3033 Two core components in cell migration are the actin cytoskeleton and integrin-based adhesion to the extra-cellular matrix, known as focal adhesions (FAs). FA turnover allows cells to move over the underlying matrix while the actin cytoskeleton directly links to the FAs providing the contractile force that generates movement. Actin filament structure is regulated by distinct isoforms of the tropomyosin (Tm) family of actin-binding proteins. Given the key relationship between adhesion and actin in cell migration, we hypothesized that Tm isoform expression may regulate adhesion structure, in turn determining downstream signalling and migration. To test our hypothesis we have employed B35 rat cells overexpressing the Tm isoform Tm5NM1 and mouse embryo fibroblasts (MEFs) derived from a Tm5NM1 knockout mouse model. Using time-lapse microscopy we determined that cells expressing Tm5NM1 display reduced random cell movement, while the Tm5NM1 -/- MEFs travel significantly further than controls. Notably, the velocity of the wild-type and knockout MEFs is identical, therefore suggesting that the Tm5NM1 -/- MEFs may be more polarized. Cells overexpressing Tm5NM1 display FAs that are twice the length of control cells and are dispersed across the ventral surface of the cell. In contrast, Tm5NM1 -/- MEFs display enhanced levels of pre-cursor adhesion formation arrayed at the leading edge of protruding membranes. To investigate why the FAs are larger in the Tm5NM1 overexpressing cells we have measured adhesion dynamics. The data demonstrate that the FAs are significantly stabilized in the Tm5NM1-expressing cells. Further the stabilization of FAs in Tm5NM1-expressing cells appears to require the presence of polymerized actin as treatment with the actin-destabilizing agent latrunculin correspondingly promotes FA turnover. We have begun to investigate how altered adhesion structure might impact integrin-mediated signalling pathways and our preliminary data indicate that the FA docking molecule p130Cas exhibits altered phosphorylation in the Tm5NM1 overexpressing cells. In a correlated finding we also observe decreased expression of the tyrosine kinase Src in these cells. Conversely, p130Cas phosphorylation and Src expression levels are unaltered in Tm5NM1-/- MEFs. Collectively, our data support Tm isoform-specific effects on FA structure, adhesion molecule activation and cell migration.
Orderly cell migration is essential for embryonic development, efficient wound healing and a functioning immune system and the dysregulation of this process leads to a number of pathologies. The speed and direction of cell migration is critically dependent on the structural organization of focal adhesions in the cell. While it is well established that contractile forces derived from the acto-myosin filaments control the structure and growth of focal adhesions, how this may be modulated to give different outcomes for speed and persistence is not well understood. The tropomyosin family of actin-associating proteins are emerging as important modulators of the contractile nature of associated actin filaments. The multiple non-muscle tropomyosin isoforms are differentially expressed between tissues and across development and are thought to be major regulators of actin filament functional specialization. In the present study we have investigated the effects of two splice variant isoforms from the same α-tropomyosin gene, TmBr1 and TmBr3, on focal adhesion structure and parameters of cell migration. These isoforms are normally switched on in neuronal cells during differentiation and we find that exogenous expression of the two isoforms in undifferentiated neuronal cells has discrete effects on cell migration parameters. While both isoforms cause reduced focal adhesion size and cell migration speed, they differentially effect actin filament phenotypes and migration persistence. Our data suggests that differential expression of tropomyosin isoforms may coordinate acto-myosin contractility and focal adhesion structure to modulate cell speed and persistence.
Abstract Focal adhesions are complex multi‐protein structures that mediate cell adhesion and cell migration in multicellular organisms. Most of the protein components involved in focal adhesion formation have been identified, but a major challenge remains: determination of the spatial and temporal dynamics of adhesion proteins in order to understand the molecular mechanisms of adhesion assembly, maturation, signal regulation, and disassembly. Progress in this field has been hampered by the limited resolution of fluorescence microscopy. Recent advances have led to the development of super‐resolution techniques including single‐molecule localization microscopy (SMLM). Here, we discuss how the application of these techniques has revealed important new insights into focal adhesion structure and dynamics, including the first description of the three‐dimensional nano‐architecture of focal adhesions and of the dynamic exchange of integrins in focal adhesions. Hence, SMLM has contributed to the refinement of existing models of adhesions as well as the establishment of novel models, thereby opening new research directions. With current improvements in SMLM instrumentation and analysis, it has become possible to study cellular adhesions at the single‐molecule level.
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In order for cells to stop moving, they must synchronously stabilize actin filaments and their associated focal adhesions. How these two structures are coordinated in time and space is not known. We show here that the actin association protein Tm5NM1, which induces stable actin filaments, concurrently suppresses the trafficking of focal-adhesion-regulatory molecules. Using combinations of fluorescent biosensors and fluorescence recovery after photobleaching (FRAP), we demonstrate that Tm5NM1 reduces the level of delivery of Src kinase to focal adhesions, resulting in reduced phosphorylation of adhesion-resident Src substrates. Live imaging of Rab11-positive recycling endosomes that carry Src to focal adhesions reveals disruption of this pathway. We propose that tropomyosin synchronizes adhesion dynamics with the cytoskeleton by regulating actin-dependent trafficking of essential focal-adhesion molecules.
In order for cells to stop moving, they must synchronously stabilize actin filaments and their associated focal adhesions. How these two structures are coordinated in time and space is not known. We show here that the actin association protein Tm5NM1, which induces stable actin filaments, concurrently suppresses the trafficking of focal-adhesion-regulatory molecules. Using combinations of fluorescent biosensors and fluorescence recovery after photobleaching (FRAP), we demonstrate that Tm5NM1 reduces the level of delivery of Src kinase to focal adhesions, resulting in reduced phosphorylation of adhesion-resident Src substrates. Live imaging of Rab11-positive recycling endosomes that carry Src to focal adhesions reveals disruption of this pathway. We propose that tropomyosin synchronizes adhesion dynamics with the cytoskeleton by regulating actin-dependent trafficking of essential focal-adhesion molecules.
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.
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