Tropomyosin Isoform Expression Regulates the Transition of Adhesions To Determine Cell Speed and Direction

Abstract

The balance of transition between distinct adhesion types contributes to the regulation of mesenchymal cell migration, and the characteristic association of adhesions with actin filaments led us to question the role of actin filament-associating proteins in the transition between adhesive states. Tropomyosin isoform association with actin filaments imparts distinct filament structures, and we have thus investigated the role for tropomyosins in determining the formation of distinct adhesion structures. Using combinations of overexpression, knockdown, and knockout approaches, we establish that Tm5NM1 preferentially stabilizes focal adhesions and drives the transition to fibrillar adhesions via stabilization of actin filaments. Moreover, our data suggest that the expression of Tm5NM1 is a critical determinant of paxillin phosphorylation, a signaling event that is necessary for focal adhesion disassembly. Thus, we propose that Tm5NM1 can regulate the feedback loop between focal adhesion disassembly and focal complex formation at the leading edge that is required for productive and directed cell movement.Among the different modes of migration that cells adopt, mesenchymal cell migration is dependent on integrin-based adhesion to the extracellular matrix (14), and the cellular mechanisms regulating integrin adhesion formation and turnover (adhesion dynamics) are integral to this process. The fate of integrin adhesions is intimately linked with filaments of polymerized actin (4). At the molecular level, actin filaments are highly dynamic, and this aspect of actin polymer biology provides an important control mechanism by which cells can organize filaments into structures with distinct properties. Tropomyosins are a multi-isoform family of actin-associating proteins that confer isoform-specific regulation of diverse actin filaments (3,16,34,35). The interdependence of integrin adhesions and actin filaments suggests that expression of actinassociated proteins such as the tropomyosins may represent a mechanism for the regulation of adhesion dynamics that determine cell migration.In migrating cells small integrin-based focal complexes form at the periphery of lamellipodial extensions (32). These complexes are characterized by their subcellular distribution, dotlike shape, dependence on Rac activity, phosphorylated paxillin, and association with the network of short, branched actin filaments at the leading edge. The focal complexes are short lived (43) but provide strong traction forces at the leading edge (2) and most likely regulate directional migration (19). Subsets of focal complexes mature into focal adhesions, structures characterized by: Rho GTPase and Rho kinase dependence, dash-like shape, high levels of paxillin and phosphorylated paxillin, and low levels of the actin-binding molecule tensin (43,44). The focal adhesions play an important role in anchoring bundles of polymerized actin stress fibers, providing the contractile force necessary for the translocation of the cell body during migration. There are at leas...

The actin-associating protein Tm5NM1 blocks mesenchymal motility without transition to amoeboid motility

“…In contrast, B35 cells stably overexpressing B5-fold higher levels of Tm5NM1 (B35.Tm5NM1) (Figure 1b) were predominantly elliptical with numerous short, pericellular protrusions ( Figure 1c). Agreeing with our earlier report that Tm5NM1 reduces migration speed of cells in 2D culture systems (Bach et al, 2009), Tm5NM1 overexpression significantly reduces migration speed within 3D collagen gels (Figure 1d). Indeed, B35.Tm5NM1 cells within 3D matrices are fundamentally stationary (speeds o0.05 mm/min represent cell oscillation rather than actual cell migration), and we observe very little translocation of cell bodies in these cultures.…”

“…Importantly, expression of this isoform can vary up to 5-fold between different tissues, with the highest levels observed in lung tissue . Furthermore, we have previously demonstrated that Tm5NM1-mediated stabilization of the actin filaments causes significant inhibition of focal adhesion disassembly (Bach et al, 2009). Given that focal adhesion turnover is required for mesenchymal migration, these results suggested that Tm5NM1 might specifically block mesenchymal migration.…”

“…Using fibroblasts derived from a mouse model lacking Tm5NM1 expression (Figure 2a), we have previously established that the absence of Tm5NM1 expression promotes increased migration persistence in 2D assays of cell migration (Bach et al, 2009). Thus, to confirm the role of Tm5NM1 in regulating mesenchymal cell migration, we assayed the behaviour of these cells in 3D collagen gels.…”

“…The tropomyosin family of actin-associating proteins has emerged as important determinant of cell migration (Bryce et al, 2003;Gupton et al, 2005;Bach et al, 2009;O'Neill 2009). This multi-isoform family of proteins primarily form lateral in register homodimers, depending on the isoform, additionally form head-to-tail polymers and lay in the major groove of polymerized actin filaments (Gunning et al, 2008).…”

“…Association of particular tropomyosin isoforms with actin filaments in turn regulates the association of molecules that determine the dynamics of actin filament turnover (Ishikawa et al, 1989;Blanchoin et al, 2001;Bryce et al, 2003). Tropomyosin isoform Tm5NM1 stimulates increased actin stress fibre formation by enhancing the stability of associated actin filaments, leading to inhibited cell migration in two-dimensional (2D) cultures (Bryce et al, 2003;Creed et al, 2008;Bach et al, 2009). Importantly, expression of this isoform can vary up to 5-fold between different tissues, with the highest levels observed in lung tissue .…”