De novo assembly of contractile myofibrils begins with the formation of premyofibrils where filaments of non-muscle myosin (NM II), and actin organize in sarcomeric patterns with Z-Bodies containing muscle-specific alpha-actinin. Interactions of muscle specific myosin (MM II) with NM II occur in a nascent myofibril stage that precedes the assembly of mature myofibrils. By the final stage of myofibrillogenesis, the only myosin II present in the mature myofibrils is MM II. In this current study of myofibril assembly, the three vertebrate isoforms of NM II (A, B, and C) and sarcomeric alpha-actinin, ligated to GFP family proteins, were coexpressed in avian embryonic skeletal and cardiac muscle cells. Each isoform of NM II localized only in the mini-A-Bands of premyofibrils and nascent myofibrils. There was no evidence of localization of NM II in Z-Bodies of premyofibrils and nascent myofibrils or in Z-Bands of mature myofibrils. Fluorescence Recovery After Photobleaching (FRAP) experiments indicated similar exchange rates in premyofibrils for NM II isoforms A and B, whereas the IIC isoform was significantly less dynamic. Fluorescence Resonance Energy Transfer (FRET) measurements of colocalized fluorescent pairs of different NM II isoforms yielded signals similar to identical pairs, indicating copolymerization of the different NM II pairs. The role of NM II may reside in establishing the future sarcomere pattern in mature myofibrils by binding to the oppositely polarized actin filaments that extend between pairs of Z-Bodies along premyofibrils prior to their transformation into mature myofibrils.
Keywords: FRAP; FRET; Myofibrillogenesis; mature myofibril; nascent myofibril; non-muscle myosin II isoforms; premyofibril; sarcomere.
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