z-bodies in the existing myofibril network, and its flexible C-terminus binds the thick filaments that are assembled independently and still scattered in the cell (Hill et al, 1986;Wang et al, 1988;Komiyama et al, 1990). Ttn facilitates the incorporation of the thick filaments into the thin filament networks and then guides the lateral alignment of thick filaments and Z-bodies to form mature nascent myofibrils (Fig.…”

Association of titin and myosin heavy chain in developing skeletal muscle.

“…Titin is periodic when the first periodic arrays of myosin form in skeletal (6,7) and cardiac (8) cells. Titin may be required for such arrays, since it is one of the few proteins unique to and ubiquitous in striated muscle.…”