LAUSR

Tropomyosin proteins form stable coiled-coil dimers that polymerize along the α-helical groove of actin filaments [1]. The actin cytoskeleton consists of both co-polymers of actin and tropomyosin and polymers of tropomyosin-free actin [2]. The fundamental distinction between these two types of filaments is that tropomyosin determines the functional capability of actin filaments in an isoform-dependent manner [3-9]. However, it is unknown what portion of actin filaments are associated with tropomyosin. To address this deficit, we have measured the relative distribution between these two filament populations by quantifying tropomyosin and actin levels in a variety of human cell types, including bone (U2OS); breast epithelial (MCF-10A); transformed breast epithelial (MCF-7); and primary (BJpar), immortalized (BJeH), and Ras-transformed (BJeLR) BJ fibroblasts [10]. Our measurements of tropomyosin and actin predict the saturation of the actin cytoskeleton, implying that tropomyosin binding must be inhibited in order to generate tropomyosin-free actin filaments. We find the majority of actin filaments to be associated with tropomyosin in four of the six cell lines tested and the portion of actin filaments associated with tropomyosin to decrease with transformation. We also discover that high-molecular-weight (HMW), unlike low-molecular-weight (LMW), tropomyosin isoforms are primarily co-polymerized with actin in untransformed cells. This differential partitioning of tropomyosins is not due to a lack of N-terminal acetylation of LMW tropomyosins, but it is, in part, explained by the susceptibility of soluble HMW tropomyosins to proteasomal degradation. We conclude that actin-tropomyosin co-polymers make up a major fraction of the human actin cytoskeleton.