Active vinculin induces an increase in FA size via its head domain
To investigate the role of different regions of vinculin in regulating FA number and size, a variety of vinculin constructs tagged to GFP or YFP were expressed in NIH3T3 cells () and their effects on FA formation were compared. Among the tested constructs were full-length vinculin (vinFL); vinculin T12 (vinT12), a constitutively active form of vinculin bearing mutations that inhibit head–tail association (Cohen et al., 2005
); vinculin LD (vinLD), which contains mutations that inhibit PIP2 binding (Chandrasekar et al., 2005
); constructs that comprise the N-terminal 880 or 258 amino acids, thus lacking the tail (vin880 and vin258, respectively); and a vinT construct (comprising amino acids 881–1066; ). All expressed constructs localized to FAs in a variety of cell types such as murine NIH3T3 fibroblasts, B16-F1 melanoma, and HeLa cells ( and not depicted). Additionally, besides localizing to FAs, vinT also colocalized with actin filaments (see and not depicted). Notably, vinT12, vin880, and vin258 induced a dramatic increase in the size and number of FAs (), and the area of the cell surface that contained FAs was approximately three- to fourfold larger than that observed for vinFL and vinLD (). Thus, although all vinculin constructs locate to adhesion sites, the size and number of these adhesions dramatically increase by preventing head–tail associations. By using vinculin fragments, this property was shown to reside within the N-terminal 258 amino acid.
Figure 1. Effect of wild-type and mutant forms of vinculin on FA growth. (A) Vinculin constructs that were expressed as fusion constructs to GFP derivatives in NIH3T3 cells: vinFL; vinculin comprising head and neck domains, amino acids 1–880 (vin880); vinculin (more ...)
Figure 7. vinT colocalization correlates with a subset of actin but not paxillin. (A) Cells expressing vinT were either labeled for actin or cotransfected with CFP–α-actinin (bottom). The intensity profiles on the right are from the area covered (more ...)
Talin and paxillin localize identically to vinculin-enlarged FAs
To identify associated molecules involved in the induction of enlarged FAs by active vinculin (vinT12) and C-terminally truncated constructs such as vin880 and vin258, image correlation analysis (ICA), which is a pixel-to-pixel comparison based on the Pearson's correlation coefficient (r
), was used. In initial experiments on cells, coexpression of CFP- and YFP-paxillin revealed r
values of ~0.8 ( B and S1, available at http://www.jcb.org/cgi/content/full/jcb.200703036/DC1
). These values reflect the virtually identical localization of two components.
Figure 2. ICA of vin880 with other FA proteins. (A) Sections of NIH3T3 cells expressing CFP- and YFP-tagged proteins as indicated. Fluorescence intensity profiles depict the area of the line drawn in image overlays. Potential direct interacting proteins talin and (more ...)
CFP- or YFP-vin880 was then coexpressed pairwise with other prominent FA regulators fused to YFP or CFP and correlation coefficients were calculated. The Pearson's correlation coefficient measures colocalization in 2D (). To produce a more visual illustration of the degree of correlation between pairs of components, fluorescence intensities in 1D line profiles drawn over FA areas were also compared (, right). Although talin and paxillin showed essentially identical colocalization with vin880 (r
= 0.72 and 0.82, respectively), the correlations of α-actinin (r
= 0.27), FAK (r
= 0.42), and a reporter for tyrosine-phosphorylated SH2-binding sites (dSH2; r
= 0.39; Kirchner et al., 2003
; Ballestrem et al., 2006
) with vin880 in FAs were low (). The paxillin result was particularly surprising because vin880 lacks the reported paxillin binding site in vinculin. The high degree of colocalization of vin880 with paxillin and talin was also observed when endogenous paxillin and talin were detected with antibodies (Fig. S2 A, available at http://www.jcb.org/cgi/content/full/jcb.200703036/DC1
; and not depicted). Thus, of the previously reported direct vinculin interaction partners, talin but not α-actinin is efficiently recruited to vin880-enlarged FAs. Also, the identical colocalization of vin880 with paxillin demonstrates that vinculin can trigger downstream events, resulting in the recruitment of paxillin independent of its interaction site located in the tail domain (also observed in vinculin null cells, see ). Moreover, the absence of FAK and dSH2 from most of the vin880-induced FAs close to the nuclei (unpublished data) suggests that FAK and tyrosine phosphorylation are not likely to play a role in the recruitment of paxillin to vin880-induced hypertrophic FAs.
Figure 6. Vinculin-induced FA hypertrophy and paxillin recruitment to FAs is not due to the potential dimerization of tailless vinculin constructs with endogenous vinculin. (A) CFP-paxillin was coexpressed with YFP-vinFL or -vin258 in vin−/− MEF (more ...)
Analysis of intensity profiles of vin880 cotransfected with FAK or dSH2 constructs revealed differences compared with the relationship between vin880 and α-actinin. Although all high intensity peaks of FAK or dSH2 correlated well with intensity peaks of vin880, high α-actinin intensity often showed no clear correlation with the intensity profile of vin880 (). Conversely, α-actinin–positive structures showed a strong correlation with actin (unpublished data). Together, these data suggest that α-actinin is unlikely to have a key role in vinculin-induced FA growth.
To assess whether vin258 and vinT12 signal via different mechanisms, their presence in FAs was correlated with the same series of proteins. Again, high correlation coefficients were only obtained between talin and paxillin ( and not depicted). These data suggest that talin and/or paxillin may play key roles in the formation of the enlarged FAs induced by active vinculin.
Vinculin regulates the clustering of integrins in FA
To determine the relationship between FA formation and receptor distribution, the colocalization of integrins with vin880 was examined in cells spread on fibronectin, the major ligand for α5β1 integrin. A YFP–α5 integrin construct colocalized with CFP-vin880 in all visible FAs (unpublished data). Integrins adopt different conformations, which can be reported by mAbs. A striking colocalization of active β1 with vin880 in FAs was observed (), which was distinct from the colocalization of total β1 with vin880. This is most obviously seen in the fluorescence intensity profile from the area of the line drawn in the image overlay (). Although the total β1 staining labeled integrins in FAs and the cell membrane, active β1 was found almost exclusively in FAs correlating highly with vin880 (r = 0.79). To examine whether these FAs link the cell to the ECM, interference reflection microscopy was used to visualize regions of the cell membrane proximal to the substratum. Indeed, FAs induced by vin880 were detected by interference reflection microscopy, demonstrating a physical association with the ECM ().
Figure 3. Vinculin regulates integrin clustering and dynamics. (A) HeLa cells overexpressing YFP-vin880 were costained with an antibody recognizing active β1 (9EG7; Bazzoni et al., 1995) and the total pool of β1 integrin (TS2/16). FAs positive for (more ...)
Vinculin constructs that induce FA enlargement have increased residency time in FAs and form tight complexes with talin and integrins
During FA size measurements, it was observed that YFP-vin258, YFP-vin880, and GFP-vinT12, all of which induced enlarged FAs, had a clearer FA pattern with a reduced cytoplasmic pool compared with GFP-vinFL ( and not depicted). The reduction of the cytoplasmic pool suggests differences in mobilities and affinities of the proteins, leading to an enhanced recruitment to FAs. To study mobilities of the different vinculin mutants in FAs, FRAP experiments were performed (; Lippincott-Schwartz et al., 2001
). The observed FRAP recoveries presented in appear to be slightly biphasic, as was previously observed for vinculin (Lele et al., 2006
). The possibility that this might be caused by the ability of vinculin to bind multiple binding partners or the enhanced fast reversible photobleaching for YFP-tagged probes used in the majority of these experiments needs further investigation. However, to avoid overinterpretation of our results, single exponential fits were used as reported previously by others with similar probes (Cohen et al., 2006
). Using such fits provided estimates of the mobile fractions (MFs) and t1/2
of recoveries. A striking twofold decrease in the MFs () and a twofold increase in t1/2
() of vinT12, vin880, and vin258 were found compared with vinFL, vinLD, and vinT. This indicates that by switching to an active conformation or exposing binding sites within N-terminal domains, vinculin changes its affinity for binding partners, resulting in an increased stability of vinculin within FAs.
Because talin binds to integrins and provides an early mechanical link to ECM proteins (Giannone et al., 2003
; Zaidel-Bar et al., 2003
), it was hypothesized that the reduction of vinT12, vin880, and vin258 mobility may be caused by the formation of a stable complex with talin and integrins. A recent paper demonstrated the delayed turnover of talin in FAs upon coexpression with constitutively active vinT12 (Cohen et al., 2006
), thus indicating their tight association in cells. Similar results were obtained for talin turnover when coexpressed with vin880 (talin t1/2
increased ~25% compared with cells coexpressing vinFL; ).
If integrins were also part of such a tight complex, we predicted that integrin subunits would turn over at similar rates to vinT12, vin880, or vin258. Indeed the MF and t1/2 of the GFP–α5 integrin chain were almost identical to YFP-vin880 (). Interestingly, YFP-paxillin, which colocalizes precisely with vin880 (), was considerably more mobile, with a t1/2 of 11 s and a MF of 70%, and did not change upon coexpression of vin880 (). These data suggest that in cells cultured on fibronectin, talin, α5β1 integrin, and active vinculin form a tight complex at points of cell–ECM contact, whereas paxillin only transiently associates with this complex.
To test whether integrin dynamics change in the presence of vinculin in FAs, YFP–α5 integrin was expressed alone or in combination with CFP-vinFL or -vin880 in vin−/− mouse embryonic fibroblasts (MEFs; Saunders et al., 2006
) and integrin turnover rates were measured using FRAP. A 40% increase in the t1/2
of α5 integrin in cells coexpressing vinFL compared with α5 alone was observed. A further 30% increase of t1/2
was observed in cells coexpressing vin880, demonstrating that integrin turnover within FAs is regulated by vinculin activity or exposure of N-terminal binding domains within vinculin ().
To confirm the apparent tight association between integrins, talin, and vin880, their interactions were tested biochemically. FN-coated beads were added to cells expressing YFP-vinFL or -vin880 and bead-bound fractions were isolated after cross-linking and detergent extraction. α5 integrin and talin were identified in all bead-bound cell fractions (). Despite the similar total levels of vin880 and vinFL expression, within the bead-bound fraction, vin880 was enriched by approximately fourfold, unlike talin, when normalized to the respective α5 integrin band intensity (). Moreover, coimmunoprecipitation experiments from cells expressing YFP-vin880 or -vinFL demonstrated the association of talin with vin880 but not vinFL (). Performing the assay after incubation with a chemical cross-linker resulted in the additional coimmunoprecipitation of the α5 integrin subunit with vin880 (1.85 ± 0.49–fold increase over control; P = 0.03, Fisher Sign test, n = 5; ). The weak α5 integrin signal observed in the coimmunoprecipitation is partly caused by the low stoichiometry of the interaction, i.e., the vast majority of talin and vinculin within the cell is not complexed with integrin at any one time, partly because of the lability of the complex and the inaccessibility of a transmembrane receptor associated with poorly soluble cytoskeletal and matrix components. This latter point means that stringent detergent conditions are required to solubilize the individual components of the complex; these conditions subsequently lead to the dissociation of the complex.
Figure 4. YFP-vin880 is enriched in FN– integrin complexes and coimmunoprecipitates talin and integrin. (A) FN–bead bound complexes isolated from NIH3T3 cells expressing either YFP-vin880 or -vinFL and immunoblotted for GFP, α5 integrin, (more ...)
Interestingly, talin coimmunoprecipitated with vin880 regardless of whether cells were in suspension or attached to the ECM (), and neither vin880 nor vinFL coimmunoprecipitated α-actinin, paxillin, or FAK (Fig. S3, available at http://www.jcb.org/cgi/content/full/jcb.200703036/DC1
). Overall, these biochemical data support the FRAP and immunofluorescence data () and indicate that the N-terminal domains of vinculin, e.g., vin880 without the C-terminal tail region, constitute a vinculin construct that forms a tight complex with talin and α5β1 integrin.
The talin–vinculin interaction is required for the vinculin-induced FA enlargement
It was observed previously that talin and paxillin but not FAK, α-actinin, or phosphotyrosine correlated highly with localization of vin880 in FAs, suggesting that the former might be involved in FA enlargement or formation. To test this possibility, paxillin and talin (talin1) were deleted by small hairpin RNA knockdown. YFP-vin880 expressed in paxillin-deficient B16 cells was still able to induce FAs of a similar size and number as those in wild-type cells (). For the knockdown of talin1, the interpretation of data was difficult, primarily because many of the talin knockdown cells rounded up and were therefore unsuitable for FA measurements (unpublished data). Therefore, to test directly the role of the talin–vinculin interaction in the formation of enlarged FAs, an A50I mutation was introduced into vin258, vin880, and vinT12. This mutation is known to reduce talin binding to vinculin in vitro (Bakolitsa et al., 2004
). Immunoprecipitations demonstrated that the A50I mutation in vin880 and vinT12 abrogated the coimmunoprecipitation of talin (). Expression of these constructs in NIH3T3 cells and subsequent analysis of FAs showed that vin258 (A50I) and vinT12 (A50I) no longer induced FA growth, whereas vin880 (A50I) exhibited greatly reduced activity (). Collectively, these experiments indicate not only that “activated” vinculin, or vinculin with exposed binding sites within its N-terminal domains (vin880 and vin258), binds to talin, but that this interaction is required for the formation of enlarged FAs. In contrast, paxillin is not required for the formation of FAs and is likely to be recruited downstream (or independently) of vinculin.
Figure 5. FA size is regulated by vinculin interaction with talin. (A) Paxillin was knocked-down using small hairpin RNA in B16 melanoma cells expressing vin880. Despite the absence of paxillin, vin880 still induced FA growth. (B) Quantification of FA number, size, (more ...)
Induction of oversized FAs and underlying signaling mechanisms are independent of endogenous vinculin
The possibility exists that both FA growth and paxillin recruitment to active vinculin could have been the result of an interaction of paxillin with endogenous vinculin that had dimerized, via an intermolecular head–tail interaction, with the expressed vinculin constructs. To test this possibility, vinculin constructs were expressed in vin−/− MEFs. Expression of tailless vinculin forms and active vinT12 in the absence of endogenous wild-type vinculin induced a two- to threefold increase of FAs (), which was abolished by the A50I mutation (). Interestingly, the induction of FAs in vin−/− MEFs by a vin880 (A50I) mutant was abrogated (compare with ), suggesting that vin880 induced the activation of endogenous vinculin to a small extent. In further experiments, the colocalization of paxillin and vin258 was analyzed in enlarged FAs of vin−/− MEFs. As outlined in , YFP-vin258 colocalized identically with CFP-paxillin, demonstrating that the highly efficient paxillin recruitment induced by tailless vinculin forms is independent of the putative dimerization of endogenous vinculin with the expressed constructs.
The similar behavior of the vinculin expression constructs in cells with and without endogenous vinculin was examined using FRAP to measure t1/2
of recovery of YFP-vinFL and YFP-vin880 (). As in cells with endogenous vinculin, the t1/2
for vin880 increased ~50%. This is in accordance with a previous paper using a vinculin head domain and active vinculin (Cohen et al., 2006
). These experiments suggest that there is little contribution of endogenous vinculin to the data presented here using C-terminal truncations or activated vinculin constructs.
vinT colocalizes with a subset of filamentous actin but not with paxillin
Paxillin and actin have been shown to bind to the vinT region (Menkel et al., 1994
; Wood et al., 1994
; Huttelmaier et al., 1997
). To elucidate possible associations of these two proteins with vinT, YFP-vinT was expressed in NIH3T3 cells and its colocalization with actin and paxillin was analyzed. The localization of vinT correlated well with actin stress fibers (r
= 0.7) but was diminished or absent in large protruding lamellipodia that were positive for α-actinin (). This strong reduction of vinT in protruding areas was not caused by the potential competition with endogenous vinculin because the phenomenon also occurred in vinculin null cells (). Furthermore, time-lapse experiments showed that it was only when large lamellipodia collapsed and started to retract that vinT became strongly associated with these structures (Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200703036/DC1
), suggesting that vinT only binds a subset of actin filaments. In contrast, CFP-paxillin was abundant in FAs of protruding cell areas () and only colocalized with vinT in retracting areas, albeit with a low correlation between their intensity profiles. Thus, paxillin localization correlates with the head region of vinculin, and there is little, if any, interaction of paxillin with its tail. vinT, however, appears to be the major domain involved in actin binding.
vinT links adhesion sites to the actin cytoskeleton
The major factor implicated in the growth of FAs has, until now, been intracellular tension mediated by the actomyosin contractile machinery (Burridge and Chrzanowska-Wodnicka, 1996
; Balaban et al., 2001
). Our data indicate that the vinculin head but not the tail induces FA growth (), suggesting that a link to actin via the C terminus of vinculin may not be required for FA growth. To examine whether the vinculin–actin interaction has the potential to modulate FAs, the association of FA growth–promoting vinculin mutants that either lacked (vin880) or retained (vinT12) the tail domain were examined in relation to actin. Approximately 75% of the internal nucleoproximal FAs (defined in this paper as >10 μm from the cell edge) induced by overexpression of vinculin mutants without tail domains were not linked to actin stress fibers (). In contrast, overexpression of vinT12, which retains its tail domain, resulted in >80% of FAs that were streaklike and linked to actin filaments (). Furthermore, in ~30% of these cells, long ropelike structures positive for both vinT12 and actin were apparently clustered together. These data suggested that the tail of vinculin forms a crucial link between FAs and the actin cytoskeleton. Because actin filaments in cells have been shown to undergo retrograde flow (Ponti et al., 2004
; Vallotton et al., 2004
; Gupton and Waterman-Storer, 2006
; Hu et al., 2007
), the possibility that vinculin dynamics are influenced by the link to actin was tested using live cell time-lapse analysis of vinT12 and the tailless vin880 ( and Videos 2 and 3, available at http://www.jcb.org/cgi/content/full/jcb.200703036/DC1
). Although vin880 was stably localized in FAs, vinT12 was transported in a retrograde manner with a mean velocity of 0.366 ± 0.112 μm/min toward the cell center upon retraction. Furthermore, vinT, which lacks a head domain, followed a retrograde flow with a mean velocity of 0.679 ± 0.295 μm/min from peripheral FAs toward the cell center ( and Video 4). F-actin retrograde flow in similar velocity ranges has been observed previously in newt lung and PtK1 epithelial cells (Ponti et al., 2004
). Thus, it is the vinT domain that links vinculin to the actin cytoskeleton, which may in turn exert forces on vinculin resulting in its relocalization outside of FAs.
The vinculin head stabilizes adhesion sites despite inhibition of actomyosin-mediated tension
Because the vinculin head region is able to form large FAs in the absence of the actin-binding tail, we reasoned that the vinculin head might be able to initiate or stabilize cell–matrix adhesions independently of the actomyosin machinery. Inhibition of Rho-kinase (ROCK) leads to the release of actomyosin-mediated tension and the disruption of actin stress fibers. As a consequence of this perturbation of the actin cytoskeleton, FAs dissolve and only focal complexes of a transient nature remain visible. The effect of the ROCK inhibitor Y-27632 in NIH3T3 cells expressing vinFL and vin880 was therefore tested. Although Y-27632 treatment resulted in the loss of essentially all adhesion sites in vinFL-expressing cells (except a few complexes at the cell periphery), a large number of adhesion sites were still apparent in cells expressing vin880 (). This was the case even when Y-27632 was used at concentrations up to 300 μM, which leads to the complete distortion of the cell morphology. Similar observations were made when vinFL- and vin880-expressing cells were treated with the actin-disrupting agent cytochalasin D (). Although quantification of adhesion sites in vin880-expressing cells revealed no change in adhesion area when cells were treated with actin-perturbing reagents, the morphology of the remaining FA structures did appear to be altered (compare with ). They were less streaklike and resembled those of β3 integrin clustering induced by switching to an active conformation through the addition of manganese, integrin-activating mutations, or talin head overexpression (Cluzel et al., 2005
). Although the clusters observed by Cluzel et al. (2005)
contained talin, they were not linked to the actomyosin machinery. This is in keeping with our model of N-terminal vinculin domains controlling the clustering of activated integrin via talin independently of linkages with the actin cytoskeleton. These results suggest that vinculin without the actin-binding tail domain is able to initiate FA formation in the absence of actomyosin-mediated forces.
Figure 8. Vin880 stabilizes adhesion sites in cells despite the inhibition of actomyosin function or disruption of actin filaments. (A, top) NIH3T3 cells expressing YFP-vinFL or -vin880 treated with 100 μM Y-27632 for 60 min. Although only small dotlike (more ...)
Together, these findings separate the vinculin head and tail regions into two distinct functional domains: a head region that binds to talin and is involved in the growth of cell–matrix adhesions associated with clustering of active integrins and a tail domain that is involved in binding actin and coupling with the mechanotransduction force machinery.