Ectopic expression and dynamics of TPM1α and TPM1κ in myofibrils of avian myotubes

From the four known vertebrate tropomyosin genes (designated TPM1, TPM2, TPM3, and TPM4) over 20 isoforms can be generated. The predominant TPM1 isoform, TPM1 alpha, is specifically expressed in both skeletal and cardiac muscles. A newly discovered alternatively spliced isoform, TPM1 kappa, containing exon 2a instead of exon 2b contained in TPM1 alpha, was found to be cardiac specific and developmentally regulated. In this work, we transfected quail skeletal muscle cells with green fluorescent proteins (GFP) coupled to chicken TPM1 alpha and chicken TPM1 kappa and compared their localizations in premyofibrils and mature myofibrils. We used the technique of fluorescence recovery after photobleaching (FRAP) to compare the dynamics of TPM1 alpha and TPM1 kappa in myotubes. TPM1 alpha and TPM1 kappa incorporated into premyofibrils, nascent myofibrils, and mature myofibrils of quail myotubes in identical patterns. The two tropomyosin isoforms have a higher exchange rate in premyofibrils than in mature myofibrils. F-actin and muscle tropomyosin are present in the same fibers at all three stages of myofibrillogenesis (premyofibrils, nascent myofibrils, mature myofibrils). In contrast, the tropomyosin-binding molecule nebulin is not present in the initial premyofibrils. Nebulin is gradually added during myofibrillogenesis, becoming fully localized in striated patterns by the mature myofibril stage. A model of thin filament formation is proposed to explain the increased stability of tropomyosin in mature myofibrils. These experiments are supportive of a maturing thin filament and stepwise model of myofibrillogenesis (premyofibrils to nascent myofibrils to mature myofibrils), and are inconsistent with models that postulate the immediate appearance of fully formed thin filaments or myofibrils.