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Focal Adhesion Kinase Stabilizes the Cytoskeleton
Ben Fabry, Anna H. Klemm, Sandra Kienle, Tilman E. Scha
¨ffer, and Wolfgang H. Goldmann*
Department of Physics, Friedrich-Alexander-University Erlangen-Nuremberg, Erlangen, Germany
ABSTRACT Focal adhesion kinase (FAK) is a central focal adhesion protein that promotes focal adhesion turnover, but the
role of FAK for cell mechanical stability is unknown. We measured the mechanical properties of wild-type (FAKwt), FAK-deficient
(FAK / ), FAK-silenced (siFAK), and siControl mouse embryonic fibroblasts by magnetic tweezer, atomic force microscopy,
traction microscopy, and nanoscale particle tracking microrheology. FAK-deficient cells showed lower cell stiffness, reduced
adhesion strength, and increased cytoskeletal dynamics compared to wild-type cells. These observations imply a reduced
stability of the cytoskeleton in FAK-deficient cells. We attribute the reduced cytoskeletal stability to rho-kinase activation in
FAK-deficient cells that suppresses the formation of ordered stress fiber bundles, enhances cortical actin distribution, and
reduces cell spreading. In agreement with this interpretation is that cell stiffness and cytoskeletal stability in FAK / cells is
partially restored to wild-type level after rho-kinase inhibition with Y27632.
INTRODUCTION
Cells that adhere to an extracellular matrix form an architec-
turally highly complex cytoskeleton. A major component of
the cytoskeleton is force-generating actomyosin stress fibers
that connect to focal adhesions (FAs). The interplay of
actomyosin stress fibers and FAs defines to a large part
the mechanical behavior of cells, such as their motility,
morphology, and contractility (1 6).
Focal adhesion kinase (FAK) is a central protein of FAs
and is known to regulate several cytoskeletal and other focal
adhesion proteins. FAK interacts with integrins (7), paxillin
(8), p130Cas (9), a-actinin (10), and other proteins that link
FAs to the actin cytoskeleton (4). The molecular details of
these interactions have not been fully characterized, but it
is generally agreed that FAK promotes a high FA turnover
through a rho-kinase (ROCK)-dependent pathway (11 14).
FAK knockout cells show high rhoA-kinase and ROCK
activity (12,13,15). Because ROCK inactivates myosin light
chain phosphatase, phosphorylates myosin light chain, and
therefore promotes actomyosin contractility, it has been
hypothesized that contractile tension in the cytoskeleton is
altered in FAK-deficient cells (10,14 18). Direct measure-
ments of traction forces, however, showed no difference
between wild-type and FAK-deficient fibroblasts (19).
With regard to the mechanical stability of cells, the role of
FAK is similarly unclear. Because FAK promotes high FA
turnover and high cell motility (12,13,19), it could be
expected that the cytoskeleton in FAK-deficient cells is
less dynamic and more rigid compared to FAK-expressing
cells. However, FAK-deficient cells show a rounded cell
morphology with a smaller spreading area, pronounced
cortical distribution of the actin cytoskeleton, and a loss of
actomyosin stress fibers (12,13), all of which are signs
of reduced cell stiffness. Treatment of FAK-deficient cells
with the ROCK-inhibitor Y27632 leads to a larger spreading
area and a reformation of stress fibers (13), which is
puzzling as this inhibitor induces the complete opposite
behavior in wild-type fibroblasts (20).
The aim of this work was first to directly measure the
impact of FAK on cell mechanics in mouse embryonic
fibroblasts (MEFs), and second to characterize how rho-
kinase contributes to the mechanical changes in wild-type
and FAK-deficient cells. Our results show that FAK is
important for maintaining cell rigidity (stiffness) through
promoting a static and highly aligned contractile cytoskel-
eton. FAK knockout leads to a pronounced speedup of cyto-
skeletal dynamics, which is independent of any decreased
FA turnover in these cells. We hypothesize that the effects
of FAK on cytoskeletal dynamics and organization are, to
a large extent, mediated through a compensatory activation
of ROCK in FAK knockout cells. In support of this hypoth-
esis, we find that treatment with the ROCK-inhibitor
Y27632 has opposite effects on cell rigidity and cytoskeletal
dynamics in FAK wild-type versus knockout cells.
MATERIALS AND METHODS
Cells and cell culture
FAK deficient (FAK / ) and FAK wild type (FAKwt) mouse embryonic
fibroblasts (MEFs) were purchased from American Type Culture Collection
(FAK / , cat. No. CRL 2644; FAKwt, cat. No. CRL 2645; ATCC,
Manassas, VA). FAKwt and FAK / cells from ATCC are reported to
carry a p53 knockout mutation (11). Moreover, FAK / cells overexpress
PYK2, a homologous protein of FAK (21). PYK2 overexpression has no
effect on cell mechanics (22), but p53 overexpression may have a small
effect (23). To rule out any of these secondary effects, we performed siRNA
downregulation experiments on differently derived MEF cells (obtained
from Dr. W. H. Ziegler, University of Leipzig, Leipzig, Germany) with
normal PYK2 and p53 expression levels. All cell lines were maintained
in low glucose (1 g/L) Dulbecco’s modified Eagle’s medium supplemented
with 10% fetal calf serum, 2 mM L glutamine, and 100 U/ml penicillin
streptomycin (i.e., Dulbecco’s modified Eagle’s medium complete
medium). This medium was also used during measurements except where
Submitted June 9, 2011, and accepted for publication September 23, 2011.
*Correspondence: wgoldmann@biomed.uni erlangen.de
Editor: Douglas Nyle Robinson.
Ó2011 by the Biophysical Society
0006 3495/11/11/2131/8 $2.00 doi: 10.1016/j.bpj.2011.09.043
Biophysical Journal Volume 101 November 2011 2131 2138 2131
stated otherwise. siRNA against FAK (siFAK) was targeted against both
splice variants of murine FAK (gene accession numbers NM 007982.2
and NM 001130409.1). The sequence was sense: R(GGG ACA UUG
CUG CUC GGA A)dTdT; antisense: R(UUC CGA GCA GCA AUG
UCC C)dTdG. siRNA was 30AlexaFluor546 labeled to assess transfection
efficiency. As a control (siControl), we used Alexa Fluor546 labeled
Allstar siRNA (Qiagen, Hilden, Germany), a nonsilencing siRNA with no
homology to any known mammalian gene. Transfection of 100,000
MEFs was performed in 35 mm wells using 6 ml HiPerFect transfection
reagent (Qiagen) with 20 nM siRNA.
Magnetic tweezer microrheology
Magnetic tweezer rheology exerts a mechanical shear stress to the cell by
applying lateral forces to magnetic beads that are connected to the cytoskel
eton through adhesion contacts on the apical cell surface (24). This
technique reports the passive mechanical properties of the cytoskeleton,
such as the elastic modulus and its time (or frequency) dependency. In brief,
superparamagnetic, epoxylated beads (4.5 mm, Dynabeads; Invitrogen,
Carlsbad, CA) were coated with fibronectin (5 mg per 1 10
7
beads; Roche,
Pleasanton, CA) at 4C for 24 h. Before measurements, the beads were
sonicated, 2 10
5
beads were added to ~10
5
subconfluent cells in a
35 mm dish, and cells were incubated with the beads for 30 min at 5%
CO
2
and 37C. Thereafter, the medium was exchanged with fresh, pre
warmed medium to remove unbound beads. Measurements were performed
on a heated inverted microscope stage at 40magnification (NA 0.6)
without CO
2
. The measuring time was limited to 30 min per dish. A sole
noid with a sharp tipped steel needle core was used to generate a defined
force on the bead.
When a step force fwas applied to a cell bound bead, it moved with
a displacement d(t) toward the tweezer needle tip (Fig. 1). Following Kasza
et al. (25) and Kollmannsberger et al. (26), we estimate the typical strain
g(t)asd(t) divided by the bead radius r, and the typical stress sas the
applied force divided by the bead cross sectional area, r
2
p. The creep
compliance J(t) in units of Pa
1
is then given by g(t)/sand is fit to the equa
tion J(t)J
0
(t/t
0
)
b
with time normalized to t
0
1 s. The prefactor, J
0
, and
the power law exponent, b, were both force dependent. The value J
0
is the
creep compliance at t
0
1 s and corresponds, apart from a negligible
correction factor (the Gamma function, G,at1 b) to the inverse magnitude
of the cell’s dynamic shear modulus evaluated at a radian frequency u
0
1
rad/s (27,28),
G0ðu0ÞþiG00ðu0Þ
¼1
J0
Gð1bÞ:
The power law exponent breflects the dynamics of the force bearing
structures of the cell that are connected to the bead (27). A power law expo
nent of b0 is indicative of a purely elastic solid, and b1 is indicative
of a purely viscous fluid. In cells, the power law exponent usually falls in
the range between 0.1 and 0.5, whereby higher values have been linked
to a higher turnover rate of cytoskeletal structures (27).
Atomic force microscopy
Atomic force microscopy (AFM) is used as an alternative method to
measure cell stiffness. A nonfunctionalized sharp tip that is in contact
with the cell for <1 s serves as a probe. The measurements are therefore
not influenced by focal adhesion formation between the probe and the
cell. Cells were seeded on fibronectin coated (50 mg/ml) cell culture dishes
(Nalge Nunc, Rochester, NY) in CO
2
independent medium (Leibovitz L 15
medium, with L Glutamine; Gibco, Invitrogen, Carlsbad, CA) for 12 h
before and during measurements. Measurements were performed on a
MFP 3D Stand Alone AFM (Asylum Research, Goleta, CA) as described
previously (22). The spring constants of the cantilevers (Bio Lever;
Olympus, Melville, NY) were determined before cell measurements using
the thermal noise method (29) and were in the range of 5.9 7.7 pN/nm.
The force mapping mode with 260 pN maximum indentation force (inden
tation depth between 30 and 100 nm) was used to measure cell stiffness
and sample height (Fig. 2 A). Force versus z piezo extension curves
(Fig. 2 C) were acquired on different positions on the sample surface. The
local shear moduli (Fig. 2 B) were obtained by fitting the extended Hertz
model to each force versus z piezo extension curve in the force map (30).
In the extended Hertz model for a conical indenter, the relationship between
the applied force Fand the resulting sample indentation dis given by
F¼2
p
E
1n2d2tanðaÞ;
where Eis the Young’s modulus, nis the Poisson’s ratio, and ais the
opening half angle of the conical cantilever tip (30,31). We assumed
an incompressible sample with a Poisson’s ratio of n0.5. Therefore,
the shear modulus Gis related to the Young’s modulus Eby G
E/[2$(1þn)] E/3. A region of 80 80 mm was scanned to obtain
10 10 or 20 20 force curves. To reduce the influence of the underlying
substrate, only regions of the cell that were at least 60% of the maximum
cell height were analyzed.
Nanoscale particle tracking
This method is used to quantify internal remodeling processes of the
cytoskeleton. Fibronectin coated fluorescent beads (4.5 mm) are bound to
confluent cells via integrins (32,33). The spontaneous movement of the
beads was tracked for 5 min. The mean squared displacement (MSD) of
bead movements followed a power law with time (t) according to
MSD D$(t/t
0
)
b
(Fig. 3). The value t
0
was set to 1 s, Dreflects an apparent
diffusivity, equivalent to the square of the distance traveled during a 1 s
interval, and the power law exponent bis a measure of the persistence,
with b1 for randomly moving beads and b2 for directed, ballistic
motion along a straight path (32).
Immunofluorescence of focal adhesions and the actin
cytoskeleton
A quantity of 10,000 50,000 cells was seeded on 5 mg/ml fibronectin
coated glass slides and incubated overnight at 37C and 5% CO
2
. Adherent
FIGURE 1 Magnetic tweezer microrheology. Bead displacement
(geometric mean from >74 cells) versus time during force steps with
increasing force magnitude. Each step lasted 1 s. Numbers above the curve
indicate the lateral pulling force (in units of nN). After 10 s, the force was
reduced to zero. The differential cell stiffness (see Fig. 4 A) and the
exponent of the creep modulus (see Fig. 4 B) was computed as described
in Kollmannsberger et al. (26).
Biophysical Journal 101(9) 2131 2138
2132 Fabry et al.
cells were fixed for 20 min with 3% paraformaldehyde and lysed for 5 min
with 0.2% Triton X 100. Cells were then blocked for 1 h in 0.5% BSA and
PBS at room temperature. Primary antibodies (1:10
6
vinculin/hVIN 1;
Sigma, St. Louis, MO) and secondary antibodies (ml:200, FITC coupled
anti mouse IgG; Jackson ImmunoResearch, West Grove, PA) were both
diluted in 0.5% BSA and PBS at given ratios and incubated for 1 h at
room temperature each. Alexa Fluor546 Phalloidin (Molecular Probes,
Eugene, OR) was added simultaneously with the second antibody for actin
staining. Samples were mounted with Mowiol (Sigma) solution. Micros
copy was carried out on a DMI6000 microscope with a 63/1.3 NA
objective (Leica Microsystems, Wetzlar, Germany). Images were acquired
with a charge coupled device camera (ORCA ER; Hamamatsu, Hamamatsu
City, Japan).
Traction microscopy
This technique measures the forces that cells exert on their surroundings by
observing the displacements of beads embedded in a flexible gel substrate
on which the cells are cultured (32,34). Traction measurements were per
formed with 6.1% acrylamide/bisacrylamide (19:1) gels with 0.5 mm green
fluorescent beads. The Young’s modulus of the gels was 12.8 50.8 kPa
as measured from the linear extension of a cylinder of gel (16 mm diameter,
50 mm length) under force. Gels were coated with 50 mg bovine collagen G
(Biochrom AG, Berlin, Germany) diluted in 50 mM HEPES overnight at
4C. 10
4
cells were seeded on the gels and incubated under normal growth
conditions. During the measurements, the cells were maintained at 37C
and 5% CO
2
in a humidified atmosphere. Cell tractions were computed
from an unconstrained deconvolution of the gel surface displacement field
(32,35) measured before and after cells were detached from the substrate
with a cocktail of 80 mM cytochalasin D and 0.25% trypsin.
Statistical evaluation
FAK knockout and FAK silencing experiments were performed on MEF
cells derived from two different cell isolations. Therefore, for statistical
evaluation, we only compared FAKwt with FAK / cells, and siControl
cells with siFAK cells. Statistical significant differences were calculated
using a Student’s unpaired ttest, assuming unequal variances. Results
were considered to be significant and marked with an asterisk for p<
0.05. All data are expressed as arithmetic mean 5standard error of the
mean, except for bead detachment experiments (expressed as cumulative
probability) and for data that show a log normal distribution, namely cell
stiffness measured with magnetic tweezers and AFM, and apparent diffu
sivity (32,36). These data are expressed as geometric mean 5geometric
standard error of the mean. Number of measurements is between 46 and
184 cells for traction measurements, between 85 and 144 cells for adhesion
strength measurements, between 611 and 1031 beads for nanoscale particle
tracking experiments, between 10 and 30 cells for AFM measurements, and
between 74 and 129 cells for magnetic tweezer measurements.
RESULTS AND DISCUSSION
MEF cells, regardless of FAK expression levels, behave me-
chanically similar to other cells: they stiffen when probed
ABC
FIGURE 2 AFM force mapping. (A) Height.
Scan range: 80 mm80 mm. Color bar range
(black to white): 0 5 mm. (B) Corresponding shear
modulus. Grayscale range (black to white):
0 20 kPa. (C) Force versus z piezo extension
data at the position indicated (red cross) in panels
Aand B. A fit of the Hertz model to the data gives
the local shear modulus at this position.
Biophysical Journal 101(9) 2131 2138
FAK Stabilizes the Cytoskeleton 2133
at high forces (Fig. 4 A). Measurements with magnetic twee-
zers show that cell stiffness was approximately twofold
higher in FAKwt versus FAK/cells. Those differences
were significant (p<0.05) at all force levels. A similar trend
was observed in siControl versus siFAK knockdown cells,
although the differences were less pronounced and were
not consistently significant at all force levels.
To test whether these differences are attributable to the
mechanical behavior of the cytoskeleton, as opposed to
differences in FAK-mediated adhesion properties of the
fibronectin-coated magnetic beads, we performed stiffness
measurements using an atomic force microscope (AFM)
(Fig. 4 D). Because the cantilever tip of the AFM was
not functionalized and was in contact with the cell surface
for <1 s, these measurements are not influenced by specific
adhesion between the cell and the probe. The AFM
measurements confirm our results obtained with magnetic
tweezers, with a notable exception that the differences
between FAK-expressing and FAK-deficient cells were
even larger than those measured by magnetic tweezers.
The shear modulus magnitude from AFM measurements
approximately matched the values obtained with magnetic
tweezers (Fig. 4,Aand D). Differences are attributable to
the uncertainty in the geometric factor that is needed to
convert forces to stress, and displacements to strain
(27,37). Moreover, the shear modulus magnitude from
magnetic tweezer measurements reflects the combined
elastic and dissipative cell properties at a timescale of 1 s,
whereas the fit of the Hertz-model to the AFM force-inden-
tation data was performed under the assumptions of a purely
elastic response and a Poisson’s ratio of 0.5. These assump-
tions are reasonable because the cell’s elastic properties
dominate over dissipative properties, the timescale of the
AFM indentation measurements was also approximately
1 s, and the Poisson’s ratio has only a moderate effect on
the result.
In the magnetic tweezer experiments, the creep compli-
ance increased with time according to a power-law with
exponent bthat was significantly increased by ~50% in
FAK /cells compared to FAKwt cells (Fig. 4 B). A
similar trend was also seen in siControl versus siFAK cells,
although the differences were not always significant at all
force levels. The larger creep exponent in the FAK-deficient
cells indicates that the cytoskeletal structures that are
connected to the magnetic beads remodel more rapidly
and show a higher turnover compared to FAK-expressing
cells. This finding is somewhat unexpected, as the focal
adhesion complex, which is part of the structure probed
by the magnetic beads, has been previously reported to
be more stable in FAK-deficient cells (11,14). As a plausible
hypothesis, we suggest that not so much the focal adhesion
AD
B
C
E
F
FIGURE 4 FAK increases cell stiffness and adhesion strength, and
reduces cytoskeletal dynamics. Cell stiffness (magnitude of the shear
modulus) (A) and exponent of the creep modulus (B) measured with
magnetic tweezer microrheology at different forces. (C) Adhesion strength
of fibronectin coated beads expressed as percentage of detached beads
versus pulling force. (D) Cell stiffness (shear modulus) measured with an
uncoated AFM tip. Apparent diffusivity (E) and power law exponent of
the mean squared displacement (F) of spontaneous movements of
FN coated beads attached to the cell surface. (Bars) Standard error.
(Asterisk) Significant (p<0.05) differences.
FIGURE 3 Nanoscale particle tracking. Cytoskeletal remodeling
dynamics was estimated from the apparent diffusivity and the power law
exponent (see Fig. 4,Eand F) of the mean squared bead displacement
(geometric mean of >611 beads) versus time.
Biophysical Journal 101(9) 2131 2138
2134 Fabry et al.
complex, but the connected cytoskeletal structures are more
dynamic and unstable in FAK-deficient cells.
To test this hypothesis, we used the nanoscale particle
tracking technique. Fibronectin-coated fluorescent beads
are connected to the cytoskeleton via integrin-type cell
surface receptors, and their movements are followed over
time for several minutes. Although the type of probe is the
same as in the magnetic tweezer experiments, the properties
being measured are not. The magnetic beads are actively
forced and thus report the passive mechanical properties
of the cytoskeleton, such as elastic modulus and its time
(or frequency) dependency. In the particle tracking experi-
ments, the beads are passive in the sense that they are not
externally forced (except thermally) and report the active
material properties of the cytoskeleton. This is because the
beads act as fiducial markers of the cytoskeleton and
move only if the cytoskeletal structures to which they are
bound also move, for instance due to cytoskeletal remodel-
ing events (38), including those that lead to an overall cell
movement, and contractile force fluctuations (32). We find
an increased apparent diffusivity of the cytoskeleton-bound
beads in the FAK-deficient cells (Fig. 4 E), which is in
agreement with our hypothesis that the cytoskeleton of
FAK-deficient cells is less stable and more dynamic.
Additional support for this hypothesis comes from the
finding that nearly 50% of the magnetic beads attached to
FAK /cells detach at forces of 10 nN, as opposed to
only 10% of the beads that detach from FAKwt cells
(Fig. 4 C). Although we do not know whether the bead
detachment occurred due to a rupture of protein bonds in
the focal adhesion complex or due to a rupture of protein
bonds in the associated cytoskeleton, previous reports of
a more stable adhesion complex in FAK-deficient cells
(11,12,14) point to the cytoskeleton as the weakest link.
We noted that the fibronectin-coated fluorescent beads
moved more persistently and with a significantly larger
exponent bwhen attached to FAK-expressing cells as
compared to FAK-deficient cells (Fig. 4 F). The bcharacter-
izes the time evolution of the MSD of the beads and was
closer to a ballistic behavior in FAK-expressing cells, as
opposed to a more random behavior in FAK-deficient cells.
Because the beads generally follow and move along a path
that is determined by the cytoskeletal architecture, the
straighter and more persistent movement of the beads
attached to FAK-expressing cells suggests a more aligned
cytoskeleton, whereas the more random bead movement in
FAK-deficient cells in turn suggests a more isotropic
arrangement of the cytoskeleton.
Passive and active cytoskeletal properties are intricately
linked by universal scaling laws (39,40): a stiffer cytoskel-
eton is usually less frequency- or time-dependent (the
power-law exponent btends toward zero) (27), remodels
more slowly on short timescales (the apparent diffusivity
Dis smaller) (38) but more persistently (the MSD-exponent
bat longer timescales is higher) (32). A comparison of the
magnetic tweezer and particle tracking data (Fig. 4) shows
that FAK/and FAKwt cells obey these scaling laws.
Fluorescent images confirm, in agreement with previous
reports (12,15), that FAKwt cells have a highly ordered
and aligned actin cytoskeleton with prominent stress fibers,
whereas FAK/cells show a more pronounced cortical
actin cytoskeleton (Fig. 5 A). Such differences are not
visible in siControl versus siFAK cells, which is consistent
with the smaller differences that we found in all of the
mechanical parameters between these cells (Fig. 4). We
attribute these smaller mechanical differences and the lack
of any discernible differences in the cytoskeletal architec-
ture between siControl and siFAK cells to an incomplete
FAK knockdown, with expression levels of ~10% of base-
line FAK in the siRNA-silenced cells (data not shown). In
this regard, FAK may behave similarly to other focal
adhesion proteins. For example, a downregulation of the
focal adhesion protein vinculin to ~10% of control level is
sufficient for proper focal adhesion formation and mechan-
ical coupling (41,42), and only cells with full vinculin
knockout show substantial mechanical impairment.
It is known that cell stiffness scales linearly with cyto-
skeletal prestress, which in turn depends on the contractile
activation, cell area (both spreading area and cross-section
area), and cytoskeletal alignment (43). To test whether the
stiffness differences in cells with different FAK expression
levels are attributable to altered prestress, we measured
the traction forces of these cells (Fig. 5,Band C). As a scalar
value of cell tractions, we computed the elastic strain energy
that is stored in the matrix below the cell (35). Strain energy
was twofold decreased in FAK/cells (Fig. 5 D), but
this was solely due to a diminished spreading area of
the FAK/cells (Fig. 5 E). When strain energy was
normalized to spreading area (corresponding to an average
surface energy density) (Fig. 5 F), no difference could be
seen between FAKwt and FAK/cells. Consistent with
this finding, the maximum tractions and average traction
magnitude in all cells types were similar (Fig. 5 B), which
is also in line with previous reports (44,45). Moreover,
siControl and siFAK cells, which did not differ in their
spreading area (Fig. 5 E), showed no difference in strain
energy (Fig. 5 D). Taken together, these findings suggest
that the smaller stiffness of FAK-deficient cells was the
result of a reduced prestress (26,46), which in turn was
predominantly caused by a decreased spreading area and
therefore increased cross-sectional area. Note that this
interpretation rests on the assumption of a similar cell
volume in FAK-expressing and FAK-deficient cells.
Decreased stress fiber expression and a more pronounced
cortical organization in the actin cytoskeleton of FAK/
cells have been reported to result from rho-kinase, which
is known to be overactive in FAK/cells (12,13,15).
We confirmed, in line with previous reports (13), that
treatment of the FAK/cells with 10 mM of the ROCK-
inhibitor Y27632 for 30 min reestablishes a more wild-type
Biophysical Journal 101(9) 2131 2138
FAK Stabilizes the Cytoskeleton 2135
fibroblast cell morphology (data not shown). Y27632-treat-
ment not only affected the organization of the actomyosin
cytoskeleton, but it partially rescued the mechanical proper-
ties of the FAK/cells toward a FAK wild-type pheno-
type. The spontaneous motion of beads bound to FAK/
cells became significantly less diffusive and more directed
after Y27632-treatment, and the cell stiffness measured by
AFM increased (Fig. 6 A). Remarkably, FAKwt cells
responded to ROCK-inhibition with Y27632 with the exact
opposite behavior: lower cell stiffness and more diffusive,
less directed bead motion.
On a cell-by-cell basis, the stiffness change in FAK/
cells after Y27632-treatment was highly correlated (r
2
¼
0.51) with the stiffness before Y27632-treatment (Fig. 6 B,
and see Fig. S1 in the Supporting Material). Soft cells
stiffened after treatment with the ROCK-inhibitor Y27632,
whereas stiff cells softened. FAKwt cells, which were
generally stiffer than FAK/cells, tended to soften after
ROCK inhibition regardless of baseline stiffness (Fig. 6 B,
and see Fig. S1). This observation, together with the fact
that ROCK is more active in FAK/cells, suggests
that cytoskeletal architecture, mechanics, and dynamics
can change in response to altered ROCK activity only
along a specific trajectory, as conceptually outlined in
Fig. 6 C. Accordingly, cell stiffness, stress fiber formation,
cytoskeletal stability, and spreading area are highest at an
intermediate ROCK activity level as is present in normal
(FAKwt) cells. At lower or higher levels of ROCK activity,
all those parameters decrease. The question then arises
how FAK and ROCK are connected such that they alter cyto-
skeletal architecture and mechanics only along a specific
trajectory.
ABC
D
E
F
FIGURE 5 Traction force magnitude is not affected by FAK. (A) Fluorescence images (actin, red; vinculin, green) of cells plated on glass. (B) Traction
maps and corresponding (C) bright field images of cells on polyacrylamide gels. (Red dashed line) Cell outlines. Strain energy (D) and spreading area (E)
are reduced in FAK / cells, but the average traction force magnitude and strain energy normalized to the spreading area (F) are not dependent on FAK
expression levels.
Biophysical Journal 101(9) 2131 2138
2136 Fabry et al.
ROCK is activated by rhoA-kinase, which in turn is regu-
lated by FAK via rho-GEFs (activator) and rho-GAPs (inac-
tivator) (18). This gives FAK a dual role that is crucial for
the spatiotemporal regulation of rhoA-kinase and ROCK
activity (3,4,18). ROCK is known to regulate intermediate
filament assembly, actin polymerization via LIM-kinase,
cortical actin distribution via adducin (47), and actomyosin
contraction via myosin light chain (MLC) phosphatase inhi-
bition and direct MLC phosphorylation (48 50).
In our experiments, it was expected that inhibition of
rho-kinase by Y27632 decreases MLC-dependent contrac-
tility and, therefore cell stiffness. FAKwt cells behaved as
expected, but FAK/cells did not. The restructuring
of the cytoskeleton toward a more mesenchymal phenotype
in FAK/cells after ROCK inhibition seemed to dominate
over any decrease in actomyosin contractility. We speculate
that this may be because ROCK activates the trans-
membrane actin binding proteins ezrin, radixin, and moesin
(ERM) that are important for the cortical distribution of
actin (51 53). Because ROCK is overactive in the FAK/
cells, it could induce an overactivation of the ERM proteins
and thereby induce the binding of cortical actin bundles to
the plasma membrane. To validate the involvement of
ERM proteins, however, further work is needed.
Regardless of the molecular mechanisms, our data show
that FAK stabilizes the actin cytoskeleton through
a ROCK-mediated pathway.
SUPPORTING MATERIAL
One figure is available at http://www.biophysj.org/biophysj/supplemental/
S0006 3495(11)01134 9.
We thank Drs. Wolfgang H. Ziegler, Staffan Johansson, Bernd Hoffmann,
Gerold Diez, Carina Raupach, Philip Kollmannsberger, Jose Luis Alonso,
Daniel Paranhos Zitterbart, and Thorsten Koch for help with experiments
and stimulating discussions.
This work was supported by grants from Bayerische Forschungsallianz;
Deutscher Akademischer Austauschdienst; Bavaria California Technology
Center; National Institutes of Health (NIH HL65960); and Deutsche
Forschungsgemeinschaft. A.H.K. has been supported by a grant from the
University of Erlangen Nuremberg.
A.H.K. and W.G. designed the study, A.H.K. and S.K. performed and
analyzed the experiments, T.S. and B.F. developed the methods, and B.F.,
A.H.K. and W.G. wrote the manuscript.
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A
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FIGURE 6 ROCK inhibitor has diverging effects on cell mechanical
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(C) Schematic representation of the proposed molecular pathway: FAK
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(Arrowheads) Expected changes of cell stiffness and cytoskeletal properties
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