© 2015. Published by The Company of Biologists Ltd | Journal of Cell Science (2015) 128, 2965-2974 doi:10.1242/jcs.172502 COMMENTARY Tropomyosin – master regulator of actin filament function in the cytoskeleton Peter W. Gunning1,*, Edna C. Hardeman1,*,‡, Pekka Lappalainen2,* and Daniel P. Mulvihill3,* ABSTRACT The discovery of Tpm in mammalian non-muscle cells in the mid- Tropomyosin (Tpm) isoforms are the master regulators of the 1970s by Elias Lazarides, naturally, led to the expectation that Tpm functions of individual actin filaments in fungi and metazoans. Tpms has a similar function in these cells compared with its role in skeletal are coiled-coil parallel dimers that form a head-to-tail polymer along muscle cells (Lazarides, 1975). Immunofluorescence experiments led the length of actin filaments. Yeast only has two Tpm isoforms, Lazarides to propose the existence of two populations of actin whereas mammals have over 40. Each cytoskeletal actin filament polymers in non-muscle cells, those that contained Tpm and those that contains a homopolymer of Tpm homodimers, resulting in a filament did not (Lazarides, 1976). In hindsight, it is difficult to understand of uniform Tpm composition along its length. Evidence for this ‘master how the discussion of the potential significance of Tpm as a core regulator’ role is based on four core sets of observation. First, spatially component of the actin filament in the non-muscle cytoskeleton and functionally distinct actin filaments contain different Tpm slowly disappeared from the literature. There are several simple isoforms, and recent data suggest that members of the formin explanations that are likely to account for this. First, Tpms are difficult family of actin filament nucleators can specify which Tpm isoform is to work with in protein chemistry experiments because, in solution, added to the growing actin filament. Second, Tpms regulate whole- they are largely unstructured and have a tendency to oligomerize and/ organism physiology in terms of morphogenesis, cell proliferation, or aggregate. Furthermore, Tpm dimers bind F-actin only with very ∼ −3 vesicle trafficking, biomechanics, glucose metabolism and organ size low affinity (Kd 10 M) (Wegner, 1979) and their efficient in an isoform-specific manner. Third, Tpms achieve these functional incorporation into an actin filament requires the formation of a outputs by regulating the interaction of actin filaments with myosin head-to-tail Tpm polymer, consisting of homopolymers of Tpm motors and actin-binding proteins in an isoform-specific manner. dimers that run along the actin filament (Tobacman, 2008). Finally, Last, the assembly of complex structures, such as stress fibers and Tpms only interact with actin through ionic interactions and, in ‘ ’ podosomes involves the collaboration of multiple types of actin essence, float above the surface of the actin polymer, making them filament specified by their Tpm composition. This allows the cell to unlike most other actin-binding proteins (von der Ecken et al., 2015). specify actin filament function in time and space by simply specifying The significance of Tpm has been brought into focus by a recent their Tpm isoform composition. analysis, which showed that evolution has selected for increasing diversity of actin filament composition and that, in the case of fungi KEY WORDS: Actin cytoskeleton, Isoforms, Tropomyosin and metazoans, Tpms have provided this diversity (Gunning et al., 2015). This Commentary examines the functional consequences of Introduction using Tpms to diversify the actin filament composition in the Tropomyosin (Tpm) is best known for its role in the regulation of cytoskeleton of fungi and metazoans, and will make the case that contraction of skeletal muscle and the heart. The contraction of these Tpms are the master regulators of cytoskeletal actin filament function striated muscles involves the synchronized movement of myosin in these organisms. We focus on four main issues: (i) the mechanism heads that are engaged with actin filaments to produce a net of assembly of actin filaments that contain homopolymers of specific translocation of the myosin thick filament with respect to the actin Tpm isoforms, (ii) the physiological function of specific populations thin filament (Geeves, 2012; Lehrer and Geeves, 2014). The actin of actin filaments that contain different Tpms, (iii) the mechanism by thin filament is composed of three core elements: a double-stranded which different Tpms direct different functional outcomes and (iv) polymer of actin, two continuous polymers of Tpm running along how different Tpm-containing filaments contribute to the assembly of each side of the actin and the troponin complex, a heteromeric large-scale actin-based structures. Where mammalian Tpms are protein complex consisting of troponin T (TnT), troponin I (TnI) specified in the text, we will use the new nomenclature (see Geeves and troponin C (TnC), which is located on each Tpm dimer et al., 2015; supplementary Table 1). (Lehman and Craig, 2008). In response to a pulse of Ca2+ the troponin complex moves the position of the Tpm polymer to Assembly of specific Tpms into actin filaments facilitate the coordinated engagement of the heads of the myosins in Most fungi and metazoan cells have the capacity to express multiple the thick filament with actins in the thin filament. isoforms of Tpm, resulting from either different gene products or different post-translational modifications. The biophysical properties of each Tpm filament and the specific way in which they 1 2 School of Medical Sciences, UNSW Australia, Sydney 2052, Australia. Institute of interact with actin can differ significantly. This cooperative interaction Biotechnology, University of Helsinki, Helsinki, 00014, Finland. 3School of Biosciences, Stacey Building, University of Kent, Canterbury, Kent CT2 7NJ, UK. with the actin polymer is crucial for Tpm function because it regulates *These authors contributed equally to this work interactions with other actin-binding proteins (e.g. myosins and ‡ cofilin) (Bryce et al., 2003), as well as the biophysical and/or dynamic Author for correspondence ([email protected]) properties of the actin filament. Different Tpms are, therefore, able to This is an Open Access article distributed under the terms of the Creative Commons Attribution impart distinct physical properties to different actin filaments and, License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed. thereby, dictate their function. As many cell types can express multiple Journal of Cell Science 2965 COMMENTARY Journal of Cell Science (2015) 128, 2965-2974 doi:10.1242/jcs.172502 Tpm isoforms, it is crucial for the viability, function and mobility of a intracellular locations is one possibility, studies in which cell to recruit the appropriate Tpm to an actin polymer at the correct cytoskeletal drugs were used have comprehensively ruled this out place and time (Bach et al., 2009). Indeed, we know that different (Schevzov et al., 1997). It was found that isoform sorting depended Tpms are sorted to different actin filament populations in many on the maintenance of polymeric structures, and that destruction of systems and cell types, but how this is brought about is not yet fully these structures in response to actin-polymerization- and understood (Vindin and Gunning, 2013). microtubule-inhibiting drugs dispersed sorted Tpm isoforms The weak binding of Tpm dimers to actin can only result in a throughout the cell (Gunning et al., 1998). Instead, the spatial continuous polymer of Tpm along an actin filament through head-to- specificity of Tpms appears to be controlled through localized tail contacts between individual Tpm dimers (Tobacman, 2008). The filament assembly (Schevzov et al., 1997). Similarly, a more recent Hitchcock-DeGregori group has long supported a model whereby study ruled out a Tpm transport mechanism that depends upon the actin–Tpm interaction is promoted by discrete actin-binding ‘sorting signals’ and demonstrated that sorting only functions in the domains that are present in the Tpm molecule (Singh and Hitchcock- context of actin–Tpm polymer formation, consistent with the notion DeGregori, 2006), thus restraining the structural conformation of the that a Tpm isoform is being held in place through assembly into actin–Tpm complex. In contrast, others support models where the isoform-specific polymeric structures (Martin et al., 2010). complementary shape and charges at the interface between the There has been growing evidence that actin organizes into coiled-coiled-coil (polymers of the coiled-coil dimer have an polymers of different conformation (Egelman and Orlova, 1995), intrinsic coiled shape) Tpm polymer and actin filament result in not only owing to differences in the actin isoforms and the discrete the Tpm dimer associating with actin with sub-micromolar affinity, nucleotide-binding states but also through different co-factor yet still allowing movement across its surface to regulate interactions interactions (e.g. Galkin et al., 2010). Subtle differences in the with other actin-binding proteins (Holmes and Lehman, 2008). shape of an actin polymer can affect the affinity of different actin- However, the precise nature of the actin–Tpm interaction is likely to binding proteins including, probably, specific Tpm isoforms to remain the subject of controversy for some time to come.