Nonmuscle tropomyosin-4 requires coexpression with other low molecular weight isoforms for binding to thin filaments in cardiomyocytes

Vertebrate tropomyosins (TMs) are expressed from four genes, and at least 1 8 distinct isoforms are generated via a complex pattern of alternative RNA splicing and alternative promoters. The functional significance of this iso form diversity is largely unknown and it remains to be determined whether s pecific isoforms are required for assembly and integration into distinct ac tin-containing structures. The ability of nonmuscle (TM-1, -2, -3, -4, -5(N M1), -5a or -5b) and striated muscle (skeletal muscle alpha-TM) isoforms to incorporate into actin filaments of neonatal rat cardiomyocytes (NRCs) was studied using expression plasmids containing TM-fusions with GFP (green fl uorescent protein) as well as with VSV- or HA-epitope tags. All isoforms, e xcept of fibroblast TM-4, were able to incorporate into the I-band of NRCs, When TM-4 was co-transfected with other low molecular weight (LMW) isoform s of TM (TM-5, TM-5a and TM-5b), it was able to incorporate into sarcomeres of NRCs. This result was not obtained when TM-4 was co-transfected with hi gh molecular weight (HMW) TMs (TM-1, TM-2 or skeletal muscle alpha-TM). The se data demonstrate that the ability of TM-4 to bind to actin filaments can be specifically influenced by its interaction with other LMW TM isoforms. In addition, cells that incorporated the muscle or nonmuscle GFP-TMs into t heir sarcomeres continued to beat and exhibited sarcomeric contraction. The se studies provide the first in vivo demonstration of synergistic effects b etween TM isoforms for binding to actin filaments. These results have impor tant implications in understanding actin filament dynamics in nonmuscle cel l systems, especially during development and in transformed cells, where al terations in the ratio of different LMW isoforms might lead to changes in t heir interactions with actin filaments. Furthermore, these studies demonstr ate that GFP-TM can be used to study thin-filament dynamics in muscle cells and actin filament dynamics in nonmuscle cells.