JCB: Article

Myosin II activity regulates vinculin recruitment

to focal adhesions through FAK-mediated
paxillin phosphorylation
Ana M. Pasapera,1 Ian C. Schneider,3,4,7 Erin Rericha,2,7 David D. Schlaepfer,5,6 and Clare M. Waterman1,7
1
Cell Biology and Physiology Center, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892
2
Institute for Research in Electronics and Applied Physics, University of Maryland, College Park, MD 20742
3
Department of Chemical and Biological Engineering and 4Department of Genetics, Development, and Cell Biology, Iowa State University, Ames, IA 50011
5
Moores Cancer Center and 6Department of Reproductive Medicine, University of California, San Diego, La Jolla, CA 92093
7
Physiology Course, Marine Biological Laboratory, Woods Hole, MA 02543

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F
THE JOURNAL OF CELL BIOLOGY

ocal adhesions (FAs) are mechanosensitive adhe- stiffness dependent. Myosin II activity promotes FAK/
sion and signaling complexes that grow and change Src-mediated phosphorylation of paxillin on tyrosines
composition in response to myosin II–mediated 31 and 118 and vinculin association with paxillin. We
cytoskeletal tension in a process known as FA matura- show that phosphomimic mutations of paxillin can spe-
tion. To understand tension-mediated FA maturation, we cifically induce the recruitment of vinculin to adhesions
sought to identify proteins that are recruited to FAs in a independent of myosin II activity. These results reveal an
myosin II–dependent manner and to examine the mech- important role for paxillin in adhesion mechanosensing
anism for their myosin II–sensitive FA association. We find via myosin II–mediated FAK phosphorylation of paxillin
that FA recruitment of both the cytoskeletal adapter that promotes vinculin FA recruitment to reinforce the
protein vinculin and the tyrosine kinase FA kinase cytoskeletal ECM linkage and drive FA maturation.
(FAK) are myosin II and extracellular matrix (ECM)

Introduction
Cells impinge force on their extracellular environments FAs are mechanosensitive organelles that recruit cyto-
during tissue morphogenesis, cardiovascular and pulmonary plasmic proteins to grow and change composition in response
function, directed cell motility, and the immune response. to mechanical tension (Chrzanowska-Wodnicka and Burridge,
Cell forces are primarily developed by myosin IIs acting in 1996; Riveline et al., 2001) in a process known as FA matura-
the actin cytoskeleton (Cai et al., 2006). Cytoskeletal forces tion. Tension driving FA maturation can be supplied either by
are linked to the ECM through transmembrane – integrin myosin II forces transmitted to FAs through the actin cytoskele-
heterodimers that cluster to form focal adhesions (FAs; Geiger ton or by external forces applied to the cell. It is thought that
et al., 2009). On their cytoplasmic face, integrin tails serve tension-driven FA compositional changes are critical to the abil-
as scaffolds for the recruitment of FA-associated proteins, ity of FAs to trigger different signaling pathways that promote
including cytoskeletal-binding and adapter proteins, and en- differentiation, division, or apoptosis (Engler et al., 2006).
zymes such as kinases, phosphatases, and small GTPases and The mechanism of tension-mediated FA maturation is not
their modulators (Zaidel-Bar et al., 2007a). These proteins well characterized. Tension on FA proteins could drive local-
contribute to FA functions in integrin-mediated signal trans- ized unfolding or conformational changes that unmask binding
duction and form the force-bearing link between the ECM sites for cytoplasmic proteins (Vogel and Sheetz, 2006). For ex-
and cytoskeleton. ample, molecular dynamics simulations suggest that directional

Correspondence to Clare M. Waterman: watermancm@nhlbi.nih.gov This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No
Mirror Sites license for the first six months after the publication date (see http://www.rupress
Abbreviations used in this paper: CB, cytoskeleton buffer; CCD, charge- .org/terms). After six months it is available under a Creative Commons License (Attribution–
coupled device; FA, focal adhesion; FAKi, FAK inhibitor; IP, immunoprecipitation; Noncommercial–Share Alike 3.0 Unported license, as described at http://creativecommons
MEF, mouse embryonic fibroblast; PY, phosphotyrosine. .org/licenses/by-nc-sa/3.0/).

The Rockefeller University Press  $30.00

J. Cell Biol. Vol. 188 No. 6 877–890
www.jcb.org/cgi/doi/10.1083/jcb.200906012 JCB 877
 force on integrin cytoplasmic tails could induce separation of
the  and  subunits (Zhu et al., 2008) to allow new protein
binding. In vitro experiments suggest that forced unfolding of
talin promotes vinculin binding (del Rio et al., 2009), whereas
stretching p130cas unmasks a tyrosine substrate for Src family
kinases (Sawada et al., 2006). However, whether these mech-
anisms operate in cells during physiological, myosin II–mediated
FA maturation is not known.
In spite of the lack of mechanistic insight, it is well
accepted that tension-mediated FA maturation involves a se-
quential cascade of compositional changes (Zaidel-Bar et al.,
2004). FAs are initiated by activation of integrin extracellular
heads’ affinity for ECM through association of their cytoplas-
mic tails with the vinculin- and actin-binding protein talin
(Tadokoro et al., 2003). Early after integrin activation, the
adapter protein paxillin is recruited by an unknown mechanism,

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and more integrins cluster into FA (Laukaitis et al., 2001; Webb
et al., 2004; Wiseman et al., 2004). Further FA growth is
accompanied by the recruitment of the actin-bundling protein
-actinin (Choi et al., 2008), which with talin (Lee et al., 2004)
may establish a link between integrins and the actin cytoskele-
ton. Myosin II is thought to transmit tension in an -actinin–
actin network to the integrin–ECM linkage. This tension promotes
elongation of an adhesion-associated actin bundle where cyto-
skeletal adapter proteins vinculin and zyxin accumulate (Choi
et al., 2008). In addition, tension on fibronectin-engaged 1 in-
tegrins promotes integrin head binding to secondary sites on
fibronectin (Friedland et al., 2009), inducing recruitment and
activation of the tyrosine kinase FAK (Shi and Boettiger, 2003;
Friedland et al., 2009). Tyrosine phosphorylation of early FA
proteins, including FAK, paxillin, and p130cas (Ballestrem et al.,
2006), then act as scaffolds for phosphotyrosine (PY)-binding
SH2 domain–containing proteins. There are also studies that show
compositional differences between small or large FAs (Zaidel-
Bar et al., 2003, 2007b; Zimerman et al., 2004), although the
order of protein addition or the requirement for tension in their
FA recruitment is not known.
To better understand tension-mediated FA maturation,
we sought proteins that are recruited to FAs in a contractility-
dependent manner and examined the mechanism for their
Figure 1. Rho kinase–mediated myosin II activity and substrate stiffness
myosin II–sensitive FA association. We find that FA localization slow MEF migration and increase adhesion size. (A) Migration rates for
of vinculin is myosin II and ECM stiffness dependent. By exam- untreated cells (control), cells treated with 20 µM blebbistatin (Blebb) or 10 µM
ining the effects of myosin II inhibition on protein interactions Y27632, or plated on 1.0 kPa compliant polyacrylamide substrates. Mean
velocity is shown above each box plot. Arrowheads indicate lower and
and phosphorylation, we deduce that the myosin II–dependent upper extreme outliers. n = number of cells. (B) Phase-contrast images of
recruitment of vinculin to FA is mediated by FAK phosphoryla- cell morphology under the same conditions as in A. Bar, 10 µm. (C) Immu-
tion of paxillin, which creates binding sites in FAs for vinculin nolocalization of PY epitopes (P-Tyr) to visualize adhesions (green) and
fluorescent phalloidin staining to visualize actin filaments (red) under the
to drive FA maturation. treatments as in A. Merged images are shown in the third column, and boxed
regions are magnified in the fourth column. Bars: (third column) 10 µm; (fourth
column) 2 µm. (D) Area of individual adhesions within PY-immunolabeled cells
Results under the conditions in A. Mean adhesion area (micrometer squared) is
shown above each box plot. n = number of adhesions.
Inhibition of myosin II activity affects
adhesion morphology and alters cell
migration Contraction was inhibited with either 20 µM of the myosin II–
To gain insight into myosin II–mediated FA maturation, we specific ATPase inhibitor blebbistatin (Ki = 0.3 µM) or 10 µM
characterized the effects of myosin II inhibition on migra- of the Rho kinase inhibitor Y27632 (Ki = 0.14 µM). As a
tion and FA morphology in mouse embryonic fibroblasts physiological approach to myosin II inhibition, MEFs were
(MEFs) adhered to coverslips coated with 5 µg/ml fibronectin. plated on fibronectin-coupled compliant substrates (1.0 kPa

 878 JCB • VOLUME 188 • NUMBER 6 • 2010

polyacrylamide gel) where myosin II activity is down-regulated
in response to mechanosensation of a compliant ECM (Pelham
and Wang, 1997). Phalloidin staining and immunolocalization
of PY epitopes (Thomas and Brugge, 1997) in control cells
showed lamellipodia in the cell front and F-actin bundles in
the center and tail, which often terminated in PY-containing
FAs (Fig. 1 C). FAs ranged in size from diffraction limited
(0.125 µm2) at the cell front to several micrometers long
(0.45 ± 0.75 µm2) in the central and rear regions (Fig. 1 D).
Because a goal of this study is to determine the compositional
changes that accompany FA size changes mediated by myosin
II, we will not classify different-sized structures as FAs, focal
complexes, or fibrillar adhesions, but will refer to all membrane
plaques marked by PY or another FA protein as adhesions.
Blebbistatin treatment inhibited actin bundles and induced a
homogeneous network, ROCK inhibition blocked central actin

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bundles, and plating on compliant substrates reduced actin
bundle length and intensity compared with controls (Fig. 1 C).
Myosin II inhibition by all three treatments decreased adhe-
sion size compared with control (Fig. 1 D), and in the case of
blebbistatin, confined adhesions to the extreme cell periphery
(Fig. 1 C). These results agree with previous studies (Totsukawa
et al., 2004; Even-Ram et al., 2007; Schaub et al., 2007). In
addition, all three treatments altered cell morphology and in-
creased migration rate compared with control (Fig. 1, A and B)
as reported previously (Totsukawa et al., 2004; Even-Ram et al.,
2007). Because myosin II ATPase and ROCK inhibition pro-
duced similar effects, in the remainder of our experiments, we
used 20 µM blebbistatin (1 h) for pharmacological inhibition of
myosin II, whereas compliant substrates were used for physi-
ological down-regulation of contractility.

Adhesion recruitment of FAK, vinculin,

zyxin, and -actinin is myosin II dependent
To determine how adhesion composition is affected by acto-
myosin activity, we performed immunofluorescence and quan-
titative image analysis of cells in which contractility was
inhibited. Because the adapter protein paxillin is a component
of newly formed adhesions (Laukaitis et al., 2001; Choi et al.,
2008), we examined the myosin II dependence of its localiza-
tion to PY-containing adhesions. In both control and blebbistatin-
treated cells, paxillin was nearly completely colocalized with
PY in all adhesions in spite of blebbistatin’s effects on adhe-
sion size, spatial distribution, and actin organization (Fig. 2 A).
Similarly, plating cells on compliant substrates did not alter
localization of paxillin to PY-containing adhesions (Fig. S1).
Thus, paxillin is present in adhesions independently of myo- Figure 2. Myosin II is required for recruitment of specific proteins to adhe-
sin II contractility, and therefore, we used it as an adhesion sions. (A) Immunolocalization of paxillin (Pxn; red) and PY epitopes (P-Tyr;
marker in further experiments. green) in untreated cells (control) or cells treated with 20 µM blebbistatin
(Blebb). Merged images are shown in the third column, and boxed regions
We performed immunofluorescence and quantitative are magnified in the fourth column. Bars: (third column) 10 µm; (fourth col-
image analysis to examine the blebbistatin sensitivity of adhe- umn) 2 µm. (B) Immunolocalization of paxillin (red) with either (in green);
sion association of several important FA proteins. We deter- talin 1 (Tln), FAK, 1 integrin (1-Int), zyxin (Zyx), vinculin (Vcl), or -actinin
(Actn) in untreated control cells (left) or cells treated with 20 µM blebbistatin
mined the ratio of intensity of fluorescence in segmented (right). Merged images are shown in the right column. Bar, 2 µm. (C) Effects
adhesions in blebbistatin-treated cells relative to controls as a of blebbistatin on adhesion localization of FA proteins expressed as the ratio
measure of the myosin II dependence of adhesion recruitment. of mean fluorescence intensities within segmented adhesions of blebbistatin-
treated relative to control cells. Mean ratios are shown above each plot.
This revealed a slight yet insignificant reduction in paxillin *, P < 0.02. Error bars indicate 95% confidence interval of the mean. n =
level after blebbistatin treatment in all experiments, ranging number of blebbistatin-treated cells/number of control cells.

Myosin II–mediated recruitment of vinculin to adhesions by paxillin • Pasapera et al. 879

 Figure 3. Vinculin is stably bound in adhesions. EGFP fusion proteins localized to adhesions were subjected to FRAP. (A) Sample fluorescence recovery curves for
vinculin, -actinin, talin 1, paxillin, FAK, and zyxin in single adhesions. Note different x-axis scales in left and right plots; fits are shown as solid lines. (B) Half-times
of fluorescence recovery. Means are shown above each plot. Vcl, vinculin; Actn, -actinin; Pxn, paxillin; Tln, talin 1; Zyx, zyxin. n = number of adhesions.

from 61 to 91% of controls (Fig. 2 C). Similar analysis revealed of both protein class (i.e., adapter, kinase, and actin binding)

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no significant difference in the levels of 1 integrin and talin in and the myosin II dependence of their adhesion localization.
paxillin-containing adhesions between control and blebbistatin- Furthermore, in MEFs, the adapter protein vinculin exhibits
treated cells (Fig. 2, B and C). In contrast, although there were particularly stable adhesion binding. Thus, we focused further
high levels of FAK, zyxin, vinculin, and –actinin in adhesions study on the mechanism of actomyosin-dependent, stable vincu-
of controls, these proteins were significantly reduced in adhe- lin association with adhesions.
sions of blebbistatin-treated cells (Fig. 2 C). Blebbistatin caused
redistribution of FAK, vinculin, and zyxin from adhesions to the Myosin II–mediated recruitment of vinculin
cytosol, whereas -actinin was relocalized to lamellipodia. to adhesions is cell type independent,
(Fig. 2 B). Together, these results indicate that the recruitment reversible, and physiologically relevant
of paxillin, talin, and 1 integrin to adhesions is independent of To characterize the generality of myosin II–dependent vinculin
myosin II activity, whereas adhesion association of FAK, zyxin, recruitment to adhesions, we examined its human cell type
vinculin, and -actinin requires myosin II contraction. specificity. Immunofluorescence analysis of osteosarcoma (U2OS),
breast epithelial (MCF-10), and endothelial (HUVEC) cell lines
Vinculin is a stably bound adhesion protein (Fig. S3) and primary foreskin fibroblasts (HFF-1; Fig. 4 A)
We next hypothesized that strong adhesion binding affinity revealed high levels of vinculin in adhesions of untreated
could override actomyosin dependence in recruitment of pro- cells but a significant reduction in vinculin in adhesions in-
teins to adhesions. To test this, we performed FRAP of EGFP- duced by blebbistatin treatment (Fig. 4 B).
tagged proteins in single adhesions and determined the mean To determine whether the blebbistatin-induced reduction
fluorescence recovery t1/2 as an estimate of the stability of of vinculin in adhesions was reversible, we analyzed the time
adhesion binding (Bulinski et al., 2001; Lele and Ingber, 2006). course of vinculin recruitment after induction of actomyosin
Our hypothesis predicts long FRAP t1/2’s for the myosin II– contractility by blebbistatin washout (Fig. 4, C and D). Immuno-
independent adhesion-associated proteins paxillin and talin fluorescence analysis showed that within 15 min after blebbi-
and short FRAP t1/2’s for the blebbistatin-sensitive adhesion statin washout, although adhesion size was still reduced compared
proteins zyxin, FAK, -actinin, and vinculin. Contrary to our with controls, vinculin was already colocalized with paxillin in
expectations, FRAP analysis revealed a broad range of mean adhesions (Fig. 4, C and D). Analysis of later time points in-
t1/2’s from <10 s to nearly 3 min with no correlation between dicated that vinculin remained colocalized with paxillin, and
myosin II dependence of adhesion association and FRAP t1/2 normal adhesion size was regained within 30–60 min (Fig. 4, C
(Fig. 3 and Fig. S2). EGFP conjugates of the myosin II– and D). Thus, blebbistatin reversibly reduces vinculin in adhe-
independent adhesion-associated proteins paxillin and talin sions, and vinculin is recruited to adhesions soon after the
had similar intermediate FRAP t1/2’s of 23 ± 3.4 s (SEM) and induction of actomyosin contraction before the completion of
25 ± 5.9 s. The myosin II–dependent adhesion proteins exhib- adhesion growth.
ited FRAP t1/2’s from very short, for EGFP-FAK (7 ± 2.1 s) We next sought to determine whether the myosin II–
and EGFP-zyxin (12 ± 2.5 s), to intermediate, for EGFP– dependent recruitment of vinculin to adhesions occurs in physi-
-actinin (36 ± 0.5 s), and to very long, for EGFP-vinculin (80 ± ologically relevant contexts. By examining vinculin localization
2.9 s), which is longer than previous reports in other cell types in cells plated on compliant ECMs, we confirmed that reduction
(Chandrasekar et al., 2005; Möhl et al., 2009; Wolfenson of contractility by ECM compliance mechanosensing reduced
et al., 2009). In addition, EGFP-vinculin often did not exhibit vinculin level in adhesions (Fig. S1). We next sought to deter-
complete fluorescence recovery after 300 s (Möhl et al., 2009; mine when in the adhesion maturation cycle vinculin is recruited
unpublished data), suggesting the presence of a stably bound to adhesions of migrating cells. Similar to blebbistatin washout,
fraction in adhesions. Thus, different adhesion proteins possess in the advancing lamellipodium of migrating cells, nascent adhe-
highly variable adhesion-binding stability, which is independent sions form independently of actomyosin contraction followed

 880 JCB • VOLUME 188 • NUMBER 6 • 2010

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Figure 4. Characterization of myosin II dependence of vinculin recruitment to adhesions. (A) Immunolocalization in human foreskin fibroblasts (HFF1)
of vinculin (Vcl; green) and paxillin (Pxn; red) in untreated (control) or 20 µM blebbistatin (Blebb)-treated cells. Bar, 10 µm. Merged, magnified images
of boxed regions are shown in the third column. Bar, 2 µm. n = number of blebbistatin-treated cells/number of control cells. (B) Effects of blebbistatin on
adhesion localization of paxillin and vinculin in the noted cell types expressed as the ratio of mean fluorescence intensities within segmented adhesions of
blebbistatin-treated relative to control cells. Mean ratios are shown above each plot. *, P < 0.02. Error bars indicate 95% confidence interval of the mean.
(C) Area of individual adhesions from paxillin immunostaining in MEF cells in untreated control cells at specific times after treatment with 20 µM blebbistatin
(15, 30, and 60 min) or washout of 20 µM blebbistatin into control media (15, 30, and 60 min w/o). Mean adhesion areas (micrometer squared) are
shown next to each box plot. Red symbols indicate the outliers at more than three interquartiles; blue symbols indicate a 95% confidence interval of the
mean. n = number of adhesions. (D) Immunolocalization of paxillin (red) and vinculin (green) and fluorescent phalloidin staining of actin filaments in cells
after treatment (Treat) for 60 min and washout (blebbistatin w/o) for 15, 30, and 60 min of 20 µM blebbistatin. Merged images are shown in the fourth
column, and boxed regions are magnified in the fifth column. Bars: (fourth column) 10 µm; (fifth column) 2 µm. (E) Images from a time-lapse dual-color
TIRF series of EGFP-paxillin (GFP-Pxn) and mCherry vinculin (mCherry-Vcl) during adhesion formation and growth in a migrating MEF cell. Time is shown
in seconds. Bar, 2 µm. (F) Normalized fluorescent protein intensity (green, paxillin; red, vinculin) in the adhesion shown in E. Horizontal lines show the
value of two times the standard deviation of the normalized background fluorescence (2× SD Vcl or Pxn Bckg); note that this is higher for mCherry because
it is much dimmer than EGFP. Arrows indicate the time when the intensity rose above these values. The time difference between arrows indicates lag time
between the accumulation of EGFP-paxillin and mCherry-vinculin at the adhesion (Pxn-Vcl lag).

by their myosin II–dependent maturation (Riveline et al., 2001; relevant contexts of ECM stiffness mechanosensing and adhe-
Choi et al., 2008). MEFs were cotransfected with mApple- or sion maturation during cell migration.
EGFP-paxillin as a marker of nascent adhesions (Choi et al.,
2008) and EGFP- or mCherry-vinculin and imaged by time- The paxillin–vinculin interaction is modulated
lapse TIRF microscopy (Fig. 4 E and Video 1). Quantitative by myosin II activity
analysis showed that the initiation of vinculin accumulation in Our results show that the vinculin-binding proteins talin
adhesions lagged behind that of paxillin by 80 ± 40 s (n = 16 (Jones et al., 1989) and paxillin (Brown et al., 1996) are re-
FAs in four cells; range 35–180 s; Fig. 4 F; Choi et al., 2008). cruited to adhesions independent of myosin II, suggesting that
The lag in vinculin accumulation relative to paxillin occurred they could serve as myosin II–sensitive scaffolds for recruit-
independently of fluorescent tag or order of image acquisition ment of subsequent proteins. To determine whether vinculin
(unpublished data). Together, these results suggest that the re- interactions with talin or paxillin were altered by myosin II
versible, myosin II–dependent recruitment of vinculin to adhe- activity, we assayed their associations by immunoprecipitation
sions occurs in a range of cell types in the physiologically (IP) from MEF lysates prepared in the presence or absence of

Myosin II–mediated recruitment of vinculin to adhesions by paxillin • Pasapera et al. 881

 Figure 5. Effects of myosin II inhibition on
the interactions between vinculin, paxillin, and
talin. IPs were performed from lysates of un-
treated control MEFs (control) or MEFs treated
with 20 µM blebbistatin (Blebb) followed by
immunoblot analysis. (A) IP with anti-vinculin
(Vcl) antibodies, immunoblot with anti-vinculin
and antipaxillin (Pxn; left), or anti–talin 1 (Tln;
right) antibodies. (B) IP with antipaxillin anti-
bodies and immunoblotting with antivinculin
antibodies. White lines indicate that interven-
ing lanes have been spliced out. (C) IP with
anti–talin 1 antibodies and immunoblotting with
antivinculin antibodies are shown.

blebbistatin. IP with antivinculin antibodies followed by prob- (Zaidel-Bar et al., 2007b), suggest that contractility enhances
ing for coprecipitation of talin or paxillin revealed association paxillin phosphorylation on Y31/118.
of both talin and paxillin with vinculin in controls (Fig. 5 A). To determine how myosin II–mediated effects on paxillin
Surprisingly, the coprecipitation of paxillin with antivinculin phosphorylation correlated with paxillin protein interactions,

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antibodies was markedly reduced in lysates of blebbistatin- we performed paxillin IPs from lysates prepared in the presence
treated cells. Similarly, although vinculin was present in IPs and absence of blebbistatin and probed for interacting proteins.
performed with antipaxillin antibodies from controls, vinculin First, probing paxillin IPs with anti-PY antibodies showed
in paxillin IPs from blebbistatin-treated cells was substantially that myosin II inhibition decreased the level of total paxillin
reduced (Fig. 5 B). In contrast, the level of talin in the vinculin PY (31%) by a similar amount as its reduction of pY31 and
IPs (or vinculin in talin IPs) was not changed by myosin II pY118 (Fig. 6 D) relative to controls. There was also a similar
inhibition (Fig. 5, A and C) in spite of the reduction in vinculin reduction to 12% and 66%, respectively, in the association of
but not of talin in adhesions of blebbistatin-treated cells, sug- either FAK or Crk with paxillin in IPs from blebbistatin-treated
gesting that these proteins form a complex in the cytoplasm. cells compared with controls (Fig. 6 D). In contrast, myosin II
IP with a nonrelated antibody (anti-EGFP) did not coprecipi- inhibition increased the paxillin–-Pix interaction to 159% of
tate vinculin (see Fig. 8 K), paxillin, or talin (not depicted). control (Fig. 6 D) and did not effect the adhesion localization
Therefore, in cell lysates, the paxillin–vinculin interaction of -Pix (Fig. S4). Together, these results show that myosin II–
is reduced by myosin II inhibition, whereas effects on talin– dependent modulation of paxillin interactions correlates with its
vinculin interaction are not detected. Thus, we focused further regulation of paxillin phosphorylation.
study on the role of paxillin in myosin II–mediated recruit- Integrin engagement promotes FAK recruitment to adhe-
ment of vinculin to adhesions. sions and activation by autophosphorylation on Y397 (pY397-
FAK; Burridge et al., 1992; Shi and Boettiger, 2003). Thus, we
Myosin II activity promotes FAK-mediated examined the effects of myosin II activity on FAK Y397 phos-
paxillin phosphorylation phorylation. Immunoblotting of lysates revealed, similarly to
Because paxillin phosphorylation is critical to cell migration ROCK inhibition in other cell types (Torsoni et al., 2005), that
and adhesion maturation (Petit et al., 2000; Zaidel-Bar et al., blebbistatin treatment or plating cells on compliant substrates
2003, 2007b; Webb et al., 2004; Nayal et al., 2006; Bertolucci substantially reduced pY397-FAK relative to control (Fig. 6 G
et al., 2008), we examined its myosin II dependence. We first and Fig. S1). Immunofluorescence showed a blebbistatin-
examined tyrosines 31 and 118 (pY31-paxillin and pY118- induced reduction in pY397-FAK that mirrored the reduction of
paxillin), whose phosphorylation mediates Crk interaction total FAK in adhesions (Fig. 2, B and C; Fig. 6, E and F; and
(Schaller and Parsons, 1995) yet also are close to the LD1 and Fig. S1). In contrast, the activity of Src, another kinase known
LD2 domains that are in part responsible for FAK and vinculin to act with FAK on paxillin Y31/118 (Turner, 2000), was not
binding (Brown et al., 1996). Immunoblotting cell lysates with affected by blebbistatin treatment, as assayed by immunoblot-
phosphospecific antibodies showed that blebbistatin reduced ting for dephosphorylation on Src Y527 (Fig. 6 G; Kmiecik and
the level of pY31- and pY118-paxillin to 45 and 24% of control Shalloway, 1987). Thus, full FAK adhesion association and
(Fig. 6 A). In contrast, phosphorylation at serine 273, which is activation requires myosin II contractility.
thought to mediate paxillin’s interaction with the Rac1 regula- Because phosphorylation of Y31/118 on paxillin is
tory complex of -Pix, Pak, and Pkl/Git1 (Turner et al., 1999; regulated by FAK (Bellis et al., 1995; Schaller and Parsons,
Nayal et al., 2006; Schmalzigaug et al., 2007), was slightly 1995), our results suggest that the blebbistatin-induced effects
enhanced to 127% of control by blebbistatin treatment (Fig. 6 D). on paxillin phosphorylation could be mediated through FAK.
Immunofluorescence and quantitative image analysis revealed Indeed, by immunoblotting cell lysates, we found that paxillin
a blebbistatin-induced slight reduction in pY31- and pY118- phosphorylation was at similar levels in lysates of cells treated
paxillin to 69% and 81% of that in control adhesions, respec- with blebbistatin, 10 µM of a small molecule FAK inhibitor (FAKi;
tively, similar to the blebbistatin-induced reduction in total PF271; Roberts et al., 2008), or both (Fig. 6 G and Fig. S5 A). In
paxillin in adhesions to 65–91% of controls (Fig. 2, B and C; addition, inhibition of Src by PP2 treatment did not further de-
and Fig. 6, B and C). These data, together with previous results crease the paxillin phosphorylation by blebbistatin but curiously

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Figure 6. Myosin II promotes tyrosine phosphorylation and interaction of paxillin, vinculin, and FAK. (A) Immunoblot of lysates of untreated MEF cells
(control) and cells treated with 20 µM blebbistatin (Blebb) using antibodies specific to paxillin (Pxn) or pY31, pY118, or pS273 paxillin (PxnY31, PxnY118,
and PxnS273). Numbers above each blot represent the mean of quantified Western blots ± 95% confidence interval (n = 4 experiments). (B) Immunolocal-
ization of paxillin (green) and pY31 or pY118 paxillin (red) in untreated control cells and cells treated with 20 µM blebbistatin. The third columns show
merged images Bar, 2 µm. (C) Effects of blebbistatin on adhesion localization of pY31 and pY118 paxillin in adhesions, expressed (also in F) as the ratio
of mean fluorescence intensities within segmented adhesions of 20 µM blebbistatin-treated relative to control cells. Antibody competition for similar epitopes
precluded comparison with effects of blebbistatin on total paxillin level in the same cells. Mean fluorescence ratio is shown above each plot. n = number of
blebbistatin-treated cells/number of control cells (also in F). (D) IP with antibodies to paxillin (left and top right) or Crk (bottom right) from lysates of untreated
control MEFs or MEFs treated with 20 µM blebbistatin followed by immunoblotting with antibodies to paxillin, PY epitopes (P-Tyr), pY31 paxillin, FAK, -Pix,
or Crk. Numbers above each blot represent the mean of quantified Western blots ± 95% confidence interval (n = 3 experiments). (E) Immunofluorescence
analysis of untreated control cells or cells treated with 20 µM blebbistatin using antibodies specific to pY397 FAK (red) or PY epitopes (green). Merged
images are shown in the third column. Bar, 2 µm. (F) Effects of 20 µM blebbistatin on adhesion localization of pY397 FAK and PY in adhesions quantified
from immunofluorescence images. *, P < 0.02. (G) Immunoblot of lysates of untreated MEF cells, cells treated with 20 µM blebbistatin, or a combination of
20 µM blebbistatin and either 10 µm PP2, 10 µm PF271 (FAKi), or all three using antibodies specific to paxillin, pY31, pY118 paxillin, FAK, pY397 FAK,
Src (c-Src), or pY527 Src (cSrcY527). Error bars indicate 95% confidence interval of the mean.

restored some blebbistatin-induced FAK inactivation (Fig. 6 G). likely express the closely related paxillin homologue Hic-5
Together, these data suggest that myosin II–dependent recruit- that also binds vinculin (Thomas et al., 1999). Therefore, we
ment and activation of FAK at adhesions mediates phosphor- performed knockdowns of paxillin and/or Hic5 by siRNA and
ylation of paxillin on Y31/118. assayed for the presence of vinculin in adhesions. 48 h after
transfection of MEFs with oligonucleotide pools, the levels
Paxillin Y31/118 phosphomimic is of paxillin and Hic-5 protein were reduced by 45% and 75%,
sufficient to promote paxillin–vinculin respectively, with even lower levels in the double knockdown
interaction and vinculin recruitment (75% for both paxillin and Hic5; Fig. 7 A). Immunofluores-
to adhesions cence revealed that cells with reduced levels of either paxillin,
We next sought to determine the requirement for paxillin and its Hic5, or both were small and poorly spread (Fig. 7 B). Double-
regulation by phosphorylation in the recruitment of vinculin to depleted cells displayed marked reduction in talin or PY-
adhesions. Unfortunately, because cells lacking talin have no containing adhesions except in cell regions containing residual
adhesions (Zhang et al., 2008), talin’s role in vinculin adhesion clustered paxillin/Hic5 (Fig. 7 C). Instead, talin in depleted
recruitment cannot be determined. In contrast, the requirement cells was evenly distributed in dim, diffraction-limited punctae,
for paxillin in vinculin adhesion recruitment is controversial PY staining was reduced, and interference reflection micros-
because vinculin is present in adhesions of embryonic stem copy confirmed the absence of localized areas of close contact
cells lacking the paxillin gene (Hagel et al., 2002), whereas with the substrate in paxillin/Hic5-depleted cells (unpublished
in smooth muscle cells, vinculin recruitment to the mem- data). Immunolocalization of vinculin revealed a similar
brane requires paxillin (Opazo Saez et al., 2004). However, localization as talin in the double-depleted cells (Fig. 7 C).
cells in these previous studies, as well our MEFs (Fig. 7 A), In addition, we were unable to find cells completely lacking

Myosin II–mediated recruitment of vinculin to adhesions by paxillin • Pasapera et al. 883

 We next sought to determine the requirement for paxillin
phosphorylation in vinculin recruitment to adhesions. To deter-
mine whether enhanced phosphorylation could induce vincu-
lin recruitment to adhesions in the absence of contractility, we
promoted tyrosine phosphorylation in blebbistatin-treated cells
by inhibition of tyrosine phosphatases with 100 µM sodium
orthovanadate (Na3VO4) or 10 µM phenylarsine oxide (unpub-
lished data). Phosphatase inhibition reversed the blebbistatin-
induced decrease in pY31-paxillin and pY397-FAK (Fig. 8 A).
In addition, IP and immunofluorescence analysis revealed that
Na3VO4 treatment rescued the blebbistatin-induced reduction
in vinculin–paxillin coprecipitation and vinculin localization in
adhesions, although it did not rescue other effects of bleb-
bistatin, including reduction in adhesion size and loss of central
adhesions (Fig. 8, B–D).
To determine whether paxillin phosphorylation was suffi­

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cient to promote vinculin recruitment to adhesions or whether
additional myosin II–dependent processes were required, we
overexpressed EGFP-tagged paxillin mutants that were either
unphosphorylatable (Y31/118F) or that mimicked phosphoryla-
tion charges (Y31/118E) at Y31/118 (Zaidel-Bar et al., 2007b)
and examined their effects on the blebbistatin sensitivity of
vinculin localization to adhesions. This revealed colocal-
ization of both EGFP-tagged paxillin mutants with vinculin
at adhesions of control cells (Fig. 8 E), with Y31/118F EGFP-
paxillin inducing larger adhesions and the Y31/118E mutant in-
ducing smaller adhesions (Zaidel-Bar et al., 2007b). Vinculin
localizing in Y31/118F EGFP-paxillin–containing adhesions
suggests either a parallel pathway for vinculin recruitment
(e.g., talin) or the effects of endogenous paxillin. Importantly,
expression of the Y31/118E EGFP-paxillin mutant rescued
the blebbistatin-induced reduction of vinculin but not zyxin
or -actinin in small, peripheral adhesions (compare Fig. 8,
E–H; with Fig. 2, B and C). FRAP analysis of mCherry vin-
culin in adhesions of cells expressing wild-type or Y31/118E
EGFP-paxillin revealed long t1/2’s of fluorescence recov-
ery (Fig. 8 I). In contrast, mCherry vinculin in adhesions of
blebbistatin-treated cells expressing Y31/118E EGFP-paxillin
displayed a significant reduction in FRAP t1/2 (Fig. 8 I), in-
dicating a labile vinculin–adhesion association. In contrast,
neither the Y31/118F EGFP-paxillin mutant nor vinculin
localized to PY-containing adhesions in blebbistatin-treated
Figure 7. Requirement for paxillin and Hic5 for adhesion formation. cells (Fig. 8 E). Corroborating the results of immunofluores-
(A) Immunoblot analysis of paxillin (Pxn; left), Hic5 (right), and tubulin (Tub;
cence, IP from lysates of blebbistatin-treated cells using anti-
both) in lysates of mock-transfected cells or cells transfected with siRNA
pools against paxillin, Hic5, or both. (B) Immunofluorescence localization GFP antibodies showed increased coprecipitation of vinculin
of Hic5 (red), paxillin (red), and vinculin (Vcl; green) in control cells or cells with EGFP-tagged Y31/118E paxillin compared with EGFP-
transfected with siRNA pools against paxillin or Hic5. (C) Immunofluores-
tagged wild-type or Y31/118F paxillin (Fig. 8 K). Expression
cence localization of paxillin (red) and vinculin, talin 1 (Tln), or PY epitopes
(P-Tyr; green) in control cells or cells transfected with siRNA pools against of both mutant and wild-type EGFP-tagged paxillins reduced
both paxillin and Hic5. (B and C) Merged images of boxed regions are the level of endogenous paxillin phosphorylation in both
magnified in third columns. Bars, 2 µm.
untreated and blebbistatin-treated cells by similar amounts,
possibly by competition between endogenous and expressed
paxillin or Hic5, and no changes in adhesions were seen after proteins for cellular kinases (Fig. 8 J). Expression of EGFP
treatment with scrambled siRNA controls (unpublished data). conjugates of any of the single paxillin tyrosine mutants (Y31E,
These results suggest that paxillin and/or Hic5 are necessary Y31F, Y118E, and Y118F) gave partial effects compared with
for formation of discrete substrate adhesions, which precludes the double mutants in both immunofluorescence and IP assays
our ability to determine the requirement for these proteins in (unpublished data). Together, these results suggest that paxil-
vinculin recruitment to adhesions. lin Y31/Y118 phosphorylation is sufficient for promoting the

 884 JCB • VOLUME 188 • NUMBER 6 • 2010

 Downloaded from http://rupress.org/jcb/article-pdf/188/6/877/1855751/jcb_200906012.pdf by guest on 31 March 2025
Figure 8. Paxillin Y31/118 phosphorylation is sufficient for promoting paxillin–vinculin interaction and labile vinculin recruitment to adhesions.
(A) Immunoblot analysis of lysates of untreated (control) and cells treated with 20 µM blebbistatin (Blebb) or with 20 µM blebbistatin and 100 µM Na3VO4
using antibodies specific to paxillin (Pxn), pY31 paxillin, pY397FAK, or FAK. (B) Comparison of cells treated with 20 µM blebbistatin or 20 µM bleb-
bistatin and 100 µM Na3VO4 and immunolabeled with antibodies to paxillin (red) and vinculin (Vcl; green). Bar, 2 µm. (C) Effects of Na3VO4 on vinculin
localization in adhesions of blebbistatin-treated cells, shown (also in G and H) as the ratio of mean fluorescence intensities within segmented adhesions
of 20 µM blebbistatin-treated cells relative to non–blebbistatin-treated cells in the presence and absence of additional Na3VO4. The mean fluorescence
ratio is shown above each plot. (D) Antipaxillin IPs from lysates of untreated control or cells treated with either 20 µM blebbistatin, 100 µM Na3VO4, and
20 µM blebbistatin or 100 µM Na3VO4 alone followed by analysis by PAGE and immunoblotting with antibodies to vinculin, paxillin, or PY epitopes.
(E–I) EGFP-conjugated paxillin (Pxn-GFP wt) or paxillin bearing mutations of tyrosines 31 and 118 to phenylalanines (Pxn Y31/118F) or glutamic acids
(Pxn Y31/118E) were expressed in MEFs and either treated with 20 µM blebbistatin or not. (E) Images of cells expressing EGFP-conjugated paxillin mutants
(green) and immunolocalization of vinculin (red) or PY epitopes (P-Tyr; red). Right columns show (also in F) merged, magnified images of the boxed regions
Bar, 2 µm. (F) Images of EGFP-conjugated Y31/118E paxillin (green) and immunolocalization of zyxin (Zyx; red) or -actinin (Actn; red) in cells treated
with 20 µM blebbistatin. (G) Effects of blebbistatin on adhesion localization of vinculin and paxillin in cells overexpressing wild type (WT) or mutant (Y31/
118E) paxillin-EGFP. (H) Effects of blebbistatin on adhesion localization of zyxin, -actinin, and paxillin in cells overexpressing Y31/118E paxillin-EGFP.
(I) FRAP analysis of mCherry-vinculin. mCherry-vinculin was expressed alone (control) or together with EGFP conjugates of wild-type or Y31/118E paxillin
in untreated cells or cells treated with 20 µM blebbistatin (+Blebb), and FRAP was performed of the mCherry vinculin fraction in adhesions. Half-times of
mCherry vinculin fluorescence recovery. Means are shown above each plot. n = number of adhesions. (J) Immunoblots of lysates of blebbistatin-treated (B)
or untreated (C) cells that had been mock transfected or transfected with the EGFP-paxillin mutants and probed with antibodies to paxillin, pY31 paxillin, or
tubulin. (K) Anti-GFP immunoprecipitates from MEFs expressing EGFP-tagged paxillins and either untreated (C) or treated with 20 µM blebbistatin (B) and
probed by immunoblotting with antibodies to vinculin and GFP. Quantification of blots is shown below. WB, Western blot. *, P < 0.02. Error bars indicate
95% confidence interval of the mean. (C, G, and H) n = number of blebbistatin-treated cells/number of control cells.

Myosin II–mediated recruitment of vinculin to adhesions by paxillin • Pasapera et al. 885

 paxillin–vinculin interaction and induction of a specific, labile results suggest that paxillin may be a critical protein, possibly
association of vinculin with immature adhesions independent in addition to talin, in the myosin II–dependent recruitment of
of myosin II activity. vinculin to adhesions in cells.
Because our results showed that FAK is critical for This study also reveals a currently unknown role for
Y31/118 paxillin phosphorylation, we finally sought to deter- paxillin phosphorylation in regulation of its interaction with
mine whether FAK activation is necessary or sufficient to vinculin in cells. We found that that in addition to vinculin,
induce the vinculin–paxillin interaction or vinculin recruit- paxillin’s interactions with FAK and Crk are promoted by
ment to adhesions independent of myosin II activity. Unfor- myosin II activity, protein interactions specific to regulation
tunately, this was difficult to assess because constitutively by paxillinY31/118 phosphorylation (Turner, 2000). This sug-
active FAK (1–100 FAK) does not target to adhesions and gests the interesting possibility that Crk signaling through
causes cell rounding (Schlaepfer and Hunter, 1996), whereas paxillin to regulate transcription may be mechanosensitive.
inhibition of FAK affects adhesion turnover (Ilić et al., 1995; We also found that myosin II activity promotes phosphory-
Webb et al., 2004). Indeed, treatment of cells with FAKi did lation of an activating tyrosine on FAK, which mediates the
not alter coprecipitation of vinculin with paxillin or recruit- myosin II–dependent phosphorylation of paxillin on Y31 and
ment of vinculin to paxillin-containing adhesions (Fig. S5). Y118. Although reduction of contractility did not completely
This is not surprising because inhibition of FAK or myosin II abrogate paxillin phosphorylation in adhesions, the phos-

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induces opposite adhesion phenotypes: FAK inhibition causes phorylation of these residues is sufficient to promote paxillin-
long-lived large adhesions (Fig. S5; Ilić et al., 1995), whereas mediated recruitment of vinculin because phosphatase inhibi-
myosin II inhibition promotes rapid turnover of tiny adhe- tion or overexpression of a phosphomimic promoted the paxillin–
sions (Fig. 1; Webb et al., 2004; Choi et al., 2008). Thus, vinculin interaction and vinculin recruitment to adhesions
although the myosin II–dependent vinculin–paxillin inter­ independent of myosin II activity. We suggest that in nascent,
action correlates with phosphorylation of FAK-regulated sites low contractility adhesions, paxillin phosphorylation may be
on paxillin, there are additional FAK-independent pathways below a threshold required to accumulate substantial vinculin,
to recruit vinculin to large and stable adhesions in the pres- with its labile binding to these sites as shown by FRAP (Fig. 8 I).
ence of cellular contractility. As actomyosin contractility begins to mature adhesions, the
increased FAK activity it promotes may amplify paxillin phos-
Discussion phorylation to the point that even low affinity vinculin binding
is sufficient for visible adhesion accumulation.
Our study has uncovered an important physiological role in Our results, together with those of others, support a
adhesion maturation for paxillin in its interaction with the speculative two-step “hand-off” model for myosin II–mediated
cytoskeletal adapter protein vinculin. To understand tension- vinculin adhesion recruitment and its role in adhesion matura-
mediated FA maturation, we sought proteins recruited to FAs in tion. We suggest that nascent adhesions form by myosin II–
a myosin II–dependent manner and examined the mechanism of independent talin recruitment to and activation of 1 inte­
their myosin II–sensitive FA association. Using pharmacologi- grin (Fig. 2; Tadokoro et al., 2003), which together with talin’s
cal inhibition of myosin II and ECM compliance to modulate actin-binding activity (Jones et al., 1989) may promote for-
cellular tension, we show that paxillin, talin, and 1 integrin mation of initial ECM–integrin–talin–actin linkages (Jiang
recruitment to adhesions is independent of myosin II activity, et al., 2003). Paxillin is also recruited to nascent, myosin II–
whereas adhesion association of FAK, zyxin, -actinin, and vin- independent adhesions by an unknown mechanism (Fig. 2;
culin is promoted by myosin II contraction. We focused on the Webb et al., 2004; Choi et al., 2008), and paxillin/Hic5 and
myosin II–dependent recruitment of vinculin to adhesions, show- talin are all required for formation of adhesion clusters (Fig. 7;
ing that it is reversible and occurs across a range of cell types and Zhang et al., 2008). Myosin II activity in an -actinin cross-
in the physiologically relevant contexts of myosin II–induced linked cytoskeleton (Choi et al., 2008) generates tension that
adhesion maturation during cell migration and ECM stiffness is transmitted to nascent adhesions. Tension across the ECM–
mechano­sensing. Although previous studies correlated vincu- integrin–talin–actin linkage promotes engagement of second-
lin accumulation to sites of applied tension on cells (Galbraith ary binding sites between 1 integrin and fibronectin to induce
et al., 2002; Möhl et al., 2009), we show the first demonstra- recruitment and activation of FAK (Friedland et al., 2009),
tion of myosin II–dependent vinculin adhesion association. By which is coupled to FAK/Src-dependent phosphorylation of
assessing the myosin II sensitivity of vinculin’s protein inter­ paxillin Y31/118 (Fig. 6; Zaidel-Bar et al., 2007b). pY31/118
actions in cell lysates, we show that the vinculin–paxillin paxillin, with its newly revealed vinculin-binding site (Fig. 5),
interaction is promoted by signaling induced by myosin II may cycle between adhesion-bound and cytosolic fractions,
contractility, whereas we did not detect myosin II–dependent inducing labile vinculin recruitment to adhesions (Fig. 8).
changes in the vinculin–talin interaction. It was recently shown Subsequent to adhesion recruitment, activation of vinculin’s
that stretching talin promotes increased vinculin binding in vitro actin-binding activity by simultaneous proximity to talin, actin,
(del Rio et al., 2009). The IPs used in our study bias detection and acidic phospholipids (Chen et al., 2006) induces activation
of myosin-dependent changes that are preserved in cell lysates, and hand off of vinculin from labile paxillin binding to these
such as covalent phosphorylation of paxillin, whereas cell lysis other partners, enhancing vinculin’s binding stability to adhe-
would clearly disrupt stretch activation of talin. However, our sions (Figs. 3 and 8) and reinforcing the cytoskeleton–ECM

 886 JCB • VOLUME 188 • NUMBER 6 • 2010

linkage. The now matured adhesion can transmit stronger and 1:20,000, respectively) for 1 h. Blots were washed three times with
forces (Galbraith et al., 2002) to promote adhesion turnover TBS-T. Protein bands were visualized using an ECL detection system (Milli-
pore) or fluorescent-labeled antibodies and imaging on a gel imaging
in cell migration (Webb et al., 2004; Zaidel-Bar et al., 2007b). system (Odyssey; LI-COR Biosciences). For analysis of ECL, digital images
High forces across the cytoskeleton–integrin link may induce a of Western blot bands were quantified with MetaMorph software (MDS
parallel pathway for vinculin recruitment by stretch activation Analytical Technologies) after performing local background subtraction
around bands of interest.
of vinculin-binding sites in talin (del Rio et al., 2009).
Questions remain about the paxillin-mediated recruitment IPs
of vinculin to adhesions. Because vinculin does not contain MEF cells were plated in 15-cm tissue culture plates precoated with 5 µg/ml
fibronectin and supplemented with 10% FBS for 16 h. Cells were treated
PY-binding SH2 domains, it is not clear how paxillin phosphory­ with myosin II inhibitors (20 µM blebbistatin) for 1 h. For EGFP-paxillin
lation induces its interaction with vinculin or whether this inter- mutants, EGFP-positive cells were cell sorted and collected by flow cytom-
action is direct. pY31/Y118 could induce a conformational etry and replated in dishes precoated with 5 µg/ml fibronectin after trans-
fection. Cells were harvested in lysis buffer, clarified by centrifugation,
change in paxillin that unmasks adjacent LD1 and LD2 domains and protein in supernatants was quantified by the Bradford assay. For IP,
involved in vinculin binding (Bertolucci et al., 2008). Addition- supernatants (1.0 mg total protein for untransfected cells and 500 µg for
ally, the alternate pathways for vinculin adhesion recruitment EGFP-transfected cells) were precleared with anti–mouse or anti–rabbit
IgG IP beads (TrueBlot; eBioscience), preclearing beads were pelleted,
that occur when FAK is inhibited (Fig. S5) remain unknown. and supernatants were incubated with 1 µg primary antibody and rotated
Future studies may help to clarify these and other questions. overnight at 4°C. The following day, the solution was incubated with

Downloaded from http://rupress.org/jcb/article-pdf/188/6/877/1855751/jcb_200906012.pdf by guest on 31 March 2025

30 µl protein A sepharose beads (TrueBlot) with rotation at 4° for 1 h.
Beads were washed three times with lysis buffer and resuspended in 30 µl
Materials and methods 2× Laemmli sample buffer. Samples were analyzed by Western blotting
using the appropriate antibodies.
Cell culture, transfection, and reagents
MEFs were obtained from American Type Culture Collection and main- Polyacrylamide substrates
tained in DME supplemented with 10% FBS (Invitrogen) at 5% CO2. For Flexible polyacrylamide substrates were generated as previously described
experiments, cells were plated on 22 × 22–mm #1.5 coverslips that had (Pelham and Wang, 1997). In brief, 22-mm coverslips were activated by
been coated with 5 µg/ml fibronectin (EMD) in PBS for 2 h at 37°C. Cells treatment with 50% 3-aminopropyltrimethyloxysilane and 0.5% glutaralde-
were plated at a low confluency (30–40%) for 16 h before experimental hyde, and each treatment was followed by extensive double-distilled H2O
manipulations. Cells were transfected by nucleofector (Lonza) by using washing. Activated coverslips were inverted on a freshly mixed solution
MEF1 solution and program T20. The following pharmacological inhibitors of 0.04% bis/7.5% acrylamide to give an adhered gel with a stiffness of
were used: 20 µM blebbistatin (Toronto Research Chemicals), 10 µM 1.0 kPa. 8 µl of acrylamide solution was sufficient to obtain a 15–20-µm-
Y-27632 ROCK inhibitor (EMD), 10 µM Src inhibitor PP2 (EMD), 100 µM thick gel. Coverslips with attached gel substrates were washed in double-
FAKi PF-271 (Roberts et al., 2008), 100 µM Na3VO4 (Sigma-Aldrich), and distilled H2O and spun dry using a custom-made coverslip spinner. To bind
10 µM phenylarsine oxide (Sigma-Aldrich). The following antibodies were fibronectin, gel substrates were activated by 2 mg/ml sulfo-SANPAH with
used: rabbit anti–pY118-paxillin, rabbit anti–pY31-paxillin, rabbit anti– two 8-min UV exposures one inch from two 10-W, 254-nm UV bulbs (UVP).
pY397-FAK, mouse anti-FAK, and rabbit anti–pY273-paxillin (Invitrogen); Activated, gel-bound coverslips were coated with 5 µg/ml fibronectin
mouse anti-Hic5, mouse antipaxillin, and mouse anti-Crk (BD); rabbit anti- (EMD) incubated for 2 h at 37°C and washed three times with PBS.
paxillin (Santa Cruz Biotechnology, Inc.); mouse antitalin clone 84d, mouse
antivinculin clone 4505, and mouse anti–-actinin (Sigma-Aldrich); anti-PY Immunofluorescence
clone 4G10 (Millipore); rabbit anti–Src-pY527 (Signal Transduction); Coverslips with bound cells were fixed/permeabilized for 1 min at 37°C
mouse anti-Src (provided by J. Brugge, Harvard University, Cambridge, in 0.01% Triton X-100, 0.25% paraformaldehyde (Electron Microscopy
MA); rabbit antizyxin (provided by M. Beckerle, Huntsman Cancer Insti- Science), and 1 mg/ml phalloidin in cytoskeleton buffer (CB; 10 mM
tute, Salt Lake City, UT); mouse anti–-Pix (Millipore); and rabbit anti-GFP MES, 3 mM MgCl2, 138 mM KCl, and 2 mM EGTA) and fixed in 3%
(Abcam). The following expression constructs were used: sources of EGFP paraformaldehyde in CB for 20 min at 37°C. After fixation, cells were
conjugates of -actinin, talin, paxillin, FAK, and zyxin were described pre- permeabilized with 0.25% Triton X-100 in CB. Free aldehydes were
viously (Hu et al., 2007). EGFP-paxillin was obtained from R. Horwitz reacted with 0.1 M glycine for 5 min, and cells were washed three times
(University of Virginia, Charlottesville, VA) and mApple-paxillin, EGFP for 10 min in TBS and blocked in blocking solution (2% BSA IgG free and
vinculin, and mCherry vinculin were provided by M. Davidson (Florida protease free; Jackson ImmunoResearch Laboratories, Inc.) in TBS-T con-
State University, Tallahassee, FL). The EGFP conjugates of paxillin and pax- taining Alexa Fluor 488 phalloidin (1:400; Invitrogen) for at least 1 h.
illin mutants were based on the original sequence for avian paxillin-EGFP Coverslips were incubated with primary antibodies diluted in blocking
(Laukaitis et al., 2001) but were generated by synthesis (Blue Heron solution overnight in a humid chamber at 4°C. After primary antibody in-
Biotechnology) and included mutations rendering dead an internal transla- cubation, cells were washed four times for 10 min in TBS-T and incubated
tion site of avian paxillin as described previously (Tumbarello et al., 2005; with fluorophore-conjugated secondary antibodies (Jackson Immuno­
Schneider et al., 2009). Research Laboratories, Inc.) diluted 1:250 in blocking solution for 1 h,
washed again, and mounted on a slide in mounting media (Dako) and
Western blot analysis sealed with nail polish.
Whole cell extracts from MEF cells were prepared in lysis buffer containing
50 mM Tris, pH 8.0, 150 mM NaCl, 5 mM EDTA, 5% glycerol, 1% Triton Microscopy and image analysis
X-100, 25 mM NaF, and 2 mM NaVO4 supplemented with 1× protease Fixed and immunolabeled cells were imaged on an inverted microscope
inhibitor cocktail (Roche) and phosphatase inhibitor cocktails I and II system (Eclipse TE-300; Nikon; Wittmann et al., 2003) with a cooled charge-
(Sigma-Aldrich). Cells were freeze thawed twice in liquid nitrogen and coupled device (CCD; Orca II; Hamamatsu Photonics) using a 60× 1.4 NA
clarified by centrifugation at 16,000 g. Proteins from supernatants were Plan Apo PH objective lens (Nikon) for cells plated on glass or a 60× 1.2 NA
quantified by the Bradford method. 10 µg of proteins were mixed with an Plan Apo violet-corrected objective lens (WI; Nikon) for cells plated on poly-
equal volume of 2× Laemmli sample buffer and separated by SDS-PAGE. acrylamide substrates. Time-lapse imaging of cell migration was performed
After electrophoresis, proteins were electrotransferred to an immobilon-P at 37°C on the same microscope using a 10× 0.5 NA PH Plan objective lens
membrane. For protein detection, membranes were blocked for 1 h at (Nikon) and 0.52 NA condenser (LWD; Nikon), and images were captured
room temperature with 5% nonfat dry milk (wt/vol) or 3% BSA for tyrosine at 5-min intervals for 24 h. Dual-color time-lapse TIRF microscopy of EGFP
phosphorylation blots in TBS-T buffer (20 mM Tris, pH 7.6, 137 mM NaCl2, and mCherry- or mApple-tagged proteins in living MEFs was performed at
and 0.1% Tween-20 [vol/vol]) and incubated overnight at 4°C with the 37°C in DME without Phenol red and supplemented with 10% FBS, 25 mM
indicated antibodies. After primary antibody incubation, blots were Hepes, and 10 U/ml oxyrase using a 100× 1.49 NA Plan objective lens
washed three times with TBS-T (10 min each) and incubated with appropri- (Nikon; Shin et al., 2010) and an inverted microscope system (TE2000E2;
ate HRP-conjugated or fluorescent-labeled secondary antibodies (1:5,000 Nikon) with an evanescent field depth of 150 nm.

Myosin II–mediated recruitment of vinculin to adhesions by paxillin • Pasapera et al. 887

 For EGFP images, the 488-nm laser was used, and for mCherry 1 integrin, zyxin, vinculin, -actinin, pY397 FAK, pY118 paxillin, or
or mApple, the 561-nm laser was used. Pairs of EGFP and mCherry or pY31 paxillin) of the same cell. The mean intensity of the fluorescence in
mApple images were captured in rapid succession at 10-s intervals using FAs was background subtracted and averaged over several cells. 95% con-
a CCD (HQ2; Hamamatsu Photonics) operated in the 14-bit readout fidence intervals were calculated for paxillin (or PY) and the FA proteins in
mode. FRAP of EGFP-tagged FA proteins was performed using a 100× control and blebbistatin-treated cells. The mean paxillin (or PY) or FA pro-
1.49 NA Plan Apo objective lens on an inverted microscope system tein intensity of the adhesions in blebbistatin-treated cells was divided by
(TE2000E2). 488-nm and 561-nm illumination provided by a 20-mW the mean intensity of the same protein in adhesions in control cells to gener-
argon laser and a 10-mW solid-state laser, respectively (CVI Melles Griot), ate the fractional FA immunofluorescence signal change after blebbistatin
was fiber optically coupled (Oz Optics) to a dual-port photostimulation treatment as reported in the figures. For vanadate treatment experiments,
epi-illuminator (Nikon), which centered and expanded the laser beams mean FA protein intensity in adhesions in the blebbistatin-treated cells was
on the objective lens back aperture to form a diffraction-limited spot at the divided by the mean FA protein intensity in cells treated with both bleb-
specimen plane. The position of the specimen was adjusted using a robotic bistatin and vanadate. Statistical significance (P < 0.02) was measured by
stage (Applied Scientific Instruments) to center the spot onto a single fluo- a two-tailed Student’s t test between control and blebbistatin-treated cells.
rescent FA. An electronic shutter (Sutter Instrument Co.) was used to limit The metric that was tested for significance was the background-subtracted
exposure of the specimen to the laser beam to the time needed for photo- intensity of the FA component divided by the background-subtracted
bleaching. For imaging, mercury arc illumination coupled to the epi-illuminator intensity of paxillin (or PY).
was electronically shuttered (Sutter Instrument Co.) and filtered (Chroma
Technology Corp.) to provide excitation specific for EGFP or mCherry. Online supplemental material
Images were acquired at 1–30-s intervals before and after photobleaching Fig. S1 shows that substrate compliance causes reduction of vinculin and
using an EM-CCD (512B; Photometrics) operated in the 14-bit mode using FAK in adhesions and reduction of FAK phosphorylation. Fig. S2 shows

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EM gain. Image frequency was adjusted depending on the fluorescence FRAP analysis of EGFP-tagged adhesion proteins. Fig. S3 shows that
photobleaching recovery rate of the fluorescent-tagged adhesion protein myosin II–mediated vinculin recruitment to adhesions is cell type indepen-
being imaged. dent. Fig. S4 shows that -Pix localization to adhesions is myosin II in-
To quantify cell migration rate, time-lapse microscopy was per- dependent. Fig. S5 shows that pharmacological inhibition of FAK does
formed at 37°C using an inverted microscope (Eclipse TE-300). Phase- not inhibit vinculin binding to paxillin or localization to adhesions. Video 1
contrast images were acquired every 5 min using a 10× 0.25 NA Plan shows time-lapse TIRF images of EGFP-paxillin and mCherry-vinculin in
objective lens and a 0.52 NA condenser (LWD). Positions of the cell an adhesion forming and growing at the leading edge of a migrating
nuclei were manually tracked over time using MetaMorph software. Instan- MEF cell. Online supplemental material is available at http://www.jcb
taneous cell velocities were calculated as the displacement (micrometers) .org/cgi/content/full/jcb.200906012/DC1.
between consecutive frames divided by the elapsed time. Area of individual
adhesions was measured from manually thresholded images of immuno- We thank Joan Brugge, Mike Davidson, and Mary Beckerle for reagents, the
localized paxillin or PY as described previously (Schneider et al., 2009). NHLBI Flow Cytometry Core Facility, William Shin, Sergei Plotnikov, and mem-
In brief, adhesion area was quantified by thresholding the paxillin or PY bers of the Waterman laboratory. Experiments in Fig. 3 were first performed
immunostaining image by eye to include only adhesion areas using Meta- in conjunction with Michelle Knowles in the Physiology Course at the Marine
Morph software. The normalized fluorescence in adhesions was calculated Biological Laboratory (Woods Hole, MA).
by dividing the background-subtracted mean fluorescence in the adhesion This work was supported by NHLBI (C.M. Waterman and A.M.
area by the background-subtracted mean fluorescence in the cytosol. Pasapera; and grant HL093156 to D.D. Schlaepfer) and the Burroughs
FRAP experiments were analyzed as follows. The intensity recovery of Wellcome Fund (E. Rericha).
the bleached region was extracted from the images, corrected for photo-
bleaching, and analysis of FRAP curves was performed as described previ- Submitted: 2 June 2009
ously (Phair et al., 2004). In brief, to extract the characteristic recovery time Accepted: 19 February 2010
for the assayed fluorescent-tagged proteins, normalized and photobleaching-
corrected curves were fit to single exponential functions. In the vinculin
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