3644 Research Article
Mechanical forces play a crucial role in controlling the integrity
and functionality of cells and tissue (Janmey and McCulloch, 2007).
External forces are sensed by cells and translated into signals that
induce cellular polarisation (Haga et al., 2007; Ingber, 2006;
Katsumi et al., 2005). Cyclic stretching of cells is commonly used
to mimic external forces exerted in the body (Bao and Suresh, 2003).
In such experiments, cells, adherent to an elastic substrate, orient
with their long axis perpendicular to the stretch direction (Jungbauer
et al., 2008). Such polarisation events require a dramatic
reorganisation of the contacts that the cell has with the extracellular
matrix (Kaunas et al., 2005; Yoshigi et al., 2005). The direct
communication between the cell and the extracellular matrix is
mediated by integrins that are associated with the actin cytoskeleton
via a multiprotein complex of regulatory molecules (Geiger et al.,
2001). Such focal adhesion (FA) sites are thought to be crucial in
the process of mechanotransduction and are involved in the
transformation of mechanical into biochemical signals (Chen et al.,
2004; Geiger et al., 2009).
Force-induced polarisation and reorganisation processes have
previously been investigated; however, many details about the
molecular mechanisms remain unclear. Little is known about the
role of microtubules (MTs) during force-induced cell polarisation,
even though they are structurally the stiffest cytoskeletal elements
and have demonstrated mechano-responsive functions. MTs grow
out to subcellular areas when forces are applied locally (Kaverina
et al., 2002; Suter et al., 1998) and their polymeric mass increases
rapidly upon single strain steps (Putnam et al., 1998).
MTs are known to regulate FA dynamics during cell migration
(Small et al., 2002). Disruption of MTs stops cell migration
(Ballestrem et al., 2000; Kaverina et al., 2000), and MT targeting
of FAs controls their local stability (Kaverina et al., 1999). Because
FAs are crucial for mechanotransduction, a detailed understanding
of FA dynamics and regulation is important.
Rho GTPases are key elements in controlling the actomyosin-
FA system and MTs play an important role in regulating their activity
(Etienne-Manneville and Hall, 2002). Disruption of MTs leads to
a stimulation of RhoA activity (Liu et al., 1998), whereas MT re-
polymerisation leads to increases of Rac1 activity (Waterman-Storer
et al., 1999). Altered activities of RhoA and Rac1 in cyclic
stretching experiments have been reported (Kaunas et al., 2005;
Liu et al., 2007); however, the results are conflicting. For example,
RhoA activity has been reported to both increase (Smith et al., 2003)
and remain unchanged (Katsumi et al., 2002; Yamane et al., 2007).
Similarly, decreases as well as increases of Rac1 activity have been
reported (Katsumi et al., 2002; Yamane et al., 2007). The
contribution of MTs in GTPase regulation during the application
of stretching forces has not been investigated.
Mechanical forces play a crucial role in controlling the integrity
and functionality of cells and tissues. External forces are sensed
by cells and translated into signals that induce various
responses. To increase the detailed understanding of these
processes, we investigated cell migration and dynamic cellular
reorganisation of focal adhesions and cytoskeleton upon
application of cyclic stretching forces. Of particular interest was
the role of microtubules and GTPase activation in the course
of mechanotransduction. We showed that focal adhesions and
the actin cytoskeleton undergo dramatic reorganisation
perpendicular to the direction of stretching forces even without
microtubules. Rather, we found that microtubule orientation is
controlled by the actin cytoskeleton. Using biochemical assays
and fluorescence resonance energy transfer (FRET)
measurements, we revealed that Rac1 and Cdc42 activities did
not change upon stretching, whereas overall RhoA activity
increased dramatically, but independently of intact
microtubules. In conclusion, we demonstrated that key players
in force-induced cellular reorganisation are focal-adhesion
sliding, RhoA activation and the actomyosin machinery. In
contrast to the importance of microtubules in migration, the
force-induced cellular reorganisation, including focal-adhesion
sliding, is independent of a dynamic microtubule network.
Consequently, the elementary molecular mechanism of cellular
reorganisation during migration is different to the one in force-
induced cell reorganisation.
Supplementary material available online at
Key words: Force, Mechanotransduction, Focal adhesion, Actin,
Force-induced cell polarisation is linked to
Alexandra M. Goldyn1,2, Borja Aragüés Rioja1,2, Joachim P. Spatz1,2, Christoph Ballestrem3,*
and Ralf Kemkemer1,*
1Department of New Materials and Biosystems, Max Planck Institute for Metals Research, 70569 Stuttgart, Germany
2Department of Biophysical Chemistry, University of Heidelberg, 69120 Heidelberg, Germany
3Wellcome Trust Centre for Cell-Matrix Research, Faculty of Life Sciences, University of Manchester, Manchester M13 9PT, UK
*Authors for correspondence (email@example.com; firstname.lastname@example.org)
Accepted 29 July 2009
Journal of Cell Science 122, 3644-3651 Published by The Company of Biologists 2009
Journal of Cell Science
Force-induced cell polarisation
In the present study, we investigated the cellular rearrangements
during stretch-induced cell polarisation. In particular, we focused
on (1) the role of MTs in FA and cytoskeletal reorganisation, and
(2) MT involvement in regulating GTPase activity under mechanical
strain. We found that the force-induced cell reorientation is mainly
dependent on FA sliding and is largely driven independently of an
intact MT network. Although the total RhoA activity increased upon
stretch, the total and local Rac1 and Cdc42 activity levels remained
unchanged regardless of the presence of intact MTs. Overall, cellular
polarisation, including mechanosensing by FAs, the reorientation
of the associated actin cytoskeleton, as well as the localised activity
of Rho GTPases, seems to be independent of a dynamic MT
Fig. 1. Stretching forces induces cell repolarisation in a microtubule-independent manner. (A)Cell reorganisation was analysed by fitting an ellipse to each cell outline
and measuring the orientation angle, , between the long axis of the cell and the stretch direction. Three still images of a time series of an 8-hour phase-contrast movie
of stretched (+) non-treated (upper row), nocodazole-treated (middle row) and taxol-treated (lower row) NIH3T3 cells illustrate the cell reorganisation. The direction of
cyclic stretch is indicated by the double-headed arrows. Scale bars: 100m. (B)Time course of the reorientation of NIH3T3 cells treated with the indicated drugs upon
uniaxial cyclic stretching of 8% at 1 Hz. The mean values for the order parameter cos2 were calculated from the orientation angle (see panel A). A value
<cos2>1 indicates a perfectly parallel orientation, –1 a perfectly perpendicular orientation and 0 a random cell orientation with respect to the stretch direction.
(C)Quantification of the maximum cell reorientation (<cos2>MAX). The value <cos2>MAXresembles the cellular mean orientation of the last 4 hours of cyclic
stretch under the indicated conditions. ‘Stretch (–)’ indicates non-stretched control conditions and ‘stretch (+)’ the application of cyclic stretch. The disruption of MTs
enhanced the cellular reorientation in comparison with non-treated cells (*P<0.001). (D)Quantification of F-actin, FA and MT orientation under the indicated
conditions after 3 hours of cyclic stretch. Cellular structures were analysed by using fast Fourier transformation (FFT) analysis of cell subareas and background
subtraction combined with threshold application to yield <cos2> values. The orientation of each analysed structure was significantly higher under non-treated,
nocodazole-treated and taxol-treated stretching conditions (+) compared with non-treated, non-stretched conditions (–) (*P<0.05). Cells treated with cytochalasin D
revealed no difference in alignment of MTs under stretch compared to the non-treated, non-stretched control (‡P>0.2). (E)MTs, FAs and actin filaments after 3 hours of
cyclic stretch. MTs were visualised by an anti--tubulin antibody, FAs were stained with an anti-paxillin antibody and actin was marked using phalloidin. ‘Stretch (–)’
indicates non-stretched control conditions and ‘stretch (+)’ the application of cyclic stretch. Actin stress fibres and FAs oriented perpendicular to the stretch direction
(double-headed arrow) under non-treated conditions and despite nocodazole or taxol treatment. MT reorientation was dependent on the orientation of the actin
cytoskeleton and did not occur in cytochalasin-D-treated cells. Scale bars: 10m.
Journal of Cell Science
network. This is in contrast to coordinated cell migration, which is
strongly dependent on MTs.
Stretching forces induce cell polarisation in a microtubule-
MTs are thought to be important in the regulation of cell polarisation.
To examine the role of MTs as well as actin in the polarisation of
cells under mechanical stress, NIH3T3 fibroblasts were exposed to
uniaxial cyclic stretching forces (1 Hz, 8% of linear stretch
amplitude). For the quantitative analysis of cell polarisation, an
ellipse was fitted to the cell outline and the orientation parameter
(cos2) was calculated as depicted in Fig. 1A, using time-lapse
phase-contrast data sets. A time series is given in Fig. 1A as an
example illustrating the reorientation for non-treated cells (see also
supplementary material Movies 1-4). The maximum mean value
of cos21 indicates a perfectly parallel orientation of the observed
cells, and the minimum of –1 indicates a perfectly perpendicular
alignment of the cells with respect to the stretch direction (Jungbauer
et al., 2008). A mean value of cos20 corresponds to a random
orientation of the cells. Consistent with previous reports (Wang et
al., 2007), we found that cyclic stretching leads to the reorientation
of fibroblasts perpendicular to the stretch axis. Cells under stretching
forces reached a maximum value of <cos2>–0.4 after a period
of 2 hours (Fig. 1B,C; supplementary material Movie 1). The
disruption of the actin cytoskeleton by cytochalasin D completely
abolished cellular reorientation (Fig. 1B,C; supplementary material
Movie 4). By contrast, neither disruption nor stabilisation of MTs
by nocodazole or taxol, respectively, inhibited stretch-induced
polarisation (Fig. 1B,C; supplementary material Movies 2 and 3).
Disruption of MTs even enhanced the level of cellular reorientation
significantly in comparison
(<cos2>noco–0.6 versus <cos2>non-treated–0.4; P<0.001). Under
non-stretched conditions, fibroblasts were randomly oriented
(supplementary material Fig. S1).
We next tested whether cellular rearrangements coincided with
cytoskeletal and adhesion site reorganisation. Under stretching
conditions, both actin filaments and FAs orientated perpendicular
with non-treated cells
Journal of Cell Science 122 (20)
to the stretching axis with <cos2> of about –0.6. This reorientation
was irrespective of MT stabilisation or disruption (Fig. 1D,E). We
also analysed how the microtubular network itself is affected by
stretching forces and found that MT reorientation reached a similar
value of alignment (<cos2>–0.6) as the actin cytoskeleton.
Although to a lesser degree, MTs stabilised by taxol also realigned
perpendicular to the stretching direction. MT reorganisation was,
however, completely blocked by the disruption of the actin
cytoskeleton with cytochalasin D (Fig. 1D,E), indicating that the
microtubular reorientation is tightly linked to the organisation of
Together, these data show that stretching induces cell polarity
perpendicular to the direction of stretching. This polarisation
depends on an intact actin network but is independent of MT
Stretch-induced oriented migration relies on intact microtubules
We next studied cell motility under stretching conditions and tracked
cells over a period of 8 hours (supplementary material Fig. S2).
Fig. 2. Stretch-induced, oriented migration relies on intact microtubules.
(A)Quantification of NIH3T3 cell-migration distance was performed by
tracking the cell nucleus every 10 minutes over 8 hours under indicated
conditions. Cyclic stretching at 1 Hz and 8% [stretch (+)] did not significantly
change the overall distance of migration compared to non-stretched cells [stretch
(–)]. Cell migration was basically blocked by stabilisation (taxol treatment) or
disruption (nocodazole treatment) of MTs. ‡No statistical difference, P>0.05.
*Significance, P<0.0001. (B)Oriented fibroblast migration was determined by
analysing the linear displacement of non-treated cells from their starting point to
their ending point. The direction of migration was perpendicular
(<cos2>–0.45±0.1) during stretching (+) and random (<cos2>0.1±0.13)
under non-stretching conditions (–) (*P<0.01).
Fig. 3. RhoA activity increases, whereas the activity of Rac1 and Cdc42 remains
constant, in response to stretching. (A)For ELISA measurements for active
RhoA, Rac1 and Cdc42 proteins, NIH3T3 cells were investigated under non-
treated, non-stretched [stretch (–)] and non-treated, cyclic stretched conditions
[1 Hz, 8%; stretch (+)] at indicated time intervals. The data set was normalised
to stretch (–) measurements. ELISA data show increased RhoA activity levels
upon cyclic stretching (*P<0.01); Rac1 and Cdc42 activity stays constant.
(B)NIH3T3 cells were transfected with either pRaichu-RhoA or pRaichu-Rac1.
FRET was determined for non-treated, non-stretched [stretch (–)], and non-
treated, cyclic stretched conditions [stretch (+)] at indicated time intervals. FRET
images were normalised to the acceptor fluorescence intensity and were
displayed using a spectral colour look-up table indicating FRET levels. FRET
measurements show that the RhoA activity level increased upon cyclic stretching
in a non-polarised fashion (stretch direction is indicated by a doubled-headed
arrow). Rac1 activity was high in protruding cell areas (arrowhead) and low in
retractions (arrow). Black squares indicate areas of analysis for Rac1 activity
gradient (supplementary material Fig. S3). Local Cdc42 activity was high in cell
protrusions and did not change upon stretching (data not shown). Scale bars:
Journal of Cell Science
Force-induced cell polarisation
Although stretching did not significantly alter the overall distance
of cell migration (150 m over the 8 hours), cells under such forces
migrated perpendicularly to the stretch axis (<cos2>–0.45±0.1;
P<0.01) as opposed to non-stretched control cells, which migrated
in a randomly oriented fashion (<cos2>0.1±0.13) (Fig. 2;
supplementary material Fig. S2). Stretch-induced oriented cell
migration was essentially blocked by stabilisation or disruption of
MTs via treatment with taxol or nocodazole, respectively.
Thus, the presence of dynamic MTs is essential for polarised
migration under stretching conditions.
Global RhoA activity increases in response to stretching,
whereas that of Rac1 and Cdc42 remains constant
Rho and Rac GTPases have a key role in FA formation, cell
polarisation and cell migration (Etienne-Manneville and Hall,
2002). To explore the regulation of GTPases in relation to the
observed changes in cell polarity, we tested their activities under
stretching conditions. Using the enzyme linked immunosorbent
assay (ELISA), we found no changes in Rac1 and Cdc42 activity,
but a twofold increase of RhoA activity (P<0.01) after 10-30 minutes
of cyclic stretching (Fig. 3A). This increase coincides with normal
stress-fibre formation observed in a large variety of cell types
(Kaunas et al., 2005; Smith et al., 2003; Yoshigi et al., 2005) (and
our unpublished data). We then used fluorescence resonance energy
transfer (FRET) reporters to analyse local activity levels of the
GTPases. Rac1 activity was usually highest in protruding
lamellipodia under both non-stretching and stretching conditions
(Fig. 3B; supplementary material Fig. S3), whereas RhoA activity
levels rose upon stretching in a non-polarised fashion throughout
the cell (Fig. 3B). The highest level of Cdc42 activity was found
in the protruding areas of the cells, and levels of local and global
Cdc42 activity did not change upon cyclic stretching stimulation
(data not shown). We next tested whether inhibition of Rac and
Rho activity influenced the polarisation of cells and the cytoskeleton
under cyclic stretching. Expression of dominant-negative Rac
(RacN17) as well as inhibition of Rho with C3 toxin reduced or
inhibited the actin-stress-fibre and cell reorientation, respectively
(Fig. 4A,B; compare with Fig. 1D,E). Assessment of MT orientation
showed that their reorientation was similar to that of F-actin in
RacN17-expressing cells (<cos2>–0.26±0.06). MTs in C3-toxin-
treated cells showed a random orientation (Fig. 4A,B). This outlines
the dependence of MT orientation on the RhoA-driven orientation
of the actin cytoskeleton. In summary, these data show that the
stretch-induced polarisation of the actin and MT cytoskeleton is
RhoA-dependent, but not Rac1- or Cdc42-dependent.
Microtubules have limited control over (localised) RhoA, Rac1
and Cdc42 GTPase activity
MT disruption is known to increase RhoA activity in adherent and
suspended cells (Liu et al., 1998; Ren et al., 1999), an observation
that we were able to confirm in our assays. The total RhoA activity
increased more than tenfold upon nocodazole treatment in non-
stretched adherent cells and 1.2-fold in non-adherent, suspended
cells (supplementary material Figs S4 and S5), suggesting that cell
adhesion mechanisms are important for reaching maximal RhoA
levels. In contrast to RhoA, overall Rac1 and Cdc42 activity levels
remained constant after treatment with nocodazole. MT stabilisation
by taxol did not show a change of RhoA, Rac1 nor Cdc42 activity
levels (supplementary material Fig. S4).
We next explored whether MTs are involved in the regulation
of total and localised GTPase activity. FRET and ELISA data
showed no additional increase of RhoA activity under stretching
conditions after the disruption of MTs (Fig. 5A,B), which is
possibly due to saturated RhoA activity as a result of nocodazole
treatment (supplementary material Fig. S4). This finding is
supported by our control experiments, because we observed a
significant increase in total RhoA activity upon nocodazole
treatment also in cells under static, non-stretched conditions
(supplementary material Figs S4 and S5). RhoA activity in cells
treated with taxol increased similarly to that in non-treated cells
upon stretching (Fig. 5A,B; compare Fig. 3A,B). Analysis of Rac1
and Cdc42 activity did not show changes in total or alterations in
local activity levels upon treatment with nocodazole or taxol under
non-stretching or stretching conditions. The highest Rac1 activity
was always found in protruding cell areas and low levels were
always in retracting areas of cells (compare Fig. 5A with Fig. 3B;
supplementary material Fig. S3). Cdc42 activity levels were
similar to those of Rac1, with higher levels in protruding areas and
lower activity in the body (data not shown).
To explore whether MT disruption or stabilisation affects
protrusion activities, we analysed membrane protrusion formation
either perpendicular (‘end’) or parallel (‘side’) to the stretch
direction (Fig. 5C). Under non-stretched conditions, protrusion
activity at cell ends and sides were similar. Cyclic stretching in the
horizontal direction doubled the protrusion activity at the ends of
the cells, whereas protrusion activity decreased by about half at the
Fig. 4. Inhibition of Rac and Rho activity influences cell and cytoskeleton
polarisation under stretching conditions. (A)MTs, F-actin and FAs in NIH3T3
cells subjected to cyclic stretch for 3 hours. MTs were stained by an anti--
tubulin antibody, FAs were visualised with an anti-paxillin antibody and actin
was marked using phalloidin. The direction of cyclic stretch is indicated by a
double-headed arrow. Cells either expressed dominant-negative Rac (RacN17)
or were treated with C3 transferase for Rho inhibition. RacN17-expressing cells
were identified by YFP co-transfection (supplementary material Fig. S6). Scale
bars: 10m. (B)MT alignment occurred perpendicular to the stretch axis and
correlates with F-actin orientation in RacN17-expressing cells (<cos2>–0.26).
MTs were randomly oriented in C3-toxin-treated stretched cells (<cos2>?0)
(*P>0.05). ‘Stretch (–)’ indicates non-stretched control conditions and ‘stretch
(+)’ the application of cyclic stretch.
Journal of Cell Science
sides of the cell regardless of MT stabilisation or disruption (Fig.
Together, these data demonstrate that a dynamic MT network is
not required to maintain localised activity of tested Rho GTPases
and associated protrusive activity of the cells upon induction of
directional stretching forces.
Focal-adhesion reorganisation under stretching occurs through
sliding mechanisms, which are independent of microtubule
The data above suggested a dramatic reorganisation of FAs together
with the actin cytoskeleton; this reorganisation seemed essentially
independent of MTs. Surprisingly, MTs had only limited control
over the activity of Rho GTPases, which does not explain why the
dramatic reorganisation of FAs is inhibited in cells without MTs
during cell migration but not during force-induced polarisation.
Therefore, we transfected cells with a GFP-vinculin construct and
examined FA dynamics closely by tracking them in time-lapse
fluorescence microscopy (Fig. 6; supplementary material Movies
5-10). In contrast to de novo FA formation and disassembly known
to be essential for cell migration, stretching forces induced a
Journal of Cell Science 122 (20)
dramatic sliding of FAs (Fig. 6, left-hand image and enlarged
inserts). The observed sliding was not disturbed by disruption nor
stabilisation of MTs. Only the orientation of existing FAs but not
the number of FAs changed during the period of stretching. Overall,
only a very small fraction of FAs was newly formed (<2%);
reorganisation occurred rather by growth, shrinking and fusion
processes of existing FAs.
These results demonstrate that, in contrast to FAs during cell
migration, the FA rearrangements during force-induced polarity are
driven by MT-independent mechanisms.
This study demonstrates that repolarisation of cells upon cyclic
stretching induces sliding of FAs and reorientation of the associated
actomyosin machinery. These polarisation events seem to be
entirely dependent on downstream RhoA signalling controlling
myosin-II function, but they are independent of an intact dynamic
Because MT depolymerisation induces the formation of actin
stress fibres and increases cell contraction via Rho activation
(Enomoto, 1996) and MT polymerisation increases Rac1 activity
Fig. 5. Microtubules have limited control over (localised) RhoA and Rac1 and
Cdc42 GTPase activity. (A)FRET measurements for RhoA and Rac1. NIH3T3
cells were transfected with either pRaichu-RhoA or pRaichu-Rac1 and treated
with taxol or nocodazole prior to the FRET measurements. FRET was
determined for non-stretched [stretch (–)] and cyclic stretching [1 Hz, 8%;
stretch (+)] conditions at indicated time intervals. FRET images were normalised
to the acceptor fluorescence intensity and are displayed using a spectral colour
look-up table indicating FRET levels. RhoA activity increased upon cyclic
stretching independent of MT stabilisation with taxol (stretch direction is
indicated by a double-headed arrow). RhoA activity was high upon disruption of
MTs (nocodazole) and did not further increase upon stretching. Under all
conditions (taxol or nocodazole treatment), Rac1 activity levels were high in
protruding cell areas (arrowhead) and low in retractions (arrow). Black squares
indicate areas of analysis for Rac1 activity gradient (supplementary material
Fig. S3). Local Cdc42 activity did not change upon application of cyclic stretch
(data not shown; refer to the Results section). Scale bars: 10m. (B)For ELISA
measurements for active RhoA, Rac1 and Cdc42 proteins, NIH3T3 cells were
treated with either taxol or nocodazole and investigated under non-stretched
[stretch (–)] and cyclic stretching conditions [stretch (+)] at the indicated time
intervals. The data set was normalised to stretch (–). ELISA data show an
increase in RhoA activity upon stretch in presence of taxol (*P<0.01). Cyclic
stretching did not further increase RhoA activity in nocodazole-treated cells
(compare with A). Rac1 and Cdc42 activity in taxol- and nocodazole-treated
cells did not change upon stretching. (C)Kymograph analysis of directional
NIH3T3 protrusion activity over a time course of 3 hours. The direction of
stretch is indicated by a double-headed arrow. To illustrate the analysis, a
nocodazole-treated cell is displayed. A line was drawn along the cell edge of a
composite phase-contrast image and the peaks were counted to yield a frequency
of membrane protrusions per hour. Cell protrusions occurring perpendicular to
the stretch axis were determined as ‘end’; cell protrusions parallel to the stretch
direction were called ‘side’. Application of stretching doubled the protrusion
activity at the ends but decreased activity by about half at the sides of cells
(*P<0.01). Independent of MT stabilisation (taxol) or disruption (nocodazole),
the rate of protrusions remained higher at ends compared with those measured at
the sides of the cells (*P<0.01).
Journal of Cell Science
Force-induced cell polarisation
(Waterman-Storer et al., 1999), we assumed that MTs would play
a crucial role during polarisation of cells stimulated by mechanical
forces. However, we found very little impact of MTs with respect
to two main aspects directing cell polarity: (1) the actin cytoskeleton
and FA rearrangements seemed similar at stretching conditions, and
(2) there was surprisingly little effect on total and local activity
levels of Rac1 and Cdc42 in cells, neither under stretching
conditions nor in the presence of reagents stabilising or disrupting
MTs. However, a certain level of RhoA activity is essential for the
polarised reorientation of cells, because blocking RhoA activity with
C3 transferase inhibited polarisation in our experiments. A moderate
increase in RhoA activity might result in an enhanced reorganisation
of actin stress fibres (Kaunas et al., 2005). By contrast, non-
physiological expression of constitutively active RhoA (RhoV14)
blocked the rearrangement of actin fibres (our unpublished
observation). This suggests that RhoA-activity modulation is
important for polarised rearrangements and disturbance leads to
impaired actin reorientation.
The slight but significantly increased level of reorientation in
cells treated with nocodazole compared to non-treated cells
suggests that MTs contribute to some extent to the observed
polarisation events by influencing RhoA activity. One way by
which MTs could contribute to the faster polarisation events might
be regulation of GEF-H1, because release of GEF-H1 from MTs
has been shown to enhance RhoA activity and actin stress-fibre
formation (Krendel et al., 2002). GEF-H1 activation, however, is
less likely to contribute to the force-induced RhoA activation per
se, because RhoA activity still increases upon force application
when MT dynamics are inhibited by taxol. Other guanine-
nucleotide exchange factors (GEFs), such as p190RhoGEF and
PDZ RhoGEF, that localise to FAs and are involved in regulating
RhoA activity (Iwanicki et al., 2008; Lim et al., 2008; Tomar and
Schlaepfer, 2009) might be potential candidates with respect to the
reorganisation of FAs and the upregulation of RhoA activity upon
We assume that the stretch-induced increased RhoA activity stems
from force transduction in FAs. A positive biomechanical feedback
loop has been suggested in which increased tension in actin stress
fibres, either by the internal actomyosin system or external stretch,
activates RhoA at FA sites and increases, in turn, stress-fibre
assembly (Besser and Schwarz, 2007). In fact, FAs and the
molecules therein are one of the primary receptors for mechanical
cues, which are then transmitted to the actin cytoskeleton (Geiger
and Bershadsky, 2002). One of the candidates might be vinculin,
which has been recently shown to provide a major link of the FA
plaque to the actin cytoskeleton (Humphries et al., 2007).
MTs target FA (Krylyshkina et al., 2003) and are vital for FA
turnover during cell migration (Kaverina et al., 2000). The observed
massive FA reorganisation during stretch-induced cell
repolarisation was not affected by the absence of MTs. We showed
that essentially all FA rearrangements, after application of
directional cyclic stretching, are accomplished by sliding and not
by de novo formation. These sliding FA rearrangements were
irrespective of the presence or absence of MTs. Therefore, it seems
probable that reorganisation of cell-matrix adhesions during
migration is controlled by molecular mechanisms that are different
from those for cell polarisation under cyclic stretching. The precise
molecular cues that are involved in FA sliding remain to be
investigated. However, one reason for the difference between
stretch-induced FA sliding and coordinated cell migration might
be that the latter depends on MT-guided shuttling of large numbers
of vesicles that fuse with the outer membrane for protrusive
activities (Schmoranzer et al., 2003). Disruption or stabilisation of
MTs would then compromise vesicular transport to some extent,
leading to the inhibition of cell migration (Schmoranzer et al.,
The cellular tensegrity model suggests that MTs build a rigid
frame that resists actomyosin contraction (Ingber, 2006; Stamenovic
et al., 2002), but it is also well known that MTs are highly dynamic
(Wehrle-Haller and Imhof, 2003). We showed that MTs are
obviously guided by actin filaments and consequently follow actin-
stress-fibre orientation, as is also observed in neuronal cells, in which
actin bundles provide guiding cues for MTs (Zhou et al., 2002). It
is likely that molecules such as ACF7, which has been shown to
regulate guidance of MTs along filamentous actin (Kodama et al.,
2003), could facilitate the microtubular reorientation.
We observed that the overall cell displacement per time unit did
not vary between non-treated non-stretched and non-treated
stretched conditions. If stretched, the cells migrated preferentially
perpendicular to the stretching direction, which resembles a one
dimensional (1D)-like random walk. By contrast, a random
migration in 2D is observed if no stretch is applied. In theory, one
would expect a twofold higher displacement of cells on static
substrates (2D migration) compared with cells on stretched
substrates (1D-like migration) considering that the mean square
displacement (MSD) is given by MSD2?d?D?t (with D the
diffusion constant, t the time and d the dimension of walk). We
attribute the deviation of our results from the expectation that the
displacement rate should be increased on stretch to the observed
higher protrusion activity perpendicular to the stretch direction,
which might lead to a higher effective diffusion constant for cells
on stretched substrates, thus resolving the apparent discrepancy.
Fig. 6. FA reorganisation occurs through a MT-independent sliding mechanism. FA dynamics was investigated in cells subjected to cyclic stretch of 8% at 1 Hz under
indicated conditions (a, non-treated; b, nocodazole-treated; c, taxol-treated). NIH3T3 cells were transfected with pGFP-vinculin and time-lapse fluorescent movies were
recorded (see supplementary material Movies 5-10). Grey-scale images on the left of each condition show single FA tracks over a time period of 115 minutes for non-
treated and 180 minutes for nocodazole- and taxol-treated cells. Enlarged areas are indicated by white boxes. Selected FAs are colour-coded: green for FAs before
stretch application (0 minutes), red for FAs after stretch (for non-treated after 100 minutes, for nocodazole and taxol after 160 minutes). Scale bars: 10m.
Journal of Cell Science
3650Journal of Cell Science 122 (20)
In conclusion, we have demonstrated that RhoA-driven actomyosin
machinery controls polarised rearrangements of the cell, their
cytoskeleton and FAs. In striking contrast to the important role of
MTs for FA assembly and disassembly during cell migration, MTs
are not required for FA sliding during cell polarisation under
mechanical stretching forces. We conclude that cell migration and
force-induced cell polarisation are directed by different molecular
Materials and Methods
Cells and plasmids
NIH3T3 (from DSMZ, Braunschweig, Germany) were cultured in DMEM (Invitrogen,
Karlsruhe, Germany) supplemented with 10% FCS (Invitrogen). pEYFP-N1 and
pECFP-N1 were from Clontech Laboratories (Saint-Germain-en-Laye, France); the
FRET probes pRaichu-Rac, pRaichu-RhoA and pRaichu-Cdc42 were a kind gift from
Michiyuki Matsuda (Itoh et al., 2002).
Cell-stretching experiments and light and fluorescent microscopy
Stretching experiments were performed as described in great detail elsewhere
(Jungbauer et al., 2008). Briefly, 50 cells/mm2were plated on fibronectin (20 g/ml)
(Sigma-Aldrich, Munich, Germany)-coated poly(dimethylsiloxane) (PDMS; Corning
Sylgard, Midland, MI) elastomeric membranes. The stretching device was mounted
on an inverted light microscope (AxioVert 200M, 10?/0.25Ph1 objective, Zeiss, Jena,
Germany) equipped with a CCD camera (PCO Sensicam, Kelheim, Germany) or an
upright light microscope (AxioImager Z1, W-Plan Apochromat 63?/1.0 VIS-IR
water-immersion objective, Zeiss) with an AxioCam CCD camera. A self-developed
software routine embedded in Image Pro 6.2 (Media Cybernetics, Bethesda, MD) or
AxioVision 188.8.131.52 (Zeiss) was used. Images for time-lapse phase-contrast movies
were acquired at 50-second or 100-second intervals for the indicated time periods
using DMEM supplemented with 10% FCS (Invitrogen) and 1% penicillin-
streptomycin (Gibco). Images for time-lapse fluorescent movies were taken every 5
minutes or 10 minutes for the indicated time periods using carbonate-free Ham’s F-
12 media with L-glutamine (Sigma) with 2% FCS (Invitrogen), 25 mM HEPES
(Sigma) and penicillin-streptomycin (Gibco). Parameters for cyclic stretching were
set to 1 Hz and 8% of linear stretch amplitude. For each experimental condition at
least three movies were acquired.
Chemical inhibitors and transfections
Concentrations of 3 M taxol, 3 M nocodazole and 1 M cytochalasin D (all Sigma)
were used and cells pre-incubated for about 30 minutes. C3 transferase (Cytoskeleton,
Denver,CO) was used according to the manufacturer’s manual. Transient transfections
were performed with Lipofectamine 2000 (Invitrogen) as recommended by the
Cell staining was performed as described previously (Humphries et al., 2007). Rabbit
monoclonal (Y113) anti-paxillin antibody and anti-Myc clone 9E10 antibody were
from Abcam (Cambridge, UK); the mouse monoclonal anti--tubulin (clone TUB2.1)
was from Sigma. The secondary antibodies (goat anti-mouse Alexa Fluor 350 and
goat anti-rabbit Alexa Fluor 568) and Alexa-Fluor-488 phalloidin were all from
Myc-tagged pRacN17 and pEYFP-N1 vectors were expressed in a 2:1 ratio and
showed coexpression efficiencies of about 95% (supplementary material Fig. S6).
The fluorescent images of fixed cells were contrast enhanced.
Analysis of the orientation of the cell, actin stress fibres,
microtubules and focal adhesions
Cell orientation (Fig. 1A) was measured as described previously (Jungbauer et al.,
2008). Briefly, phase-contrast images in order of their acquisition were taken and the
cell outline of each single cell was marked. An ellipse was fitted to each cell outline.
The orientation angle, , of the long axis of the ellipse with respect to the stretch
direction was measured (Fig. 1A). The mean values for the order parameter cos2
were calculated from the orientation angle and <cos2> denotes the mean value
of the orientation parameter. A value of <cos2>0 corresponds to cells that were
randomly oriented, <cos2>1 if all cells were parallel oriented, and <cos2>–1
if they were perpendicularly oriented with respect to the stretch axis. The time series
of nocodazole-treated stretched NIH3T3 cells in Fig. 1A were contrast enhanced.
The value for the maximum cell reorientation <cos2>MAXresembles the mean
orientation value of the last 4 hours of cyclic stretch under the indicated condition.
For analysis of actin-stress-fibre and MT orientation (Fig. 1D; supplementary
material Fig. S7A) a self-developed software macro embedded in ImageJ
(http://rsb.info.nih.gov/ij/) was used. Subareas of a cell were analysed by texture
analysis via a fast Fourier transformation (FFT) in analogy to Kemkemer et al.
(Kemkemer et al., 2000). In brief, each cell picture was divided into many small
squares (64?64 pixels). For each square, a FFT was performed and the resulting
image was further analysed to measure the mean orientation of fibres within the square.
The measured angles of orientation of actin stress fibres or MTs within the analysed
square were plotted into the image, where the x-axis of the image indicates the direction
of stretch or an arbitrary x-axis in the non-stretched case (supplementary material
Fig. S7A). The mean value (<cos2>) of all analysed cell subareas was calculated
and yielded in a mean orientation value for actin stress fibres or MTs of a single cell.
The angle for FA orientation (Fig. 1D; supplementary material Fig. S7B) was measured
with ImageJ and expressed as a <cos2> value. Briefly, FAs were separated from
the background by using the threshold tool of ImageJ and a mask of FAs was generated
(supplementary material Fig. S7B). The orientation of the mostly elliptic FAs was
determined using the measurement tool in ImageJ. The obtained angles were then
transformed into the mean value (<cos2>) for each single cell. In total, 20-30 cells
from three independent experiments were evaluated for actin (stress) fibre, MT and
FA sliding was analysed using Manual Tracking in ImageJ. A minimum of three
cells was analysed for each condition.
Analysis of migration and cell protrusive activity
Migration was measured by tracking the cell nucleus using ImageJ. For each
experimental condition, a minimum of ten cells was analysed. Images were captured
every 10 minutes over 8 hours from at least three independent experiments. The
orientation during cell migration was determined by analysing the linear displacement
of a cell from its starting point to its ending point; the obtained angle () of the straight
line with respect to the stretch axis was transformed into cos2 values (Fig. 2B).
Membrane protrusive activity was quantified by kymograph analysis of ImageJ as
described previously (Ballestrem et al., 2000; Humphries et al., 2007). Briefly, the
single images were lined up on a time scale in order of their acquisition. The resulting
composite phase-contrast picture allowed us to continuously follow the translocation
of recorded structures over time. Membrane protrusions were identified by their
characteristic centripetal movement, beginning at the lamella edge. A line was drawn
along the cell edge and the peaks were counted to yield a frequency of membrane
protrusion per hour. Dynamics of ten cells from at least three independent experiments
were studied at intervals of 50 seconds over the time course of 3 hours (Fig. 5C).
FRET reporters were transfected and cells were plated on the elastic membranes as
explained above. The filter sets used were: for CFP: 54HE; YFP: 46; FRET: BP
535/30, DBP 464/32, and 547/43, excitation 54HE (all from Zeiss). Cells were
illuminated with a lambda-DG4 Xenon lamp (Sutter Instrument Company, Novato,
CA) and imaged in carbonate-free Ham’s F-12 media with L-glutamine (Sigma) with
2% FCS (Invitrogen), 25 mM HEPES (Sigma) and penicillin-streptomycin (Gibco).
Following stretching, a set of three images – (1) excitation (Ex) CFP/emission (Em)
CFP; (2) ExCFP/Em YFP (FRET filter); (3) ExYFP/Em YFP – was captured at the
indicated time intervals. The function of the FRET constructs was tested to report
GTPase activity by addition of serum to starved cells. The calibration experiments
showed a similar outcome as originally published (Itoh et al., 2002; Zaidel-Bar et
For image analysis, the PixFRET plug-in (Feige et al., 2005) for ImageJ was used.
Calculation of FRET images was done as described in detail previously (Ballestrem
et al., 2006). Briefly, FRET was calculated from all recorded channels for pixels
above background levels. The FRET values were corrected for the bleed-through of
the CFP and YFP channel and normalised to the acceptor fluorescence intensity;
bleaching of CFP and YFP was negligible under the settings we chose for the
experiments. FRET images were displayed using a spectral colour look-up table
indicating FRET levels.
Rac1-, Cdc42- and RhoA-activity assay
The Rac1-, Cdc42- or RhoA-activity assay was performed with a commercially
available ELISA Kit (Cytoskeleton) and experiments were performed as described
in the manufacturer’s manual. Luminescence (RhoA, Rac1) readout or
absorbance(OD490 nm)(Cdc42) for evaluation of Rho GTPase activities was detected
with a fluorescence spectrophotometer (Tecan, Crailsheim, Germany). The obtained
values were normalised to one for the non-treated non-stretched condition.
Data were expressed as means ± s.e.m. OriginLab 8.0 software (OriginLab
Cooperation, Northampton, MA) were used for statistical analysis (Student’s t-test
and ANOVA). Differences were considered as statistically significant when the
calculated P-value was less than 0.05.
The authors thank Simon Jungbauer, Melih Kalafat and Christine
Mollenhauer for technical assistance; Mohammed Tasab, Charles
Streuli and Richard Segar for discussion and proof reading. C.B.
acknowledges BBSRC (BB/GG004552/1) and Wellcome Trust (grant
077100) for funding. This publication and the project described herein
were also partly supported by the NIH Roadmap for Medical Research
(PN2 EY 016586) and by the Excellence Cluster ‘CellNetwork’ of the
Journal of Cell Science
Force-induced cell polarisation
University of Heidelberg. J.P.S holds a Weston Visiting Professorship
at the Weizmann Institute, Department of Molecular Cell Biology. The
support of the Max Planck Society is highly acknowledged. Deposited
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Journal of Cell Science