Logo of molcellbPermissionsJournals.ASM.orgJournalMCB ArticleJournal InfoAuthorsReviewers
Mol Cell Biol. 2009 Mar; 29(6): 1506–1514.
Published online 2009 Jan 5. doi:  10.1128/MCB.00857-08
PMCID: PMC2648248

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


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 actin-associated 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, dot-like 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 least three distinct classes of stress fibers observed in migrating cells (20, 27). Dorsal stress fibers are inserted into focal adhesions at the ventral surface of the cell. The distal end of the dorsal fibers can associate with a second type of actin fiber, the transverse arcs that run parallel to the leading edge and are not directly connected to focal adhesions. Ventral stress fibers have focal adhesions at either end and can be established following the contraction of two dorsal stress fibers and the associated transverse arc to form one actin bundle (20).

Increased ventral stress fibers and focal adhesions are characteristic of nonmotile cells, in contrast, cell migration depends on focal adhesion turnover at the leading edge, allowing the formation of newly protruding regions of membrane and focal complex formation (28, 39). While the precise mechanism of focal adhesion turnover is incompletely understood, activation and phosphorylation of Src kinase, p130Cas, and paxillin (13, 39, 45) have all been implicated in focal adhesion turnover. A biphasic relationship between cell adhesion and cell speed suggests that conditions that alter the turnover rate of focal adhesions (either too much or too little) can reduce cell speed (18, 22).

In cells with a fibroblastic phenotype, increased levels of acto-myosin contractility promote focal adhesion transition to fibrillar adhesions (also known as ECM contacts) (6, 7): elongated, thin, central arrays of dots or elongated fibrils that characteristically contain tensin but low levels of phosphorylated paxillin (29, 44, 45) and bind fibrils of fibronectin parallel to actin bundles (23, 29). These adhesions are formed by ligand-occupied fibronectin integrin receptor translocation from focal adhesions along bundles of actin filaments toward the cell center, and the process is dependent on an intact actin cytoskeleton and myosin activity (29). Receptor translocation stimulates matrix reorganization by transmitting cytoskeleton-generated tension through the integrin receptors onto the surrounding matrix (25, 29). The rate of receptor translocation is apparently independent from the rate of cell migration (29). However, the cytoskeletal tension that causes the fibrillar adhesion formation is also reported to decrease paxillin phosphorylation (45). Since phosphorylated paxillin is required for the generation of new focal complexes (45), conditions which switch the balance of adhesion in favor of fibrillar adhesion should presumably result in significantly reduced paxillin phosphorylation, leading to reduced focal adhesion turnover and correspondingly decreased cell migration.

The cytoskeletal tropomyosin Tm5NM1 is a broadly distributed isoform (37) that alters cell shape (34), localizes to and promotes stress fibers that are resistant to actin depolymerizing drugs (9), enhances myosin IIA activation and recruitment to stress fibers, and inhibits cell migration (3). Therefore, we hypothesized that Tm5NM1 expression might determine cell migration by coordinating actin-dependent transition toward a predominance of focal adhesions and fibrillar adhesions. Using overexpression, knockdown, and genetic knockout models, we demonstrate that Tm5NM1 inhibits cell migration by promoting selective stabilization of focal adhesions and transition to fibrillar adhesions via the regulation of paxillin phosphorylation.


Cell culture, drugs, and antibodies.

B35 cell clones overexpressing Tm5NM1 (B35/Tm5NM1) and Tm3 (B35/Tm3) have been previously described (3, 9, 34). γ-Tm knockout mouse lines and primary mouse embryo fibroblasts (MEF/Tm5NM1−/−) have also been described (35; T. Fath et al., unpublished data). All animal experiments were performed in accordance with institutional and National Health and Medical Research Council of Australia guidelines. B35 cell lines were transfected by using Lipofectamine 2000 (Invitrogen), MEFs using a Nucleofector (Amaxa) and an MEF2 Nucleofector kit (Integrated Sciences) and human-specific Tm5NM1 small interfering RNA (siRNA) oligonucleotides (9) were transfected by using TransIT siRNA transfection reagent (Mirus). Latrunculin A was purchased from Sigma-Aldrich (St. Louis, MO). Latrunculin assay of actin filament disassembly was performed as previously described (21), except that cells were plated on polylysine-coated eight-well chamber slides. Antibodies were purchased from the following companies: antipaxillin, anti-p130Cas, and antifibronectin (BD Transduction Laboratories); CY5-phalloidin (Sigma-Aldrich); anti-Src (Upstate Biotechnology); anti-phospho-Y118 paxillin (Biosource International/Invitrogen); horseradish peroxidase-conjugated anti-mouse and anti-rabbit antibodies (Amersham Pharmacia Biotech); Alexa 488- and CY3-conjugated donkey anti-mouse and donkey anti-rabbit antibodies (Jackson Immunoresearch); and tensin (Santa Cruz Biotechnology).


Immunofluorescence was carried out as previously described (1). Where indicated, cells were grown on coverslips pretreated with poly-l-lysine (50 μg/ml) and then coated with a solution of fibronectin (20 μg/ml) and laminin (10 μg/ml) for 2 h at 37°C. Immunostained cells were mounted by using FluorSave mounting reagent (CalBioChem/Merck Sharp & Dohme, Australia). Images of fixed cells were captured by using either a Spot II-cooled charge-coupled device (CCD) digital camera (Diagnostic Instruments) and an Olympus BX50 microscope with a 60× (numerical aperture [NA], 0.65 to 1.25) oil objective or an ORCA ERG cooled CCD camera (Hamamatsu/SDR Clinical Technology, Australia) and an Olympus IX81 inverted microscope, with 60× (NA, 1.35) and 40× (NA, 1.00) oil objectives, using fluorescence filters as follows: BP360-370/LP420 (DAPI [4′,6′-diamidino-2-phenylindole]), BP460-495/BP510-550 (Alexa 488 and green fluorescent protein [GFP]), BP530-550/BP575-625 (TRITC [tetramethyl rhodamine isothiocyanate] and CY3), BP490-500/BP515-560 (yellow fluorescent protein [YFP]), and EX620/EM700 (CY5).

Image analysis.

Images were pseudocolored and overlaid by using Metamorph V6.3 software (Molecular Devices, Sunnyvale, CA). Final micrograph images and gray level adjustments were prepared in Adobe Photoshop. Unless otherwise indicated, scale bars represent 25 μm. Image quantitation and measurement procedures were carried out using Metamorph V6.3 software. Focal adhesion quantitation was performed following background subtraction, high-pass filtering, calibration, and thresholding. Ratio images were prepared by using background-subtracted images. The phosphopaxillin image was divided by the sum of phosphopaxillin and paxillin staining, i.e., phosphopaxillin/(phosphopaxillin + paxillin), and the numerator was multiplied by a constant to obtain an optimal range. Final ratio images were pseudocolored using the gradient map function of Adobe Photoshop. Line scans were performed on 5-pixel-width lines. The relative phosphorylation of paxillin at individual focal adhesions was determined by drawing polygons around individual focal adhesions in the paxillin images and obtaining the pixel-by-pixel intensities. Polygons were then transferred to the matching phosphopaxillin image, and the pixel intensity data were collected. The total phosphopaxillin pixel intensities divided by the sum of the total phosphopaxillin and paxillin intensities was calculated by using Microsoft Excel. Only paxillin-positive focal adhesions at the cell periphery were included for analysis; adhesions at the center of the cell were excluded.

Protein extraction and immunoblotting.

Conditions of protein extraction and immunoblotting were carried out as previously described (8). Protein concentrations were determined by using a BCA protein assay kit (Pierce Biotechnology, Rockford, IL), and protein concentrations were equalized prior to loading on gels. Computer-assisted densitometry of protein bands on autoradiographs was achieved by using NIH ImageJ 1.34S software.

Live cell imaging.

Cells were grown in 35-mm glass-bottom dishes (MatTek, Ashland, MA) in phenol-red free media containing 25 mM HEPES (pH 7.2), and images captured by using an ORCA ERG cooled CCD camera (Hamamatsu/SDR Clinical Technology) and Olympus IX81 inverted microscope equipped with an environmental chamber heated to 37°C. For assay of focal adhesion dynamics, glass-bottom dishes were coated with fibronectin and laminin (24). Images were captured every 2 min for 90 min.

Migration analysis.

Transmitted light images were captured every 5 min for 3 h (40× objective). Cells undergoing division or apoptosis were excluded from analyses, and random migration analyses were performed on sparsely plated cultures. After image capture, nuclear translocation was tracked in time-lapse stacks using Metamorph V6.3 software. For wounding assays, confluent cultures were scratched with a needle, and cells were tracked as they migrated into the scratch. Only cells at the wound edge were tracked, and apoptotic or mitotic cells were excluded from analysis. Cell track (reoriented to zero in migration traces) and velocity and persistence ratio (i.e., the ratio of vectorial distance traveled to the total path length described by the cell) (40) calculations were performed using Microsoft Excel. MEF/Tm5NM1−/− cells transfected with either empty vector (YFP) or plasmid encoding YFP fused with Tm5NM1 (YFP.Tm5NM1) (30) were similarly imaged and tracked. After background subtraction of the first time point for each stack of YFP.Tm5NM1 images, fluorescence intensity line scans were performed on lines drawn across the cell and oriented perpendicular to the YFP-Tm5NM1-positive stress fibers, using Metamorph V6.3 software. Cells with fluorescence intensity peaks equal to or greater than 250 arbitrary units and displaying distinct Tm5NM1-positive stress fibers were selected for analysis.

Statistical analysis.

All error bars on histograms in the figures show the standard error of the mean (SEM). Statistical comparison of two means was performed by using a Student t test.


Adhesion regulation.

Based on the prominent actin stress fibers and inhibited migration seen in B35 neuroblastoma cell culture models expressing exogenous Tm5NM1 (3), these cells were predicted to have increased focal adhesion formation. Immunostaining with paxillin antibodies revealed that focal adhesions are significantly longer and more numerous than controls, with a greater concentration of focal adhesions obvious in the central regions under the cell (Fig. 1A to C). In contrast, cells overexpressing a second tropomyosin isoform, Tm3, do not show the same increases in focal adhesions (Fig. 1A to C). This confirms the isoform-specific role of Tm5NM1 since both isoforms associate with actin stress fibers (34, 37) but clearly cause distinct focal adhesion phenotypes. siRNA-mediated downregulation of the exogenous human-derived Tm5NM1 sequence further confirmed that the increased focal adhesion numbers and distribution are specifically due to exogenously expressed Tm5NM1. Approximately 40% reduction in total exogenous Tm5NM1 protein (see Fig. S1 in the supplemental material) was sufficient to stimulate a marked diminution of the centrally located focal adhesions (Fig. (Fig.1D),1D), accompanied by reduced numbers of focal adhesions per cell (Fig. (Fig.1E1E).

FIG. 1.
Tm5NM1 overexpression promotes adhesions. (A) B35 neuroblastoma cells immunostained with paxillin antibodies. Focal adhesions are indicated by arrows. (B and C) Histograms show lengths and numbers of focal adhesions, expressed relative to control cells ...

The increased centrally located focal adhesions in the Tm5NM1 cells suggested an increased transition to fibrillar adhesions. Colocalization with tensin is considered to be a hallmark of fibrillar adhesions (29, 47); moreover, fibrillar adhesions are reported to bind fibrils of fibronectin parallel to actin bundles (23, 29). Thus, we examined the distribution of both tensin and fibronectin in the cells. Coimmunostaining for paxillin and tensin demonstrates clear organization of tensin in the Tm5NM1 cells, observed as linear arrays of punctate staining, that coaligns with paxillin staining (Fig. (Fig.2A).2A). In contrast, control cells display punctate tensin staining randomly throughout the cytoplasm. The same tensin distribution was observed in cells either immunostained for tensin alone or when the fluorescently tagged secondary antibodies used to detect the coimmunostained molecules were inverted (data not shown), confirming the specificity of the tensin staining pattern. Providing further evidence that these structures are fibrillar adhesions, coimmunostaining of cells for F-actin and fibronectin shows linear fibronectin staining parallel to actin stress fibers across the ventral surface of the cells overexpressing Tm5NM1 (Fig. (Fig.2B2B).

FIG. 2.
Enhanced fibrillar adhesion formation in cells overexpressing Tm5NM1. (A) Cells plated onto fibronectin- and laminin-coated coverslips, fixed and coimmunostained for paxillin and tensin. The two right-hand panels show a merged image of the costained cells. ...

Actin stabilization and ventral stress fiber generation.

The arrangement of the actin filaments is strikingly different between controls and cells expressing elevated exogenous Tm5NM1. The controls display an actin meshwork at the leading edge and transverse arc filaments behind the lamellipodial region (Fig. (Fig.3A).3A). In contrast, the Tm5NM1 cells display a predominance of ventral stress fibers, seen as F-actin-rich cables traversing the cell body (Fig. (Fig.3A).3A). This is a tropomyosin isoform-specific effect, since Tm3 does not cause the same actin stress fiber formation (see Fig. Fig.3B)3B) despite the fact that Tm3 is a stress fiber-associating isoform. Control cells costained for paxillin display focal complexes associated with the actin meshwork (Fig. (Fig.3A).3A). Behind the lamellipodium focal adhesions are oriented perpendicularly to the transverse arcs; however, the very bright staining of the transverse arcs makes it difficult to discern any dorsal-type stress fibers emanating from these focal adhesions (Fig. (Fig.3A).3A). In contrast, the Tm5NM1 cells display prominent focal adhesions aligned with the ends of the ventral stress fibers (Fig. (Fig.3A).3A). Moreover, paxillin-positive dots arrayed along the ventral stress fibers mark the fibril-type adhesions in the Tm5NM1 cells (Fig. (Fig.3A3A).

FIG. 3.
Actin filaments are stabilized by Tm5NM1. (A) Cells plated on fibronectin and laminin and coimmunostained with CY5-phalloidin to detect filamentous actin and paxillin antibodies. Panels on the right-hand side show merged images of the coimmunostained ...

Tm5NM1 association with actin filaments causes the filaments to be more resistant to actin depolymerizing drugs (9), suggesting that Tm5NM1 may alter the dynamics of actin filament turnover, thus resulting in stabilized filaments. Increased actin filament stability promotes both increased stress fibers and increased cell size (21), consistent with the phenotypes stimulated by Tm5NM1 overexpression (3, 9, 36). We therefore compared the rate of actin stress fiber disassembly after exposure to the actin sequestering drug Latrunculin A, as previously described (21). After 15 min, control cells displaying actin stress fibers are reduced by 60% (Fig. 3B and C). In contrast, there is ∼20% reduction in Tm5NM1 cells at the same time point, increasing to only 40% reduction at 30 min (Fig. 3B and C). Correlated with these data, the Tm5NM1 cells maintain a well-spread phenotype even after 30 min of treatment with Latrunculin A, and by contrast the control cells have already shrunk significantly by 15 min. Confirming that this represents a tropomyosin isoform-specific regulation of actin stability, latrunculin treatment of cells overexpressing Tm3 causes the same rapid shrinkage and rounding of the cell body, as seen with the control cells (Fig. (Fig.3B),3B), thus supporting isoform-specific regulation of actin dynamics.

Reduced focal adhesion turnover.

Since cell migration is determined by both cell speed and cell direction, we assessed these parameters by measuring intrinsic migration as previously described (10). Cells with elevated Tm5NM1 expression exhibited significantly reduced directional persistence and velocity (Fig. 4A to C). By comparison, the Tm3-overexpressing cells show no change in persistence (Fig. (Fig.4C).4C). Since both too few and too many adhesions can reduce cell speed (18, 22), the reduced velocity of the Tm3 cells (Fig. (Fig.4B)4B) may reflect the reduced numbers of adhesions formed in these cells. To test whether Tm5NM1 expression alters cellular polarization in response to an external directional cue, we next performed scratch wound healing experiments, as previously described (5). Classical tests of cell polarization by measuring the orientation of the microtubule organizing center were not possible since the control cells displayed little evidence of microtubule organizing center orientation toward the wound (see Fig. S2 in the supplemental material), a phenomenon previously reported for other cell types (42). Instead, individual cells were tracked for the first 3 h as they migrated into the wound (Fig. (Fig.4D).4D). Both control and Tm5NM1 cells display a higher average persistence ratio while migrating toward the wound (B35, 0.73 ± 0.02; Tm5NM1, 0.64 ± 0.03) than in the random migration assay (Fig. (Fig.4C),4C), indicating that both cell types are responding to the directional cue. While the difference between the average control and Tm5NM1 persistence ratios is statistically significant (P < 0.05), to more precisely understand the behavior of these cells, we calculated the cumulative persistence as the cells migrated into the wound. This analysis revealed that the two cell types have identical trajectories immediately after wounding, but the Tm5NM1 cells begin to show increasingly reduced directional persistence with time (Fig. (Fig.4E).4E). Notably, this does not correlate directly with changes in cell speed since analysis of the cumulative cell speeds shows that the Tm5NM1 cells move consistently more slowly throughout the time course (Fig. (Fig.4E).4E). Thus, these data suggest that elevated levels of Tm5NM1 cause both reduced cell speeds and reduced directional migration.

FIG. 4.
Tm5NM1 reduces cell persistence and speed. (A) Migration traces for control B35, B35/Tm5NM1, and B35/Tm3 cells. (B and C) Histograms showing average (B35, n = 17; Tm5NM1, n = 34; Tm3, n = 24) speed and persistence ratios, respectively, ...

We next determined the focal adhesion dynamics underlying these distinct migration phenotypes by measuring adhesion dynamics in cells grown under the same conditions as for the assays of intrinsic migration. To measure focal adhesion turnover, we performed time-lapse imaging of cells transfected with YFP-tagged p130Cas, since p130Cas is a critical regulator of focal adhesion turnover (39). YFP.p130Cas-positive adhesions exhibit strikingly increased stability in the Tm5NM1 cells compared to the parental control cells (Fig. (Fig.5A).5A). Comparison of the focal adhesion life history plots (YFP.p130Cas-positive focal adhesion length over time) graphically demonstrates the changes in p130Cas-positive focal adhesion lengths in the control cells and the contrasting stability in the Tm5NM1 cells (Fig. (Fig.5B).5B). Quantitation of the percentage time in which adhesions were observed to be either in a growing or shrinking state confirmed that focal adhesion dynamics are significantly reduced in the Tm5NM1 cells (Fig. (Fig.5C5C).

FIG. 5.
Tm5NM1 expression inhibits focal adhesion turnover. (A) Cropped regions from time-lapse series of cells transfected with YFP.p130Cas. Stable adhesions are indicated by arrows, a newly formed adhesion is indicated by an arrowhead, and adhesions that turn ...

Suppression of adhesion signaling pathways.

The phosphorylation of adhesion-associated molecules such as p130Cas has emerged as a critical regulator of their role in focal adhesion turnover (39, 45). Since p130Cas positive adhesions are more stable in the Tm5NM1 cells, it was possible that this reflected a change in p130Cas phosphorylation. P130Cas can be detected as at least two distinct phospho-forms by Western blotting (8), and we find that there is a significant loss of hyperphosphorylated p130Cas (p-p130Cas) in cells overexpressing Tm5NM1 (Fig. (Fig.6A).6A). The major kinases that phosphorylate p130Cas are the Src family kinases (33), and Src kinase activity is also required for focal adhesion turnover (39). Accordingly, we find significantly decreased Src protein expression levels in cells with elevated Tm5NM1 (Fig. (Fig.6B).6B). Contrasting these data, cells overexpressing Tm3 show a predominance of hyperphosphorylated p130Cas and no loss of Src kinase expression (Fig. 6A and B).

FIG. 6.
Downregulation of adhesion signaling pathways. (A and B) Western blot analysis of p130Cas (A) and Src protein (B) expression. Relative levels of hyperphosphorylated (pp130Cas, upper form) and hypophosphorylated (p130Cas, lower form) p130Cas and total ...

Another major substrate of Src family kinases is paxillin, a molecule recently suggested to function as a switch that regulates adhesion phenotypes (45). Interestingly, total paxillin is significantly reduced in cells with elevated Tm5NM1 expression (Fig. (Fig.7A).7A). This was surprising, given the large number of paxillin-positive focal adhesions in the Tm5NM1 cells; however, there is a notable level of perinuclear paxillin in the control cells that is not seen in the Tm5NM1 cells (Fig. (Fig.1A).1A). It therefore appears that while total levels are reduced in the Tm5NM1 cells, the paxillin present in these cells is predominantly more associated with the focal adhesions than in the control cells. Src kinase phosphorylates paxillin on tyrosine residue 118 (31) and, importantly, Y118 phosphorylation of paxillin is significantly reduced in the Tm5NM1 cells (Fig. (Fig.7A).7A). We next examined the spatial activation of phosphorylated paxillin, since phosphorylated paxillin is reported to be specifically activated at focal adhesions, and this subcellular localization facilitates paxillin function in adhesion turnover (45). Thus, control cells should have higher levels of phosphorylated paxillin at peripheral focal adhesion sites, consistent with the rapid focal adhesion dynamics observed in these cells (Fig. (Fig.5A),5A), while reduced focal adhesion turnover in the Tm5NM1 cells predicts reduced focal adhesion-associated phosphorylated paxillin. Immunostaining confirms that there is a conspicuously higher level of phosphorylated paxillin at the focal adhesions in the control versus Tm5NM1-overexpressing cells (Fig. 7B and D). Fluorescence ratio imaging highlights the high levels of phosphorylated paxillin in the peripheral adhesions of the control cells (seen in red hues in the inset in Fig. Fig.7C)7C) and the lower levels in the peripheral adhesions of the Tm5NM1 cells (seen in blue and green hues in the inset in Fig. Fig.7C).7C). Semiquantitative calculation of the ratio of phosphorylated paxillin at individual focal adhesions confirms that phosphorylation is significantly reduced in cells with elevated levels of Tm5NM1 (Fig. (Fig.7E7E).

FIG. 7.
Reduced paxillin tyrosine phosphorylation in cells with elevated Tm5NM1 expression. (A) Western blot analysis of phosphorylated and total paxillin levels and tubulin to demonstrate equal loading. The histogram shows the ratio of phosphopaxillin divided ...

To summarize, the preceding data suggest that Tm5NM1 stabilizes the ventral actin stress fibers, causing tension-dependent inhibition of paxillin phosphorylation and corresponding focal adhesion stabilization. In turn, this reduces both cell speed and directional migration.

Focal complexes and directional migration.

A key role for Tm5NM1 in regulating adhesion dynamics predicts that deletion of the Tm5NM1 gene product should have adhesion effects opposite to the overexpression of Tm5NM1. Given that focal adhesion turnover is thought to be required for de novo focal complex formation, reduced Tm5NM1-mediated focal adhesion stability may be predicted to result in increased formation of focal complexes. We therefore assessed adhesion phenotypes in primary MEFs derived from a Tm5NM1 gene knockout model (MEF/Tm5NM1−/−) (35). This revealed a remarkable increase in cells displaying focal complexes (Fig. 8A and B). Transfection with dominant-negative Rac (GFP.RacN17) as described previously (1), caused a significant reduction in the number of cells displaying these adhesions (Fig. 8C and D), confirming their identity as focal complexes. Next, if Tm5NM1 plays a significant role in determining paxillin phosphorylation, it is expected that the loss of Tm5NM1 expression should result in the opposite affect to elevated Tm5NM1 expression, that is, cause increased paxillin phosphorylation. Indeed, there is significantly increased phosphorylated paxillin in the MEF/Tm5NM1−/− cells (Fig. (Fig.8E8E).

FIG. 8.
Focal complex stimulation and enhanced paxillin phosphorylation in cells lacking Tm5NM1 expression. (A) Paxillin staining of wild-type (MEF) and knockout (MEF/Tm5NM1−/−) cells. Focal complexes are indicated by arrowheads (inset i). (B) ...

Our earlier data suggest Tm5NM1 may regulate directional persistence. Related to this, focal complexes are emerging as regulators of directional migration (19). Thus, we assayed directional persistence in the cells lacking Tm5NM1 expression. These cells have significantly increased directional persistence in assays of random cell migration (Fig. 9A and C), despite having identical velocity to wild-type control cells (Fig. (Fig.9B).9B). The response of the MEF/Tm5NM1−/− cells to an external directional cue was next measured by in vitro scratch wound healing assays. Calculation of the cumulative persistence ratios as the cells migrated into the wounds over the first 6 h reveals that the MEF/Tm5NM1−/− cells maintain their enhanced directional persistence, even in response to an externally derived migration cue (Fig. 9D to E). Finally, the direct role of Tm5NM1 in cell migration was separately confirmed by performing rescue experiments with YFP-tagged Tm5NM1. Since Tm5NM1 has dose dependent effects on cell migration (3), only cells scoring average YFP intensity levels above a predetermined threshold were included for analysis. Velocity is significantly inhibited in this population of cells (<40% of the control cell velocity), rendering the cells essentially nonmotile (Fig. (Fig.9F9F).

FIG. 9.
Tm5NM1 expression regulates cell persistence. (A) Migration traces for wild-type MEF and MEF/Tm5NM1−/− cells. (B and C) Histograms showing average velocities and persistence ratios, respectively (MEF, n = 27; MEF/Tm5NM1, n = ...


The rate of focal adhesion formation and disassembly is a key step in the regulation of mesenchymal cell migration. In the present study we investigated the role of the tropomyosin family of actin-associating molecules in determining the dynamics of cell adhesions. We have established a Tm5NM1 isoform-specific switch to stabilized stress fibers and focal adhesions, increased fibrillar adhesions, loss of polarized cell migration, and reduced cell velocity. These affects are accompanied by a significant loss of phosphorylated paxillin. Collectively, our data suggest that tropomyosin regulation of the actin cytoskeleton is an important determinant of adhesion dynamics in cell migration.

A previous study found that exogenous skeletal muscle α-tropomyosin drove increased focal adhesion turnover and correspondingly increased cell migration in nonmuscle cells (17), in contrast to the adhesion-stabilizing, migration-inhibitory function that we have established for the Tm5NM1 isoform. Together, these studies indicate that there are isoform-specific tropomyosin effects on adhesion dynamics and cell migration. Elevated Tm5NM1 expression results in increased stress fiber formation, due to increased actin filament stability. Treatments that cause microtubule depolymerization similarly cause stress fiber formation (11) and increased focal adhesions (26). Therefore, the cross talk between the actin and microtubule filament systems may determine the final focal adhesion disassembly rate, and actin regulators such as Tm5NM1 are likely to play a key role in this process.

Notably, elevated levels of Tm5NM1 expression cause increased myosin IIA association with actin filaments, both in vitro and in vivo (3), and myosin IIA is required for contractility and the regulation of cell migration (12). Moreover, the transition to fibrillar adhesions is dependent on integrin association with the actin cytoskeleton (41) and requires acto-myosin contractility (47). Thus, we propose that enhanced myosin IIA localization to the actin stress fibers mediated by Tm5NM1 expression causes a tension-dependent decrease in focal adhesion turnover and increased transition to fibrillar adhesions. These adhesion sites promote fibrillogenesis and thereby contribute to matrix reorganization (29, 41). Therefore, our data suggest that Tm5NM1 expression may play a role both in the regulation of cell migration and in matrix reorganization, two critical adhesion-dependent cellular processes.

Our results demonstrate a previously unrecognized role for tropomyosins in the regulation of directional persistence. Focal complex formation favored in the absence of Tm5NM1 expression resulted in increased directional persistence. Focal complexes are reported to contain higher levels of αvβ3 integrin receptors, while focal adhesions have a predominance of α5β1 fibronectin receptors (46). Previous studies suggest that β1 integrin engagement supports random migration and, in contrast, β3 integrin engagement supports persistent movement (10), further the dynamics of one receptor subtype may influence the surface presentation of the other receptor subtype (40). Increased fibrillar adhesions in cells with elevated Tm5NM1 expression suggests a potential link between Tm5NM1 expression and the surface expression of fibronectin receptors in these cells. Potentially, Tm5NM1-mediated stabilization of focal adhesions may result in decreased αvβ3-dependent focal complex assembly (40) and reduced directional persistence. Disabling of this feedback loop in cells lacking Tm5NM1 expression then may result in increased focal complex formation and enhanced directional persistence.

The tropomyosins are a multi-isoform family that confers distinct structural and functional properties to actin filaments (15). Their expression is highly temporally and spatially regulated, both at the whole-tissue level and within individual cells, during discrete biological processes. Moreover, the tropomyosin isoform expression profile is profoundly changed during malignant progression of cancer cells (38). Therefore, these proteins are well placed to act as key players that integrate adhesion and actin filament dynamics to determine morphologically regulated processes such as cell migration.

Supplementary Material

[Supplemental material]


This study was supported by the University of Sydney Bridging Support Grants Scheme (G.O. and P.G.) and a New Staff grant (G.O.), a Cancer Institute NSW Research Infrastructure Grant (G.O. and P.G.), the Oncology Children's Foundation (C.T.T.B.), National Health and Medical Research Council (NHMRC) grants 117409 (P.G.) and 512251 (G.O. and P.G.), and a Career Development Award from the NSW Cancer Council (G.O.). P.G. is a Principal Research Fellow of the NHMRC.

We acknowledge excellent technical assistance of Judy Shao and Andrew Madry. We thank Kathy Kamath for live imaging protocols and Kat Gaus for assistance with ratio imaging.


Published ahead of print on 5 January 2009.

Supplemental material for this article may be found at http://mcb.asm.org/.


1. Bargon, S. D., P. W. Gunning, and G. M. O'Neill. 2005. The Cas family docking protein, HEF1, promotes the formation of neurite-like membrane extensions. Biochim. Biophys. Acta 1 746143-154. [PubMed]
2. Beningo, K. A., M. Dembo, I. Kaverina, J. V. Small, and Y. L. Wang. 2001. Nascent focal adhesions are responsible for the generation of strong propulsive forces in migrating fibroblasts. J. Cell Biol. 153881-888. [PMC free article] [PubMed]
3. Bryce, N. S., G. Schevzov, V. Ferguson, J. M. Percival, J. J. Lin, F. Matsumura, J. R. Bamburg, P. L. Jeffrey, E. C. Hardeman, P. Gunning, and R. P. Weinberger. 2003. Specification of actin filament function and molecular composition by tropomyosin isoforms. Mol. Biol. Cell 141002-1016. [PMC free article] [PubMed]
4. Carragher, N. O., and M. C. Frame. 2004. Focal adhesion and actin dynamics: a place where kinases and proteases meet to promote invasion. Trends Cell Biol. 14241-249. [PubMed]
5. Cau, J., and A. Hall. 2005. Cdc42 controls the polarity of the actin and microtubule cytoskeletons through two distinct signal transduction pathways. J. Cell Sci. 1182579-2587. [PubMed]
6. Chen, W. T., E. Hasegawa, T. Hasegawa, C. Weinstock, and K. M. Yamada. 1985. Development of cell surface linkage complexes in cultured fibroblasts. J. Cell Biol. 1001103-1114. [PMC free article] [PubMed]
7. Chen, W. T., and S. J. Singer. 1982. Immunoelectron microscopic studies of the sites of cell-substratum and cell-cell contacts in cultured fibroblasts. J. Cell Biol. 95205-222. [PMC free article] [PubMed]
8. Cowell, L. N., J. D. Graham, A. H. Bouton, C. L. Clarke, and G. M. O'Neill. 2006. Tamoxifen treatment promotes phosphorylation of the adhesion molecules, p130Cas/BCAR1, FAK, and Src, via an adhesion-dependent pathway. Oncogene 257597-7607. [PubMed]
9. Creed, S. J., N. Bryce, P. Naumanen, R. Weinberger, P. Lappalainen, J. Stehn, and P. Gunning. 2008. Tropomyosin isoforms define distinct microfilament populations with different drug susceptibility. Eur. J. Cell Biol. 87709-720. [PubMed]
10. Danen, E. H., J. van Rheenen, W. Franken, S. Huveneers, P. Sonneveld, K. Jalink, and A. Sonnenberg. 2005. Integrins control motile strategy through a Rho-cofilin pathway. J. Cell Biol. 169515-526. [PMC free article] [PubMed]
11. Danowski, B. A. 1989. Fibroblast contractility and actin organization are stimulated by microtubule inhibitors. J. Cell Sci. 93255-266. [PubMed]
12. Even-Ram, S., A. D. Doyle, M. A. Conti, K. Matsumoto, R. S. Adelstein, and K. M. Yamada. 2007. Myosin IIA regulates cell motility and actomyosin-microtubule crosstalk. Nat. Cell Biol. 9299-309. [PubMed]
13. Fincham, V. J., and M. C. Frame. 1998. The catalytic activity of Src is dispensable for translocation to focal adhesions but controls the turnover of these structures during cell motility. EMBO J. 1781-92. [PMC free article] [PubMed]
14. Friedl, P. 2004. Prespecification and plasticity: shifting mechanisms of cell migration. Curr. Opin. Cell Biol. 1614-23. [PubMed]
15. Gunning, P., G. O'Neill, and E. Hardeman. 2008. Tropomyosin-based regulation of the actin cytoskeleton in time and space. Physiol. Rev. 881-35. [PubMed]
16. Gunning, P. W., G. Schevzov, A. J. Kee, and E. C. Hardeman. 2005. Tropomyosin isoforms: divining rods for actin cytoskeleton function. Trends Cell Biol. 15333-341. [PubMed]
17. Gupton, S. L., K. L. Anderson, T. P. Kole, R. S. Fischer, A. Ponti, S. E. Hitchcock-DeGregori, G. Danuser, V. M. Fowler, D. Wirtz, D. Hanein, and C. M. Waterman-Storer. 2005. Cell migration without a lamellipodium: translation of actin dynamics into cell movement mediated by tropomyosin. J. Cell Biol. 168619-631. [PMC free article] [PubMed]
18. Gupton, S. L., and C. M. Waterman-Storer. 2006. Spatiotemporal feedback between actomyosin and focal-adhesion systems optimizes rapid cell migration. Cell 1251361-1374. [PubMed]
19. Harms, B. D., G. M. Bassi, A. R. Horwitz, and D. A. Lauffenburger. 2005. Directional persistence of EGF-induced cell migration is associated with stabilization of lamellipodial protrusions. Biophys. J. 881479-1488. [PMC free article] [PubMed]
20. Hotulainen, P., and P. Lappalainen. 2006. Stress fibers are generated by two distinct actin assembly mechanisms in motile cells. J. Cell Biol. 173383-394. [PMC free article] [PubMed]
21. Hotulainen, P., E. Paunola, M. K. Vartiainen, and P. Lappalainen. 2005. Actin-depolymerizing factor and cofilin-1 play overlapping roles in promoting rapid F-actin depolymerization in mammalian nonmuscle cells. Mol. Biol. Cell 16649-664. [PMC free article] [PubMed]
22. Huttenlocher, A., M. H. Ginsberg, and A. F. Horwitz. 1996. Modulation of cell migration by integrin-mediated cytoskeletal linkages and ligand-binding affinity. J. Cell Biol. 1341551-1562. [PMC free article] [PubMed]
23. Hynes, R. O., and A. T. Destree. 1978. Relationships between fibronectin (LETS protein) and actin. Cell 15875-886. [PubMed]
24. Kamath, K., and M. A. Jordan. 2003. Suppression of microtubule dynamics by epothilone B is associated with mitotic arrest. Cancer Res. 636026-6031. [PubMed]
25. Katz, B. Z., E. Zamir, A. Bershadsky, Z. Kam, K. M. Yamada, and B. Geiger. 2000. Physical state of the extracellular matrix regulates the structure and molecular composition of cell-matrix adhesions. Mol. Biol. Cell 111047-1060. [PMC free article] [PubMed]
26. Kaverina, I., O. Krylyshkina, and J. V. Small. 1999. Microtubule targeting of substrate contacts promotes their relaxation and dissociation. J. Cell Biol. 1461033-1044. [PMC free article] [PubMed]
27. Kaverina, I., K. Rottner, and J. V. Small. 1998. Targeting, capture, and stabilization of microtubules at early focal adhesions. J. Cell Biol. 142181-190. [PMC free article] [PubMed]
28. Laukaitis, C. M., D. J. Webb, K. Donais, and A. F. Horwitz. 2001. Differential dynamics of alpha 5 integrin, paxillin, and alpha-actinin during formation and disassembly of adhesions in migrating cells. J. Cell Biol. 1531427-1440. [PMC free article] [PubMed]
29. Pankov, R., E. Cukierman, B. Z. Katz, K. Matsumoto, D. C. Lin, S. Lin, C. Hahn, and K. M. Yamada. 2000. Integrin dynamics and matrix assembly: tensin-dependent translocation of α5β1 integrins promotes early fibronectin fibrillogenesis. J. Cell Biol. 1481075-1090. [PMC free article] [PubMed]
30. Percival, J. M., J. A. Hughes, D. L. Brown, G. Schevzov, K. Heimann, B. Vrhovski, N. Bryce, J. L. Stow, and P. W. Gunning. 2004. Targeting of a tropomyosin isoform to short microfilaments associated with the Golgi complex. Mol. Biol. Cell 15268-280. [PMC free article] [PubMed]
31. Petit, V., B. Boyer, D. Lentz, C. E. Turner, J. P. Thiery, and A. M. Valles. 2000. Phosphorylation of tyrosine residues 31 and 118 on paxillin regulates cell migration through an association with CRK in NBT-II cells. J. Cell Biol. 148957-970. [PMC free article] [PubMed]
32. Rottner, K., A. Hall, and J. V. Small. 1999. Interplay between Rac and Rho in the control of substrate contact dynamics. Curr. Biol. 9640-648. [PubMed]
33. Ruest, P. J., N. Y. Shin, T. R. Polte, X. Zhang, and S. K. Hanks. 2001. Mechanisms of CAS substrate domain tyrosine phosphorylation by FAK and Src. Mol. Cell. Biol. 217641-7652. [PMC free article] [PubMed]
34. Schevzov, G., N. S. Bryce, R. Almonte-Baldonado, J. Joya, J. J. Lin, E. Hardeman, R. Weinberger, and P. Gunning. 2005. Specific features of neuronal size and shape are regulated by tropomyosin isoforms. Mol. Biol. Cell 163425-3437. [PMC free article] [PubMed]
35. Schevzov, G., T. Fath, B. Vrhovski, N. Vlahovich, S. Rajan, J. Hook, J. E. Joya, F. Lemckert, F. Puttur, J. J. Lin, E. C. Hardeman, D. F. Wieczorek, G. M. O'Neill, and P. W. Gunning. 2008. Divergent regulation of the sarcomere and the cytoskeleton. J. Biol. Chem. 283275-283. [PubMed]
36. Schevzov, G., P. Gunning, P. L. Jeffrey, C. Temm-Grove, D. M. Helfman, J. J. Lin, and R. P. Weinberger. 1997. Tropomyosin localization reveals distinct populations of microfilaments in neurites and growth cones. Mol. Cell Neurosci. 8439-454. [PubMed]
37. Schevzov, G., B. Vrhovski, N. S. Bryce, S. Elmir, M. R. Qiu, G. M. O'Neill, N. Yang, N. M. Verrills, M. Kavallaris, and P. W. Gunning. 2005. Tissue-specific tropomyosin isoform composition. J. Histochem. Cytochem. 53557-570. [PubMed]
38. Stehn, J. R., G. Schevzov, G. M. O'Neill, and P. W. Gunning. 2006. Specialisation of the tropomyosin composition of actin filaments provides new potential targets for chemotherapy. Curr. Cancer Drug Targets 6245-256. [PubMed]
39. Webb, D. J., K. Donais, L. A. Whitmore, S. M. Thomas, C. E. Turner, J. T. Parsons, and A. F. Horwitz. 2004. FAK-Src signalling through paxillin, ERK and MLCK regulates adhesion disassembly. Nat. Cell Biol. 6154-161. [PubMed]
40. White, D. P., P. T. Caswell, and J. C. Norman. 2007. alpha v beta3 and alpha5beta1 integrin recycling pathways dictate downstream Rho kinase signaling to regulate persistent cell migration. J. Cell Biol. 177515-525. [PMC free article] [PubMed]
41. Wierzbicka-Patynowski, I., and J. E. Schwarzbauer. 2003. The ins and outs of fibronectin matrix assembly. J. Cell Sci. 1163269-3276. [PubMed]
42. Yvon, A. M., J. W. Walker, B. Danowski, C. Fagerstrom, A. Khodjakov, and P. Wadsworth. 2002. Centrosome reorientation in wound-edge cells is cell type specific. Mol. Biol. Cell 131871-1880. [PMC free article] [PubMed]
43. Zaidel-Bar, R., C. Ballestrem, Z. Kam, and B. Geiger. 2003. Early molecular events in the assembly of matrix adhesions at the leading edge of migrating cells. J. Cell Sci. 1164605-4613. [PubMed]
44. Zaidel-Bar, R., M. Cohen, L. Addadi, and B. Geiger. 2004. Hierarchical assembly of cell-matrix adhesion complexes. Biochem. Soc. Trans. 32416-420. [PubMed]
45. Zaidel-Bar, R., R. Milo, Z. Kam, and B. Geiger. 2007. A paxillin tyrosine phosphorylation switch regulates the assembly and form of cell-matrix adhesions. J. Cell Sci. 120137-148. [PubMed]
46. Zamir, E., and B. Geiger. 2001. Molecular complexity and dynamics of cell-matrix adhesions. J. Cell Sci. 1143583-3590. [PubMed]
47. Zamir, E., M. Katz, Y. Posen, N. Erez, K. M. Yamada, B. Z. Katz, S. Lin, D. C. Lin, A. Bershadsky, Z. Kam, and B. Geiger. 2000. Dynamics and segregation of cell-matrix adhesions in cultured fibroblasts. Nat. Cell Biol. 2191-196. [PubMed]

Articles from Molecular and Cellular Biology are provided here courtesy of American Society for Microbiology (ASM)
PubReader format: click here to try


Save items

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...


  • Gene
    Gene records that cite the current articles. Citations in Gene are added manually by NCBI or imported from outside public resources.
  • GEO Profiles
    GEO Profiles
    Gene Expression Omnibus (GEO) Profiles of molecular abundance data. The current articles are references on the Gene record associated with the GEO profile.
  • HomoloGene
    HomoloGene clusters of homologous genes and sequences that cite the current articles. These are references on the Gene and sequence records in the HomoloGene entry.
  • MedGen
    Related information in MedGen
  • PubMed
    PubMed citations for these articles
  • Substance
    PubChem chemical substance records that cite the current articles. These references are taken from those provided on submitted PubChem chemical substance records.

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...