A Role for the Juxtamembrane Cytoplasm in the
Molecular Dynamics of Focal Adhesions
Haguy Wolfenson1, Ariel Lubelski2, Tamar Regev1, Joseph Klafter2,3, Yoav I. Henis1*, Benjamin Geiger4*
1Department of Neurobiology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv, Israel, 2School of Chemistry, Sackler Faculty of Exact Sciences, Tel Aviv
University, Tel Aviv, Israel, 3Freiburg Institute for Advanced Studies (FRIAS), University of Freiburg, Freiburg, Germany, 4Department of Molecular Cell Biology, Weizmann
Institute of Science, Rehovot, Israel
Focal adhesions (FAs) are specialized membrane-associated multi-protein complexes that link the cell to the extracellular
matrix and play crucial roles in cell-matrix sensing. Considerable information is available on the complex molecular
composition of these sites, yet the regulation of FA dynamics is largely unknown. Based on a combination of FRAP studies in
live cells, with in silico simulations and mathematical modeling, we show that the FA plaque proteins paxillin and vinculin
exist in four dynamic states: an immobile FA-bound fraction, an FA-associated fraction undergoing exchange, a
juxtamembrane fraction experiencing attenuated diffusion, and a fast-diffusing cytoplasmic pool. The juxtamembrane
region surrounding FAs displays a gradient of FA plaque proteins with respect to both concentration and dynamics. Based
on these findings, we propose a new model for the regulation of FA dynamics in which this juxtamembrane domain acts as
an intermediary layer, enabling an efficient regulation of FA formation and reorganization.
Citation: Wolfenson H, Lubelski A, Regev T, Klafter J, Henis YI, et al. (2009) A Role for the Juxtamembrane Cytoplasm in the Molecular Dynamics of Focal
Adhesions. PLoS ONE 4(1): e4304. doi:10.1371/journal.pone.0004304
Editor: Neil Hotchin, University of Birmingham, United Kingdom
Received November 11, 2008; Accepted December 12, 2008; Published January 28, 2009
Copyright: ? 2009 Wolfenson et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: The research is funded by the National Institutes of Health through the NIH Roadmap for Medical Research, Nanotechnology Center for Mechanics in
Regenerative Medicine (PN2 EY016586); the VW Foundation; the German Israeli collaboration; the Juvenile Diabetes Research Foundation. None of these
organizations have any impact in the study design.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: YoavHe@tauex.tau.ac.il (YH); email@example.com (BG)
Integrin-mediated cell adhesion to the extracellular matrix
(ECM) occurs through specialized multi-molecular complexes
termed focal adhesions (FAs) [1–4]. The mechanisms underlying
the dynamic regulation of FA assembly and reorganization are
critical to FA function in tissue scaffolding and cell signaling, thus
affecting processes such as cell migration, wound repair and tissue
morphogenesis [5–8], as well as survival, growth and differenti-
Thus far, information concerning the regulation of FA dynamics
is scarce. Studies based on image correlation spectroscopy provided
a measure of the coupling between adhesion components and actin
, and fluorescent speckle microscopy was employed to explore
interactions between F-actin and FA-associated molecules ,
revealing an apparent hierarchical flow of FA proteins and actin
through FAs. Another recent study demonstrated that paxillin
dynamics in FAs are regulated by FA assembly/disassembly,
location in the cell and treadmilling . These reports contributed
to the understanding of the dynamic relationships between actin
and FA proteins. In addition, several fluorescence recovery after
photobleaching (FRAP) studies have addressed the dynamic
properties of numerous FA proteins [13–22]. These studies, which
indicated that FAs are molecularly dynamic sites, have mostly
estimated a single FRAP halftime, without providing detailed
mechanistic analyses of the processes involved.
To address this issue, we investigated the membrane association
dynamics of fluorescence-tagged FA-associated proteins (paxillin,
vinculin and b3-integrin) within and around FAs. To this end, we
employed highly sensitive FRAP studies combined with in silico
experiments and mathematical modeling to fit the data to
fluorescence recovery by diffusion, exchange, or combination of
both. Our results reveal the existence of several sub-domains, each
characterized by distinct mechanisms controlling the dynamics of
FA-associated proteins. Of particular importance is the novel
notion that that there exists a juxtamembrane cytoplasmic region
surrounding FAs, characterized by higher concentrations of FA
proteins (e.g., paxillin and vinculin), whose diffusion is attenuated
in this environment. The dynamics in this area differ from the
exchange-dominated dynamics of FA-bound molecules, and from
the diffusion-based dynamics seen in the cytoplasm. These findings
lead us to suggest a new model for the regulation of FA steady-
FA plaque proteins display several dynamically distinct
To characterize the dynamic properties of plaque proteins at
various cellular locations, we performed FRAP studies on HeLa-
JW cells expressing paxillin-YFP or mCherry-vinculin (Figure 1A).
These cells display relatively stable FAs that do not undergo
structural reorganization on the FRAP timescale employed (up to
160 s). Combining a small illumination spot (focusing the Gaussian
laser beam to 1.14 or 1.86 mm2) , a high-intensity bleaching
beam (capable of achieving photobleaching in 2 ms), and a high-
sensitivity photomultiplier, we were able to perform FRAP studies
at high spatial and temporal resolution (4 ms), enabling discrim-
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Figure 1. Typical curves demonstrating differences in paxillin and vinculin FRAP rates at different locations within the cell. (A) HeLa-
JW cells expressing paxillin-YFP or mCherry-vinculin, plated on 20 mg/ml fibronectin-coated cover slips. (B–E) Typical FRAP curves of paxillin-YFP or
free GFP in HeLa-JW cells. Cells were subjected to FRAP experiments 24–48 h after plating at 37uC, using a 636 objective (see Experimental
Procedures). The temporal resolution (dwell time per channel) was 6 ms for the short time scale (3-s experiments), 120 ms for the long timescale
FRAP studies on paxillin-YFP (60-s experiments), and 320 ms for the long timescale experiments on mCherry-vinculin (160-s experiments). Solid lines
denote the best fit of a nonlinear regression analysis, fitting to a lateral diffusion process ; the resulting t and Rfvalues are shown. (B) FRAP of
paxillin-YFP in the cytoplasm results in fast, complete recovery. (C) Free cytoplasmic GFP recovers instantaneously on the FRAP experimental
timescale; therefore, fitting by non-linear regression was not possible. (D) FRAP of paxillin-YFP in FAs (3 s timescale). Lower Rfand slower recovery
relative to cytoplasmic paxillin were observed. (E) FRAP of paxillin-YFP in FAs (60 s timescale) resulted in higher Rfand longer t, as compared to (D).
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ination between dynamic subpopulations of FA proteins at various
locations both within and outside FAs.
We initially measured paxillin-YFP in the cytoplasm, focusing
the laser beam at least 1 mm away from the ventral or dorsal
membranes. These measurements yielded full recovery, with a
short characteristic fluorescence recovery time t (the time required
to attain half of the recoverable fluorescence) (Figures 1B and 1F).
Knowing the Gaussian radius of the beam (0.77 mm with a 636oil
objective), one can calculate the diffusion coefficient D ,
obtaining D=4.0 mm2/s (see also Supporting Information). This
result is close to the value of D determined by correlation
spectroscopy for the cytoplasmic population of Lyn-GFP , and
suggests relatively free cytoplasmic diffusion of paxillin-YFP. Yet,
some restrictions on paxillin-YFP diffusion are likely to exist (see
Discussion), since free cytoplasmic GFP (Figure 1C) exhibited even
faster recovery (beyond the experimental timescale).
FRAP measurements taken on the same timescale (3 s), focusing
the beam on the plasma membrane at FAs, resulted in t values
higher than those measured in the cytoplasm (Figures 1D and 1F),
and a reduced mobile fraction (Rf) of 0.4960.02 (n=48). Both
effects can occur due to interactions of diffusing molecules with
immobile entities [26,27]. The particular type of effect depends on
the FRAP timescale relative to the dissociation/association rates:
stable interactions (i.e., long complex lifetimes relative to t) would
reduce Rf, while transient interactions would increase t, since each
fluorescent molecule fluctuates between bound and unbound states
during the measurement [26,27]. Therefore, the higher t of
paxillin-YFP at FAs on the 3 s timescale indicates the existence of a
population that interacts transiently with FAs. However, the
reduction in Rfindicates the existence of another subpopulation,
with relatively stable interactions during the 3 s FRAP experiments.
Since such interactions could become transient at longer times, we
increased the FRAP timescale to 60 s, to allow sufficient time for
additional paxillin-YFP molecules to dissociate. This would lead to
an increase in Rfand in t. Indeed, under these conditions, Rf
increased to 0.8160.02 (n=61) (Figure 1E), accompanied by a 25-
fold increase in t (Figures 1E and 1F). A further increase in the
FRAP timescale yielded no additional effects, suggesting that the
least several minutes. Thus, apart from the fast-diffusing cytoplas-
mic population, the FA-associated paxillin-YFP consists of several
subpopulations: one showing intermediate recovery on a 3 s
timescale, another characterized by slow recovery, and a third
which is immobile. In order to ensure that the slow recovery on the
long FRAP timescale is not due tolateral diffusion within the FA
itself, we employed the same beam size to bleach whole FAs by
focusing the laser beam on small enough FAs that fitted entirely
within the beam. Under these conditions, recovery of fluorescence
cannot be from within the bleached FA. The recovery rates
measured were similar to those in the standard experiments on
larger FAs (not shown), validating that the recovery is from pools
outside the FA, in keeping with the finding (Figure 2) that the
recovery on the long timescale occurs mainly by exchange.
A similar pattern of dynamic subpopulations was observed for
vinculin. Cytoplasmic vinculin displayed very fast recovery
(Figure 1F); the fraction of mCherry-vinculin at FAs with an
intermediate FRAP rate was somewhat faster than paxillin-YFP
(Figure 1F), with a similar Rf(0.5060.01, n=31); on the longer
timescale, vinculin at FAs exhibited Rfsimilar to paxillin (80%),
albeit with 2.5-fold slower kinetics (Figure 1F).
In contrast to paxillin and vinculin, the transmembrane FA
protein b3-integrin was essentially immobile in FAs on the
timescale of our measurements (up to 5 min; data not shown), in
accordance with previous reports . This immobility, on a
timescale close to the FA lifetime (10–30 min ), indicates that
the recovery of the plaque proteins in FAs is not by lateral
movement of large integrin-associated complexes within FAs, but
rather by exchange with cytoplasmic plaque proteins.
Different mechanisms govern the dynamics of fast- and
slow-recovering subpopulations in FAs
FRAP beam-size analysis is a method recently developed by us
[23,30] to explore the membrane interactions of proteins that
exchange between association with the plasma membrane and the
cytoplasm. FRAP of intracellular proteins that interact with
membrane-associated structures such as FAs can occur by diffusion
and/or by exchange with cytoplasmic pools, and is therefore
amenable tothesameanalysis[23,30]. To characterizetherecovery
modes of paxillin and vinculin subpopulations at FA sites, we
employed FRAP beam-size analysis , using 636 and 1006
fit within an FA (Figure 2). If FRAP occurs solely by diffusion, t is
proportional to the bleached area tD=v2/4D, where tDrepresents
the characteristic diffusion time, and v is the laser beam’s Gaussian
radius . In this case, the ratio between the recovery times
obtained with the two objectives, t(636)/t(1006), should be 1.63
(the measured ratio between the illuminated areas). When FRAP
occurs by exchange, t reflects the chemical relaxation time, which is
independent of the bleached area, i.e., t(636)/t(1006)=1 [23,30].
Intermediate t ratios suggest mixed recovery, in which the faster
process plays a greater role [23,30].
Forboth paxillinandvinculin,the interaction dynamicsoftheFA
subpopulations that recover at intermediate (3 s timescale) or slow
rates (60 or 160 s timescale) differ markedly (Figure 2). On the short
timescale, t(636)/t(1006) yielded 1.59 for paxillin and 1.58 for
vinculin (Figures 2A and 2B), suggesting recovery mainly by
diffusion. This enables the calculation of D from the t values,
yielding D=0.9 and 1.15 mm2/s for paxillin and vinculin,
respectively. These values are ,4-fold lower than those obtained
for the same proteins in the cytoplasm (Figures 1B and 1F),
suggesting that they experience diffusion-attenuating interactions in
the juxtamembrane cytoplasmic region above the FAs. The slow-
recovering populations yielded t(636)/t(1006)=1.09 and 1.05 for
paxillin and vinculin, respectively (Figures 2C and 2D); the fact that
these values are significantly below 1.63 indicates a major
contribution of exchange to the recovery. This situation precludes
an accurate translation of t to D; however, calculation of D and the
exchange rate by fitting to models of FRAP by diffusion plus
exchange (see below) yields D values similar to those measured for
the fast-recovering populations (Supporting Information, Table S1).
To further support the notion of different recovery modes
suggested by the FRAP beam-size analysis, we employed a
complementary approach based on in silico simulations of a system
modeled to resemble the dynamics of plaque proteins in FAs, as
determined in the FRAP experiments (Figure 3). Based on the FRAP
data (Figures 1 and 2), the system includes a population that recovers
Note that fitting for t ignored the first 6 points after the bleach, which correspond to the recovery phase shown in (D). (F) Average t values for the
subpopulations of paxillin and vinculin. Note the different timescales shown in each panel. Results represent the mean6SEM of 40–60
measurements, each conducted on a single FA within each cell and on different cells. In general, paxillin and vinculin displayed analogous patterns of
dynamic subpopulations; the only major difference lay in the slow-recovering FA populations, where vinculin recovery was ,2.5-fold slower.
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by diffusion in the cytoplasm (3D), and an FA-associated population
that separates into two subpopulations, one undergoing exchange
during the measurement, and the other which is immobile on this
timescale (Figure 3A). The simulation data were generated using a
constant time random walk (CTRW) algorithm [31,32], and the
results fitted to recovery by diffusion , exchange, or two
subpopulations recovering by diffusion and exchange (see Supporting
Information and Figure S1 for derivation of the analytical
expressions). If the rate of diffusion in the cytoplasm is much faster
than the rate of exchange (as is the case for paxillin and vinculin;
Figure 2), the contribution of exchange is negligible at the short
timescale required for diffusion, and the situation can be approxi-
mated by two populations, one recovering by diffusion and the other
by exchange (see Eq. 18, Supporting Information). Although the
diffusion in the cytoplasm is in 3D, the lateral diffusion equation 
(Eq. 14) is still valid, since in FRAP experiments involving
fluorescence collection from a restricted confocal plane, the 3D
diffusion is projected into a 2D space (see Supporting Information).
This finding is demonstrated by fitting the 3D simulated data on the
short timescale, to the analytical expressions for 2D diffusion and/or
exchange (Figure 3B). The lateral diffusion equation (Eq. 14) showed
an excellent fit to the simulated data, yielding the correct t value
entered in the 3D simulation (Figure 3B). The analytical expression
for two subpopulations undergoing diffusion and exchange (Eq. 18)
exchange alone (Eq. 17) did not fit well at all (Figure 3B). Extending
the timescale to cover the slower exchange process (Figure 3C) shows
a good fit only to the analytical expression for two subpopulations
undergoing diffusion and exchange (Eq. 18).
We then proceeded to fit the experimental FRAP data to the
different models (Figure 4). In FAs, the fraction that recovers on
the short timescale (3 s) did not fit exchange, but was well-fitted by
pure diffusion (Figure 4A). The fit was not significantly improved
by combining diffusion and exchange (data not shown). The slow-
recovering fraction (60 s timescale) was best fitted by the analytical
expression for two subpopulations undergoing diffusion and
exchange (Figure 4B). Similar results (not shown) were obtained
for vinculin. These findings are in accord with the FRAP beam-
FRAP dynamics of plaque proteins differ at the proximal
and distal FA ends
The ‘‘proximal end’’ of an FA (the edge pointing towards the
attached actin bundle) and the ‘‘distal end’’ often display distinct
Figure 2. FRAP beam-size analysis reveals two different recovery modes inside FAs. FRAP experiments were conducted on HeLa-JW cells
expressing paxillin-YFP or mCherry-vinculin, as described in Figure 1. Two beam sizes were generated using 636and 1006objectives, and t values
were determined with each. The ratio between areas illuminated by the two beams was 1.6360.03 (n=59); this ratio is expected for FRAP by lateral
diffusion, whereas a ratio of 1 is expected for recovery by exchange . (A) t values derived from FRAP experiments on a short timescale (3 s). For
both paxillin and vinculin, the t(636) differed significantly from the t(1006) value of the same protein (**, p=261027; Student’s t-test). (B) t(636)/
t(1006) ratios derived from (A). The t ratio for the 3 s measurements yielded 1.59 for paxillin and 1.58 for vinculin, close to the 1.63 ratio expected for
lateral diffusion (solid line) (p=0.24 and 0.15 for paxillin and vinculin, respectively; Student’s t-test). (C) t values from FRAP experiments on long
timescales (60 or 160 s). For both proteins, the t(636) and t(1006) values of the same protein were similar (p=0.38; Student’s t-test). (D) t(636)/
t(1006) ratios derived from (C). The t ratios (1.09 for paxillin, 1.05 for vinculin) differed significantly from the 1.63 value for diffusion (p=4*10223and
4*10225for paxillin and vinculin, respectively; Student’s t-test). These values imply a major contribution of exchange to the recovery, as they are close
to the ratio of 1 predicted for pure exchange (broken line). Bars in (A) and (C) represent means6SEM of 30–60 measurements. In (B) and (D), SEM of
the t(636)/t(1006) ratios were calculated using bootstrap analysis.
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assembly/disassembly kinetics . Thus, FA ‘‘migration’’ can
occur due to treadmilling, manifested by extension at the proximal
end and dissociation at the distal one. To examine whether this
asymmetry is reflected in paxillin and vinculin dynamics, we
performed FRAP measurements at the two FA ends. Relatively
long adhesions (.5 mm) were chosen to discriminate between
On the short timescale, we found no differences between the
dynamics at the two ends (Figure 5C). Furthermore, diffusion
remained the major recovery mechanism, as indicated by beam-
size analysis (data not shown). However, on the longer timescale
(60 s for paxillin, 160 s for vinculin), FRAP revealed marked
differences in dynamics at the proximal and distal ends. At the
former, FRAP rates and Rfvalues for paxillin and vinculin were
similar to those measured at the centers of smaller adhesions
(compare Figures 1E and 1F with Figures 5A and 5C). Beam-size
analysis suggested that exchange remained the major FRAP
mechanism. At the distal end, the recovery rate was extremely
slow, enabling extraction of only Rf(,25%) (Figures 5B and 5C).
Thus, the dynamics of the slow-recovering population depend on
the location within FAs, with faster exchange occurring at the
proximal end. The distribution of exchange rates correlates with
the localization of actin stress fibers tips in FAs , which is lower
at the distal end (Figure 5D). The correlation with actin density is
in line with theoretical models proposing that the force exerted by
the actomyosin machinery regulates FA dynamics [35,36] (see
Variable dynamics of FA plaque proteins and b3-integrin
in the vicinity of FAs
To examine whether the FA edge defines a sharp boundary for
FA protein dynamics, we performed FRAP measurements of
paxillin-YFP, mCherry-vinculin, and GFP-b3-integrin at varying
distances from this edge, moving sideways within the plane of the
ventral membrane in increments of 1.54 mm, equivalent to the
laser beam diameter (Figure 6A). Although these proteins are
concentrated in FAs, they are also present at lower densities
outside FAs (Figures 6C and 6E). For b3-integrin, a dramatic
change from immobility on our 1–3 minute timescale to high
apparent mobility (Rf=0.85) was observed in regions immediately
adjacent to the FAs (region 1; Figure 6B); calculation of D from t
yields D=0.18 mm2/s. A gradual decrease in t (higher D values)
was found as distances from the FA edge increased (D=0.20 and
0.25 mm2/s at distances of 1.54 mm and .5 mm from the edge,
Similar experiments with paxillin-YFP and mCherry-vinculin
demonstrated that at all locations outside FAs, these proteins
exhibited complete recovery (Rf=1) on the short timescale (3 s),
Figure 3. 3D simulation of plaque protein dynamics and fitting
to analytical expressions for different FRAP mechanisms. (A)
Schematic representation of the model for plaque protein dynamics.
The particles (molecules of FA plaque proteins) undergo fast 3D
diffusion (random walk) in a cubic volume, and reversible binding to
one of the volume boundaries (stripes). The bound molecules are
assumed to be laterally immobile, to mimic FAs on the experimental
timescale. A fraction of the bound molecules can, however, undergo
exchange (slow, relative to the diffusion) with the free molecules. (B–C)
FRAP simulations and fittings to different mechanisms. A CTRW
algorithm [31,32] was used to simulate FRAP experiments in a system
modeled after paxillin and vinculin dynamics in FAs. The simulation
parameters were chosen to resemble those of the FRAP experiments
(Figures 1 and 2). Since the Rfvalues of both proteins at FAs on the
short timescale were around 0.50 (Figure 1D), 50% of the particles were
denoted as undergoing 3D diffusion. Based on the increase in Rfto
,0.80 on the longer timescale (Figure 1E), the remaining 50% of the
particles were divided, with 30% undergoing exchange, and 20%
immobile. t for diffusion (tD) was introduced (in arbitrary units; au) as
100. To simulate a ,60-fold slower exchange rate (see Figure 4), we
introduced 1/b=tD660=100660 au, i.e., b=1.6761024, where b
represents the dissociation rate constant. (B) Short timescale. Under
such conditions, the contribution of slow exchange was negligible, and
it was sufficient to consider only the 3D diffusion. This was evident from
the excellent fit of the data to the analytical expression for FRAP by
lateral diffusion  (Eq. 14; red line). Fitting to exchange (Eq. 17; blue
line) was much worse (Average Squared Deviation - ASD - values are
shown). Fitting to two subpopulations (Eq. 18) did not improve the fit
(data not shown). The parameters derived by fitting to lateral diffusion
were similar to those introduced in the simulation [panel (B)]. Thus, the
analytical expression for lateral diffusion  (Eq. 14) could be used to
fit 3D diffusion. (C) Long timescale. At this timescale, the contribution of
exchange becomes significant. The situation could be approximated by
two subpopulations, one recovering by fast diffusion, and one by slow
exchange (Eq. 18). Diffusion (red) or exchange (blue) alone did not fit.
However, the analytical expression for two populations (Eq. 18; green)
yielded a good fit. The fitted parameters were in agreement with those
introduced in the simulation [Panel (C)].
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Figure 4. FRAP of paxillin in FAs fits recovery of two subpopulations by diffusion and exchange. FRAP data for paxillin-YFP in FAs (636
objective; experimental design as in Figure 1) was fitted to the analytical expressions for FRAP by lateral diffusion, by exchange, or by diffusion and
exchange (two subpopulations; Figure 3). To improve the signal-to-noise ratio, 40–60 FRAP curves were averaged in each panel by summing up the
intensities of each individual curve, starting from the bleach point for synchronization. To normalize the intensities, the pre-bleach level of each curve
was given a value of 1. (A) FRAP on the 3 s timescale was well-fitted by diffusion. The diffusion equation  (Eq. 14, Supporting Information) yielded
a good fit, while exchange (Eq. 17) was not well-fitted (see ASD values in lower panels). The t value derived from this fit (0.15 s) was similar to that
obtained by fitting each individual curve to lateral diffusion and averaging (Figure 1F; 0.16 s). The values obtained in the same manner for vinculin are
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Figure 5. Paxillin and vinculin display different dynamics at the FA proximal and distal ends. FRAP experiments were carried out as
described in Figure 1, focusing the beam on the two FA ends. (A) A typical FRAP curve of paxillin-YFP at the proximal FA end (60 s timescale). This
curve is similar to curves obtained in the middle of smaller FAs on the same timescale (Figure 1E). (B) A typical curve at the distal FA end (60 s
timescale). Fast recovery by diffusion exists, but the ensuing exchange is very slow, preventing determination of t. To eliminate the contribution of
the fast recovery, the first 6 points after the bleach were ignored in the fitting. (C) Average t values of paxillin-YFP and mCherry-vinculin at the two FA
ends. On the short timescale (left panels), which represents 3D diffusion (Figures 1, 2 and 4), the t values were similar at both ends, resembling those
at the centers of smaller FAs. On the long timescale (right panels), there were marked differences between the two: at the proximal end, significant
recovery was observed at rates resembling those at smaller adhesions, while at the distal end, recovery was too slow to be measured. (D) Paxillin co-
localizes with actin at the FA proximal end. HeLa-JW cells co-expressing paxillin-YFP and mCherry-actin were visualized by fluorescence microscopy
(see Experimental Procedures). Co-localization was visible at the proximal edge (solid arrow), but not at the distal edge (dashed arrow). Scale bar: 10
depicted in Table S1. (B) FRAP of the slow-recovering fraction (60 s timescale). The fit obtained for the combination of diffusion and exchange (two
subpopulations; Eq. 18) was better than the fit for each mechanism separately (see ASD values in lower panels). The fit yields t=0.15 s, similar to the
0.16 s value obtained for the fast-diffusing population, and b=0.11 s21(i.e., 1/b=9.09 s). Due to the much slower exchange, the process can be
approximated as one subpopulation recovering by diffusion, and the other by slow exchange (see Results).
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suggesting the absence of the slow-recovering subpopulation
observed in FAs. Beam-size analysis (Supporting Information,
Figure S2) indicated that adjacent to the edge or one beam
diameter away, FRAP of paxillin and vinculin occurred by an
apparent mixture of diffusion and exchange. The transition from
recovery by diffusion in FAs on the 3 s timescale (Figures 2 and 4)
to FRAP with the increasing contribution of exchange (near FAs),
suggests a faster exchange rate relative to diffusion in the latter
case , as validated by fitting to recovery by diffusion and
exchange (Supporting Information, Table S1). The faster
recoveries of paxillin and vinculin near FAs, as compared to
values within FAs (Figure 6D) suggest fewer binding sites and/or
weaker transient binding (exchange) to membrane-associated
protein complexes, resulting in less attenuation. These interactions
appear to generate a gradient that decreases with distance from
the FA. Indeed, at large distances from the FA (.5 mm), the
Figure 6. The rate of paxillin recovery outside FAs changes with distance from the FA edge. FRAP experiments were conducted as
described in Figure 1. (A) Typical HeLa-JW cells expressing paxillin-YFP are shown, demonstrating clear, highlighted FAs with large differences in
fluorescence intensity between adhesion and non-adhesion areas. Scale bar: 10 mm. FRAP experiments were performed at defined distances from the
FA edge (right panel; scale bar: 1 mm). The numbers represent the position of the bleached regions relative to the FA. The locations of the bleached
regions are defined as follows: 0 – inside the FAs; 1 – immediately adjacent to the FA edge; 2 – one beam diameter (1.54 mm) from the FA edge; and 3
– regions .5 mm from the FA edge. (B) Average t values at the various locations for GFP-b3-integrin. While b3-integrin was virtually immobile at FAs, it
was mobile outside the FA region, and displayed a gradient of recovery rates. (C) Relative average fluorescence intensities of GFP-b3-integrin at the
various locations. Fluorescence intensities were quantified by using the FRAP instrumentation under non-bleaching conditions. The intensity of GFP-
b3-integrin fluorescence inside FAs (location 0) was normalized to 1. (D) Average t values at the various locations of paxillin-YFP and mCherry-
vinculin. A gradient of recovery rates was observed for both paxillin and vinculin as a function of the distance from the FA edge. (E) Relative average
fluorescence intensities of paxillin-YFP and mCherry-vinculin at the various locations. The respective intensities of each protein at location 0 were
normalized to 1.
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mobility-restricting interactions fade away, leading to t values
resembling those observed in the cytoplasm (Figure 6D), and to
recovery by diffusion (as in the cytoplasm; Table S1).
In the present study, we demonstrate that FA plaque proteins
are characterized by four distinct dynamic populations, which
define three spatial domains (Figure 7). The interchanges and
cross-talk among these domains can play critical roles in regulating
FA stability and matrix adhesion. The three domains include: (i)
the cytoplasm (.1 mm deep), where paxillin and vinculin display
very rapid FRAP rates, though slower than those of free GFP
(Figure 1); (ii) the juxtamembrane region surrounding FAs (50% of
the FA-associated molecules), where the plaque proteins display
attenuated diffusion at "intermediate" rates (t=0.12–0.16 s); and
(iii) the FA domain, consisting of two subpopulations, one (30% of
the molecules) that undergoes slow exchange (4–9 s; Figures 1, 2
and 4), and another (20%) which is essentially immobile on the
experimental timescale, representing molecules stably associated
The diffusion of paxillin and vinculin in the cytoplasm occurs at
somewhat slower rates as compared to free GFP (Figure 1). This
mild retardation is most likely due to their transient association
with slower-diffusing or immobile proteins/structures, since the
size of paxillin-YFP (93 KDa) or mCherry-vinculin (144 KDa) is
still smaller than the size reported to result in steric constraints on
cytoplasmic protein diffusion [37,38].
The combination of FRAP beam-size analysis (Figure 2) and
mathematical modeling (fitting to analytical expressions for
diffusion, exchange or both in combination; Figure 4) demon-
strates a two-step fluorescence recovery mechanism at FAs,
composed of relatively fast diffusion (intermediate recovery rate
on the 3 s timescale), followed by slow exchange. Therefore,
FRAP measurements on the short timescale, where the contribu-
tion of slow exchange is negligible, fit recovery by diffusion
(Figures 2 and 4). On a longer timescale (60–160 s) diffusion is
completed during the initial phase of the recovery, and a major
contribution is provided by exchange. Earlier studies on two-step
recovery [22,39] assumed a D value, and fitted only the exchange
parameters. Here we show that when the rates of the two processes
are well-separated, it is possible to approximate the data as two
populations, one recovering by relatively fast diffusion, and the
other by exchange (Figure 3). Fitting to the analytical expression of
this model (Eq. 18, Supporting Information) enables derivation of
the dynamic parameters characterizing each process (Figure 4 and
To explain the attenuated (but still rather fast) diffusion of
paxillin and vinculin around FAs, we propose a novel "juxtamem-
brane domain" or zone (Figure 7). The molecules within this
domain are cytoplasmic, and show recovery by diffusion (Figures 2
and 4), albeit their diffusion rate is reduced relative to the remote
cytoplasmic population. The attenuated diffusion rates of FA
plaque proteins in this domain may be attributed to transient
interactions, either among themselves, with the membrane
cytoskeleton, or with other structures. Such transient interactions
are capable of increasing the local density of the plaque proteins in
the juxtamembrane region, provided that the target proteins (e.g.,
integrins) that bind them are concentrated in FAs (Figure 7). This
juxtamembrane domain may act as an intermediary between the
fast-diffusing cytoplasmic pool and the FA-associated population,
able to exchange with both. This could be advantageous for the
fast regulation of FA formation and reorganization, particularly for
the FA stress response . It was shown that locally applied rapid
Figure 7. A schematic representation of the three spatial domains defined by FA protein dynamics. The results presented in this article
point to the presence of three spatial domains within and around FAs, with distinct molecular dynamics: (i) the cytoplasm, characterized by fast-
diffusing FA plaque proteins; (ii) the juxtamembrane region surrounding FAs, which extends into the z-axis as well as laterally (shaded area), where
the plaque proteins display attenuated diffusion; (iii) the FA domain, containing two subpopulations of molecules, one associated with the FA surface
and undergoing exchange ( ), and an immobile, FA-bound subpopulation. The model is not drawn to scale for demonstrative purposes, and
specifically the distance along the z axis is exaggerated in order to clearly depict molecules with a higher local concentration in this region. The
juxtamembrane zone (shaded area) is actually adjacent (just above) the FAs, and has a gradient nature that decreases gradually with the distance
from the FA both in the XY plane and in the z direction (see text). Depending on the polar density of actin stress fiber tips in FAs, the plaque proteins
at the two FA ends (proximal, high actin fiber density; distal, low density) display different exchange rates. We propose that the density of integrins,
which is higher at the FA region but is also not negligible outside FAs, attracts FA plaque proteins by both direct and indirect binding, leading to a
density gradient of FA plaque proteins, as well as to distinct domains and dynamic populations.
Regulating Adhesion Dynamics
PLoS ONE | www.plosone.org9 January 2009 | Volume 4 | Issue 1 | e4304
stress induces FA elongation and increase in area, accompanied by
recruitment of new molecules, within approximately one minute
. Such a quick response may be facilitated by the
juxtamembrane domain, where the immediate availability of FA
plaque proteins would eliminate the need to recruit them from the
remote cytoplasmic pool. Notably, Figure 6 demonstrates that the
juxtamembrane domain also extends to the sides of the FAs. In
areas adjacent to the sharp borders of the FA (locations 1 and 2 in
Figure 6), paxillin and vinculin displayed unique dynamic
properties characterized by a gradient of recovery rates, with a
mixed contribution of diffusion and exchange. The exchange rates
at these locations were considerably higher than at FAs (b, the
dissociation rate constant, ranges from 2 to 10 s21for both
proteins; Table S1). However, at distances .5 mm from FAs, the
dynamic properties were similar to those of the cytoplasmic
population (i.e., fast recovery by diffusion). A parallel gradient
(reduced density and faster diffusion as the distances from FAs
increased), appears to exist for b3-integrins as well (Figure 6C),
suggesting that transient association with b3-integrins underlies the
gradient of recovery rates observed for paxillin and vinculin, thus
extending the juxtamembrane domain laterally.
We therefore propose a structural-dynamic model compatible
with these results (Figure 7). In this model, integrins immobilized
by binding to ECM components in FAs  provide a focal point
for recruitment of FA plaque proteins. Outside FAs, integrins
display significant mobility, while within FAs (Figure 6B) they are
nearly immobile relative to the FA lifespan (minutes or more),
suggesting very stable interactions with the ECM. Plaque proteins
at the FA site interact locally with the cytoplasmic faces of the
integrins, of which ,20% are immobile (i.e., tightly bound to
immobile targets). This may reflect heterogeneity in their binding
sites on integrins, since multiple sites enabling direct or indirect
binding can result in a variety of dissociation rates . The
existence of a spectrum of exchange rates is exemplified by the
differing dissociation rate constants of the different plaque proteins
(e.g., b=0.11 s21for paxillin, and b=0.025 s21for vinculin).
The dynamics of FA plaque proteins are also affected by their
location within the FA itself. We detected differences between the
exchange rates at the FA proximal and distal regions. At the
proximal end, paxillin and vinculin display much faster exchange
rates than at the distal end (Figure 5). Several factors could
contribute to this behavior, taking into consideration the polarity
of FAs, which is mainly affected by mechanical forces exerted via
the attached actin stress fibers. This polarity is also manifested in
the varying densities of phospho-paxillin, which affect FA
assembly, turnover and stability . As suggested by the co-
localization of actin and paxillin within the FA, the forces exerted
by actin stress fibers may differ at either end of the adhesion,
resulting in variations in the exchange dynamics of FA plaque
proteins (Figure 7).
In the current study, we examined FAs at steady-state, yet our
results may also shed new light on two models previously
constructed to explain the regulation of FA size [35,36]. Both
models theorize different flux rates of plaque proteins at the
proximal and distal ends of FAs, a prediction corroborated by our
observation of different exchange rates at these locations. The two
models also suggest that at steady-state, plaque proteins from the
proximal end should move laterally toward the distal end.
However, we found no evidence for such a mechanism on the
timescale of our measurements, since lateral diffusion within FAs
did not contribute significantly to the recovery (see Results). An
additional feature not included in these models is our novel finding
of the existence of a juxtamembrane domain surrounding the
adhesion (Figure 7), which may have important implications for
FA dynamics. The availability of plaque proteins in the area
surrounding the adhesion enables the treatment of the process in
these models as kinetically limited (e.g., involving the dissociation
rate of plaque proteins), rather than diffusion-limited.
We propose that juxtamembrane domains may be relevant not
only to FAs, but also to other membrane-associated complexes.
Signaling initiated by transmembrane growth factor receptors, for
example, requires the binding of various components of the
‘‘signaling cascade’’ to the activated membrane receptors. Future
challenges include defining the parameters that regulate the
structure and function of the FA juxtamembrane domain, and
directly testing the existence of and the roles played by
juxtamembrane domains in other systems, such as clusters of
growth factor receptors (e.g., EGF receptor) or cell-cell adhesions.
Materials and Methods
Reagents and plasmids
Hanks Balanced Salt Solution (HBSS), phosphate buffered
saline (PBS) and fibronectin were supplied by Sigma-Aldrich.
Fugene transfection reagent was purchased from Roche.
Cell Lines and transfections
The HeLa-JW and REF52 cell lines expressing either YFP-
tagged paxillin or GFP-tagged b3-integrin were previously
described [42,43]. For experiments involving transient expression
(mCherry-vinculin), cells were transfected with Fugene 6 (Roche).
All cell lines were cultured in Dulbecco’s Modified Eagle’s
Medium (DMEM) supplemented with 10% fetal calf serum
(FCS), 100 U/ml penicillin, 100 mg/ml streptomycin, and 4 mM
glutamine. All cell culture components were provided by
Biological Industries, Beit Haemek, Israel.
Fluorescence recovery after photobleaching (FRAP)
FRAP studies were conducted on live cells expressing the
various fluorescence-tagged FA-related proteins. The cells were
taken for FRAP experiments 24–48 h after being plated on glass
cover slips coated with 20 mg/ml fibronectin. Measurements were
taken in HBSS supplemented with 20 mM Hepes, pH 7.2, at
37uC. An argon ion laser beam (Innova 70C; Coherent) was
focused through a fluorescence microscope (AxioImager D.1; Carl
Zeiss MicroImaging, Inc.) to Gaussian spots of 0.6060.01 (plan-
apochromat 1006/1.4 NA oil immersion objective), 0.7760.01
mm (plan-apochromat 636/1.4 NA oil immersion objective), or
1.1760.02 mm (C-apochromat 40x/1.2 NA oil immersion
objective); experiments were conducted with each beam size
[beam-size analysis [23,44]]. The ratio between the illuminated
areas was 2.2960.02 (n=39) using the 636and 406lenses, and
1.6360.02 (n=59) using the 1006and 636lenses. After a brief
measurement at monitoring intensity (488/528 nm, 1 mW), a
5 mW pulse (2–10 ms) was used to bleach 50–75% of the
fluorescence in the spot. The time course of the fluorescence
recovery was tracked by the attenuated monitoring beam. The
apparent characteristic fluorescence recovery time t and the
mobile fraction Rf were extracted from the FRAP curves by
nonlinear regression analysis, fitting to a lateral diffusion process
 or exchange process.
Co-localization of paxillin and actin by fluorescence
HeLa-JW cells stably expressing paxillin-YFP were plated on 20
mg/ml fibronectin-coated cover slips. One day after plating, cells
were transfected with 1 mg of a plasmid encoding mCherry-actin.
After 24 h, cells were fixed with 4% paraformaldehyde, mounted
Regulating Adhesion Dynamics
PLoS ONE | www.plosone.org10January 2009 | Volume 4 | Issue 1 | e4304
with gel/mount containing anti-fading agents (Biomeda, Foster
City, CA, USA) and visualized using a Zeiss Axio Imager D.1
fluorescence microscope with a 636/1.4 NA objective. Fluores-
cence images were recorded using OED capture software with a
CoolSNAP HQ-M CCD camera (Photometrics).
Detailed descriptions of the models and simulations, as well as
derivations of the various analytical expressions, are presented in
the Supporting Information.
Calculations of SEM for the beam-size ratios and the t ratios in
Figures 2 and S2 were performed using bootstrap analysis, a
preferred method for ratio estimation . The t values from the
FRAP experiments using the 636 and 1006 lenses were
resampled with replacement using Excel, and average values from
each group of resampled data were extracted. For each lens, 100
average samples were generated in this way, followed by division
of the 636 resampled data by the 1006 resampled data. The
group of 100 ratio values was then analyzed, using SPSS for
average and SEM values. Estimation of the goodness-of-fit of the
FRAP data to the various analytical expressions (Figures 4 and 5)
was performed by calculating the Average Squared Deviation
(ASD) value of each fit. The squared deviations between the
calculated points (from the fit) and the simulated/experimental
data were summed up, and then divided by the number of
calculated points to obtain the ASD value.
Found at: doi:10.1371/journal.pone.0004304.s001 (0.84 MB EPS)
1st figure for the Supporting Information
Found at: doi:10.1371/journal.pone.0004304.s002 (0.60 MB EPS)
2nd figure for the Supporting Information
Found at: doi:10.1371/journal.pone.0004304.s003 (0.09 MB
1st table for the Supporting Information
Y.I.H. is the incumbent of the Zalman Weinberg Chair in Cell Biology.
B.G. is the incumbent of the Professor Erwin Neter Chair in Cell and
J.K. is the incumbent of the Heinemann Chair in Physical Chemistry.
Conceived and designed the experiments: HW AL JK YIH BG. Performed
the experiments: HW AL TR. Analyzed the data: HW AL. Wrote the
paper: HW JK YIH BG.
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