Colocation of Tpm3.1 and myosin IIa heads defines a discrete subdomain in stress fibres

Co-polymers of tropomyosin and actin make up a major fraction of the actin cytoskeleton. Tropomyosin isoforms determine the function of an actin filament by selectively enhancing or inhibiting the association of other actin binding proteins, altering the stability of an actin filament and regulating myosin activity in an isoform specific manner. Previous work has implicated specific roles for at least 5 different tropomyosin isoforms in stress fibres, as depletion of any of these 5 isoforms results in a loss of stress fibres. Despite this, most models of stress fibres continue to exclude tropomyosins. In this study we investigate tropomyosin organisation in stress fibres using super resolution light microscopy and electron microscopy with genetically tagged, endogenous tropomyosin. We show that tropomyosin isoforms are organised in subdomains within the overall domain of stress fibres. Tpm3.1/3.2 co-localises with non-muscle myosin IIa/IIb heads and are in register but do not overlap with non-muscle myosin IIa/IIb tails. Furthermore, perturbation of Tpm3.1/3.2 results in decreased myosin IIa in stress fibres, which is consistent with a role for Tpm3.1 in maintaining myosin IIa localisation in stress fibres.