Our study has uncovered an important physiological role in adhesion maturation for paxillin in its interaction with the cytoskeletal adapter protein vinculin. To understand tension-mediated FA maturation, we sought proteins recruited to FAs in a myosin II–dependent manner and examined the mechanism of their myosin II–sensitive FA association. Using pharmacological inhibition of myosin II and ECM compliance to modulate cellular tension, we show that paxillin, talin, and β1 integrin recruitment to adhesions is independent of myosin II activity, whereas adhesion association of FAK, zyxin, α-actinin, and vinculin is promoted by myosin II contraction. We focused on the myosin II–dependent recruitment of vinculin to adhesions, showing that it is reversible and occurs across a range of cell types and in the physiologically relevant contexts of myosin II–induced adhesion maturation during cell migration and ECM stiffness mechanosensing. Although previous studies correlated vinculin accumulation to sites of applied tension on cells (Galbraith et al., 2002
; Möhl et al., 2009
), we show the first demonstration of myosin II–dependent vinculin adhesion association. By assessing the myosin II sensitivity of vinculin’s protein interactions in cell lysates, we show that the vinculin–paxillin interaction is promoted by signaling induced by myosin II contractility, whereas we did not detect myosin II–dependent changes in the vinculin–talin interaction. It was recently shown that stretching talin promotes increased vinculin binding in vitro (del Rio et al., 2009
). The IPs used in our study bias detection of myosin-dependent changes that are preserved in cell lysates, such as covalent phosphorylation of paxillin, whereas cell lysis would clearly disrupt stretch activation of talin. However, our results suggest that paxillin may be a critical protein, possibly in addition to talin, in the myosin II–dependent recruitment of vinculin to adhesions in cells.
This study also reveals a currently unknown role for paxillin phosphorylation in regulation of its interaction with vinculin in cells. We found that that in addition to vinculin, paxillin’s interactions with FAK and Crk are promoted by myosin II activity, protein interactions specific to regulation by paxillinY31/118 phosphorylation (Turner, 2000
). This suggests the interesting possibility that Crk signaling through paxillin to regulate transcription may be mechanosensitive. We also found that myosin II activity promotes phosphorylation of an activating tyrosine on FAK, which mediates the myosin II–dependent phosphorylation of paxillin on Y31 and Y118. Although reduction of contractility did not completely abrogate paxillin phosphorylation in adhesions, the phosphorylation of these residues is sufficient to promote paxillin-mediated recruitment of vinculin because phosphatase inhibition or overexpression of a phosphomimic promoted the paxillin–vinculin interaction and vinculin recruitment to adhesions independent of myosin II activity. We suggest that in nascent, low contractility adhesions, paxillin phosphorylation may be below a threshold required to accumulate substantial vinculin, with its labile binding to these sites as shown by FRAP (). As actomyosin contractility begins to mature adhesions, the increased FAK activity it promotes may amplify paxillin phosphorylation to the point that even low affinity vinculin binding is sufficient for visible adhesion accumulation.
Our results, together with those of others, support a speculative two-step “hand-off” model for myosin II–mediated vinculin adhesion recruitment and its role in adhesion maturation. We suggest that nascent adhesions form by myosin II–independent talin recruitment to and activation of β1 integrin (; Tadokoro et al., 2003
), which together with talin’s actin-binding activity (Jones et al., 1989
) may promote formation of initial ECM–integrin–talin–actin linkages (Jiang et al., 2003
). Paxillin is also recruited to nascent, myosin II–independent adhesions by an unknown mechanism (; Webb et al., 2004
; Choi et al., 2008
), and paxillin/Hic5 and talin are all required for formation of adhesion clusters (; Zhang et al., 2008
). Myosin II activity in an α-actinin cross-linked cytoskeleton (Choi et al., 2008
) generates tension that is transmitted to nascent adhesions. Tension across the ECM–integrin–talin–actin linkage promotes engagement of secondary binding sites between β1 integrin and fibronectin to induce recruitment and activation of FAK (Friedland et al., 2009
), which is coupled to FAK/Src-dependent phosphorylation of paxillin Y31/118 (; Zaidel-Bar et al., 2007b
). pY31/118 paxillin, with its newly revealed vinculin-binding site (), may cycle between adhesion-bound and cytosolic fractions, inducing labile vinculin recruitment to adhesions (). Subsequent to adhesion recruitment, activation of vinculin’s actin-binding activity by simultaneous proximity to talin, actin, and acidic phospholipids (Chen et al., 2006
) induces activation and hand off of vinculin from labile paxillin binding to these other partners, enhancing vinculin’s binding stability to adhesions ( and ) and reinforcing the cytoskeleton–ECM linkage. The now matured adhesion can transmit stronger forces (Galbraith et al., 2002
) to promote adhesion turnover in cell migration (Webb et al., 2004
; Zaidel-Bar et al., 2007b
). High forces across the cytoskeleton–integrin link may induce a parallel pathway for vinculin recruitment by stretch activation of vinculin-binding sites in talin (del Rio et al., 2009
Questions remain about the paxillin-mediated recruitment of vinculin to adhesions. Because vinculin does not contain PY-binding SH2 domains, it is not clear how paxillin phosphorylation induces its interaction with vinculin or whether this interaction is direct. pY31/Y118 could induce a conformational change in paxillin that unmasks adjacent LD1 and LD2 domains involved in vinculin binding (Bertolucci et al., 2008
). Additionally, the alternate pathways for vinculin adhesion recruitment that occur when FAK is inhibited (Fig. S5) remain unknown. Future studies may help to clarify these and other questions.