Interplay between Cytoskeletal Stresses and Cell
Adaptation under Chronic Flow
Deepika Verma2, Nannan Ye2, Fanjie Meng1, Frederick Sachs1, Jason Rahimzadeh2, Susan Z. Hua1,2*
1Department of Physiology and Biophysics, SUNY-Buffalo, Buffalo, New York, United States of America, 2Department of Mechanical and Aerospace Engineering, SUNY-
Buffalo, Buffalo, New York, United States of America
Using stress sensitive FRET sensors we have measured cytoskeletal stresses in a-actinin and the associated reorganization of
the actin cytoskeleton in cells subjected to chronic shear stress. We show that long-term shear stress reduces the average
actinin stress and this effect is reversible with removal of flow. The flow-induced changes in cytoskeletal stresses are found
to be dynamic, involving a transient decrease in stress (phase-I), a short-term increase (3–6 min) (Phase-II), followed by a
longer-term decrease that reaches a minimum in ,20 min (Phase-III), before saturating. These changes are accompanied by
reorganization of the actin cytoskeleton from parallel F-actin bundles to peripheral bundles. Blocking mechanosensitive ion
channels (MSCs) with Gd3+and GsMTx4 (a specific inhibitor) eliminated the changes in cytoskeletal stress and the
corresponding actin reorganization, indicating that Ca2+permeable MSCs participate in the signaling cascades. This study
shows that shear stress induced cell adaptation is mediated via MSCs.
Citation: Verma D, Ye N, Meng F, Sachs F, Rahimzadeh J, et al. (2012) Interplay between Cytoskeletal Stresses and Cell Adaptation under Chronic Flow. PLoS
ONE 7(9): e44167. doi:10.1371/journal.pone.0044167
Editor: Scott A. Weed, West Virginia University, United States of America
Received April 10, 2012; Accepted July 30, 2012; Published September 19, 2012
Copyright: ? 2012 Verma 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: This work was supported by National Institutes of Health (NIH) grant DK77302 (S.Z.H.). F.M. was supported by NIH HL054887 (F.S.). The funders had no
role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: email@example.com
Renal epithelial cells experience a wide range of shear stress due
to urinary flow. Through various adaptation mechanisms cells
adjust their cytoskeleton structure, adhesion assembly and cell-cell
interactions, thereby accommodating the mechanical challenge
[1,2]. This remodeling minimizes local stresses and affects
membrane transport and other cell functions [3,4]. Exposure of
renal tubule cells to chronic flow results in reorganization of the
cytoskeleton and an increase in the formation of cell-cell junctions
[5,6]. While the molecular mechanisms of shear stress transduction
remain unclear, flow shear stress appears to induce conformational
changes in a variety of cytoskeleton proteins . These, in turn,
can activate biochemical pathways that affect cell morphology,
migration and growth [8,9,10].
The spatial and time-dependent distribution of external force
applied to the cells is inherently non-uniform because the
cytoskeleton is a heterogeneous and anisotropic collection of
dynamically cross-linked proteins. Stress varies with the intrinsic
elasticity of individual proteins, the dynamics of cross-links, and
the degree of prestress [11,12,13,14,15]. The adaptation of cells to
mechanical stimuli occurs over multiple time scales . At short
time scales, external force induces rapid and reversible changes in
cytoskeletal stresses associated with elastic stretching of load-
bearing proteins [8,16]. This is followed by conformational
changes of the proteins . With longer term stimulation, the
cytoskeleton undergoes chronic rearrangements [14,17,18,19].
The response of cells to short term (,seconds) mechanical
stimuli has been studied by various means [20,21,22,23]. Applying
force to integrin receptors by a pipette or using ligand-coated
magnetic beads attached to the cell surface causes cell stiffening via
changes in focal adhesion assembly at the stimulation sites [20,24].
This increase in stiffness can extend to periods lasting minutes
. We have previously shown that a pulsatile shear stress results
in reversible changes in stress within a-actinin, and these changes
decay gradually with multiple challenges. Thus the adaptation
begins at time scale of seconds (,45 sec) . Although cells may
use multiple sensors to activate adaptation mechanisms, the
earliest adaptation response appears to be consistently towards
increased stiffness. However, epithelial cells in the kidney
experience chronic (long term) fluid shear stress (in the range of
minutes to hours) and experience a large variation in mechanical
forces. The dynamics of adaptation to flow involves polymeriza-
tion and depolymerization of F-actin, changes in cross linking, and
correlated changes in migration and growth [26,27]. With the
development of our genetically coded force sensitive fluorescent
probes [28,29], we are able to directly measure the variation of
stress in specific proteins and visualize the accompanying changes
in cytoskeleton structure in real time and begin pinpointing which
proteins are involved in adaptation.
In this study, we have measured actinin stresses and simulta-
neously observed corresponding cytoskeletal anatomy in Madin
Darbey Canine Kidney (MDCK) cells subjected to chronic fluid
shear stress, using the stress sensitive FRET sensor referred to as
spectrin repeat stress sensitive FRET (sstFRET) . Our study
reveals that long-term (,3 hrs) exposure to fluid shear stress
produces three distinct phases of cytoskeletal stress variation, each
lasting for minutes. This process was inhibited in the presence of
Gd3+and GsMTx4, blockers of mechanosensitive ion channels
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Co-localization of actinin-sstFRET with F-actin in MDCK
We expressed the FRET probe, actinin-sstFRET (Fig. 1a), in
MDCK cells and then subjected the cells to a constant flow in a
microfluidic chamber. The sensitivity of this FRET sensor has
been previously characterized using DNA springs in solution and
is on the order of 5 pN. The sensor was also expressed in cells and
it does not affect physiological function of the host protein  (see
Materials and Methods section for further details on the FRET
stress sensor). Actinin-sstFRET co-localizes with F-actin, which
allows us to use the FRET probe as a label for F-actin to monitor
actin translocation. Colocalization is shown by expressing actinin-
sstFRET in MDCK cells (Fig. 1b) and subsequently staining actin
with phalloidin-Alexa Fluor 568 (Fig. 1c). As a control, we also
transfected the free sstFRET cassette itself, i.e., without its linkage
to actinin, and the fluorescence image in Fig. 1d shows that free
probe is uniformly distributed in the cytosol (Fig. 1d).
Dynamics of cytoskeletal stresses under chronic flow
We followed changes in stress in cells subjected to a steady shear
flow (0.74 dyn/cm2) for up to 3 hrs and found that the average
stress decreased and then stabilized. Figure 2a shows time
dependent changes in the FRET ratio averaged over an entire
cell under flow; the (red) line marks the start of flow. Remarkably,
we consistently observed three distinct phases. In Phase-I, marked
t1–t2 in Fig. 2a, there was a rapid increase in FRET ratio (a
decrease in stress) lasting 1–2 min. This was followed by a
decrease in FRET (an increase in stress) that took ,3–6 min to
reach a minimum (Phase-II, t2–t3). This was followed by a long
term (,15 min) increase in FRET (decrease in stress) (Phase-III;
t3–t4). Following this, the FRET ratio stabilized or slightly
decayed under the continuous flow lasting up to 3 hrs (t.t4).
These dynamics were consistently observed in repeated experi-
ments (n.10) and the statistics of the peak FRET ratio change for
each Phase (left axis) and the time period (right axis) for three
phases are shown in (Fig. 2c). In contrast, for cells under no-flow
condition, the FRET ratio varied only slightly with time (Fig. 2b)
(its statistics are also shown in Fig. 2c).
Corresponding to the various phases shown in Fig. 2a, Figs. 2d,e
show the simultaneously acquired FRET images (Fig. 2d, movie
S2) and the underlying reorganization of F-actin (Fig. 2e, movie
S1). The image in Fig. 2e corresponding to Phase-I (Fig. 2e, t1–t2)
did not show significant actin redistribution indicating that the
decrease in stress at this stage is due to the deformation of actinin
proteins themselves; this is consistent with our previous results
. The short-term increase in stress during Phase-II is
associated with an inward contraction of F-actin. It is character-
ized by a shrinking nucleus and an increase in the density of F-
actin around the nucleus (Fig. 2e, t2–t3). No such ‘ring formation’
around the nucleus was observed in cells under a static
environment (movie S3, S4). Phase III, which was accompanied
by a large decrease in stress, was found to be associated with an
observable dissociation of actin, Fig. 2e (t3–t4). Longer times
following t4 are characterized by actin reassembly, resulting in F-
actin bundles at the cell perimeter (Fig. 2e, t5). Although the above
results are shown for a shear stress of 0.74 dyn/cm2, cells
responded similarly to stresses as low as 0.3 dyn/cm2but the
magnitude of responses was reduced (data not shown).
The spatiotemporal changes in cytoskeletal stress is further
analyzed by mapping time-dependent changes in FRET ratio and
F-actin distribution across a given region of interest (red windows
in Figs. 2d,e), and this analysis is shown in Fig. 2f and 2g,
Figure 1. Actinin-sstFRET sensor construction and expression in MDCK cells. a: Schematic of actinin-sstFRET sensors inserted in actin
cytoskeleton. The sensor consists of Cerulean as donor, Venus as acceptor, and a linker (spectrin repeat domain), and is inserted close to the middle of
actinin. b: Fluorescence image of actinin-sstFRET in MDCK cells. c: Fluorescence image of F-actin stained with phalloidin-Alexa Fluor 568, showing co-
localization of actinin-sstFRET with actin. d: Fluorescence image of MDCK cells expressing free sstFRET, showing uniformly distributed fluorescence
molecules in cytosol. The scale bars represent 10 mm.
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Figure 2. Shear stress adaptation and actin reorganization. a: Time dependent change of average FRET ratio in the upper cell in (d and e),
showing that changes in actinin stress occur in three distinct phases under shear stress of 0.74 dyn/cm2. b: Typical variation of average FRET ratio for
cell under static condition in the chamber. c: Statistics of FRET peak changes for three phases (n=7) and variation under static conditions (n=8) (red,
left axis); and statistics of time period for three phases and to saturation (n=7) (gray, right axis). (*) indicates peaks observed under flow. d,e:
Respective FRET ratio and CFP images of two cells subjected to shear stress at times indicated in (a). The FRET ratio is shown in 16 color map, where
blue indicates a low FRET ratio and higher tension; red indicates a higher FRET ratio and lower tension. The numbers in brackets indicate the duration
under flow. The arrow on the left of (d) indicates flow direction. The scale bar represents 10 mm. f, g: Time dependent FRET distribution along the
window in (d), and time dependent actin intensity along the window in (e). These distributions are correlated with various phases in (a), which is also
replotted for ease of discussion.
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respectively. (For convenience, the FRET ratio in Fig. 2a is also
shown juxtaposed with Fig. 2f,g). As seen in Fig. 2g, there is no
significant change in F-actin density or redistribution over the
course of phase-I (t1–t2), representing the earlier stage of
deformation of actinin proteins. A decrease in FRET ratio
(increase of stress) corresponding to phase-II (t2–t3) is seen in
Fig. 2f as a color change from warm (red/yellow) to cool (blue/
green); the accompanying inward movement of enhanced actin
density is marked by the black arrows in Fig. 2g indicating
contraction of the actin network around the nucleus by the end of
phase-II. The correlated increase in stress and actin contractile
motion suggests that the cytoskeletal tension is responsible for the
increase in F-actin concentration around the nucleus. Notice that
the dense actin ring persists in Phase-III (t3–t4, Fig. 2g), however,
the mean cytoskeletal stress decreases to a minimum during this
time (Fig. 2f). This observation suggests that contraction triggers F-
actin disassociation in cells. At t5, there is a high actin density at
the cell periphery (Fig. 2g) marking actin reassembly under flow
We showed that flow-induced cytoskeleton reorganization is a
reversible process at short times . To examine whether
reorganization can be reversed at longer times, we applied a steady
flow (0.74 dyn/cm2) for 3 hrs and then stopped the flow. By
measuring the changes in stresses and the structure we found that
cytoskeleton reorganization is also reversible over a time scale of
hours. Figure 3a shows the average stress in a cell measured as the
FRET ratio and Fig. 3b shows the simultaneously recorded F-actin
structure. Figure 3a shows that flow causes an increase in FRET
ratio that returns reversibly to its initial state upon removal of flow.
The corresponding images in Fig. 3b show that under flow (blue
arrows) the actin fibers gradually disappear, but they reappear
after the flow is stopped. It shows that the flow induced softening
and correlated cytoskeleton reorganization is reversible; the
parallel F-actin bundles were restored (Fig. 3b) and the cells
returned to a state of higher stress (lower FRET) when the flow
was stopped (Fig. 3a).
Inhibition of mechanosensitive channels diminishes
Previous studies have suggested that shear stress may activate
mechanosensitive channels (MSCs) and trigger Ca2+entry that
affects contractile forces and cytoskeleton structure [34,35]. To
examine the role of MSCs we measured the average FRET ratio
under flow, with and without MSC blockers: 100 mM Gd3+or
5 mM GsMTx4, the latter being the only known specific blocker of
MSCs. The shear stress-induced reduction in cytoskeletal stresses
was blocked by both Gd3+and GsMTx4 (Fig. 4a). Under static
condition the inhibitors had no obvious effects on the cells (Fig. 4b).
Figures 4c,d show the CFP images and FRET ratio, respectively,
of the cell in flow condition in the presence of Gd3+at various
times (Fig. 4a, also see movie S5, S6). Under shear there were no
distinct phases of stress variation (Fig. 4a) or any associated F-actin
accumulation around the nucleus in the presence of MSC
inhibitors. We have also observed that shear stress of 1 dyn/cm2
caused Ca2+increase in MDCK cells and the response can be
blocked by the same amount of MSC inhibitors (Fig. S1),
suggesting the involvement of Ca2+permeable MSCs in the
To examine actin itself we stained F-actin using phalloidin in
fixed cells. Figure 5a,b shows cells with no flow versus those
subjected to a shear stress of 0.74 dyn/cm2for 3 hrs, respectively.
They clearly show that actin bundles are randomly distributed
throughout the cells cultured under static condition (Fig. 5a). In
contrast, Fig. 5b shows that shear stress rearranges the F-actin
bundles to the cell periphery. This flow-induced F-actin reorga-
nization was abolished in the presence of 100 mM Gd3+. This is
shown in Fig. 5d,e where abundant F-actin can be seen in the
cytosol without and with flow, respectively, and the results were
consistent across the experiments (n=7) (Fig. 5c,f). Thus MSC-
mediated signaling is involved in the reorganization of the
cytoskeleton induced by shear stress.
Shear stress reduces cell mobility via MSC dependent
To determine whether the F-actin rearrangement induced by
shear stress affects mobility, we analyzed cell movement with a 2D
map of the nucleus location with and without flow. Under static
(no flow) conditions the cells were motile (Fig. 6a), and the time
lapse images (Fig. 6d) show typical movements. A shear stress of
0.74 dyn/cm2markedly reduced the cell motility (Fig. 6b) and
adding 100 mM Gd3+lifted the restriction on mobility (Fig. 6c).
Some cells under flow showed active expansion and contraction at
the edges, but the position of the nucleus remained fixed. We
suggest that this lack of migration is the result of reduced actin
driven traction forces since the basal stress fibers disappeared and
F-actin is peripheral under flow condition.
Tubular fluid flow in the kidney causes drag on the apical
surface of epithelial cells and these forces modulate cytoskeletal
stress. This in turn activates multiple intracellular signaling
pathways causing reorganization of cytoskeleton and cell remod-
eling. Our experiments allowed us to dissect the reorganization
into several distinct stages (Fig. 2a). The process seems to involve a
Ca2+influx through MSCs.
The initial decrease in cytoskeletal stresses is consistent with our
previous findings that sudden change in shear stress causes
reversible compression in a-actinin. Repetitive application results
in progressive adaptation . The shear stress amplitude in this
study is an order of magnitude smaller than in our previous study
indicating that we are far from saturation in these experiments. F-
actin shows no significant change within this time suggesting that
this process only involves the conformational changes of the
linking proteins or deformation of actinin itself (Fig. 2e). This
adaptation to mechanical stress has also been observed with
magnetic beads attached to the cell surface .
The shear-induced increase in cytoskeletal stress and corre-
sponding inward contraction of F-actin with time appears to be an
essential process for reorganization of the cytoskeleton since this is
followed by extended reduction in cytoskeletal stresses and
redistribution of F-actin to the periphery. Similar increase in
cytoskeletal stress (as in Phase-II) has also been observed using
magnetic beads at early stages of cell adaptation . Recently,
Martin, et. al. reported that pulsed apical contractions powered by
actomyosin pull the cytoplasm inward towards the nucleus .
This contractile force is known to facilitate actin polymerization
via actin binding proteins at focal adhesions . Thus, the
observed inward movement of actin during phase II (Fig. 2e,g)
indicates the contraction of actin network that triggers the
subsequent actin reassembly. Our results thus suggest that such
actin-myosin contraction can be activated by flow shear stress so
that the net effect of fluid shear stress acts via body stress in the
Long term adaptation seems to involve Rho GTPases and its
downstream targets. Rho-associated kinase (ROCK) is a key
regulator for cytoskeleton reorganization, focal adhesion develop-
ment and cell motility [38,39,40]. In MDCK cells, application of
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20 mM Y27632, a Rho-ROCK inhibitor, caused disassociation of
F-actin, and blocked the cytoskeleton remodeling in flow (Fig. S2).
Our results suggest Rho GTPases may participate in flow induced
actin reorganization. It has been reported that applying a
mechanical force to the cell surface of MDCK cells leads to Rho
translocation , and Rho-kinase regulates myosin light chain
phosphorylation that increases cell contractility and alters the
formation of actin stress fibers [19,39]. The observed contractile
force in phase-II (Fig. 2d,e) seems to be the result of Rho mediated
myosin contraction that facilitates the actin restructuring. In
addition, flow-induced Rho activation facilitates cytoskeleton
reorganization in endothelial cells [19,27,41,42,43] with an initial
transient inactivation that is followed by an increase peaking at
,60 minutes . Although this time scale (in the range of 5–
60 min) is similar to our observation for actin disassociation and
reassembly under flow (Fig. 2e), the cytoskeletal dynamics are
different for endothelial and epithelial cells probably because they
have different cytoskeletal prestress and organization .
The whole adaptation process can be initiated by MSCs since
changes in structure and motility were blocked by Gd3+and
GsMTx4. A similar effect has been reported with Gd3+for stresses
from magnetic beads . Flow-induced MSC activation and
Ca2+transients have been observed in MDCK cells [34,44]. We
have also shown that shear stress causes a transient Ca2+response
in MDCK cells and the Ca2+influx can be blocked by MSC
inhibitors Gd3+and GsMTx4 (Fig. S1). The time dependence of
the Ca2+response shows that intracellular Ca2+peaked within
1 min in flow, preceding the contractile actin ring formation.
These observations suggest a connection between Ca2+increase
and downstream signaling pathways, such as Rho GTPases
activation. The reorganization, however, does not require a
continued elevation of Ca2+, but the transient elevation appears to
lead to activation of a long lived later messenger such as Rho or
possibly altered prestress in the cytoskeleton.
In conclusion, fluid shear stress regulates cytoskeletal dynamics
triggered by Ca2+permeable MSCs and progressing through a
series of structural adaptations, leading to a reduction of
cytoskeletal tension. The process is illustrated schematically in
Fig. 7. This remodeling is important for the cell to minimize the
stress that it experiences and/or to optimize its structure to resist
the external force so as to minimize the internal stress gradients.
Materials and Methods
Actinin-sstFRET sensor construction
The sstFRET stress sensor was constructed with Cerulean (a
CFP) as the donor, Venus (an improved version of YFP) as the
acceptor, and a spectrin repeat domain as the linker that was
Figure 3. Reversible cytoskeleton reorganization under fluid shear stress. a: Changes of average FRET ratio in a cell subjected to shear
stress of 0.74 dyn/cm2for 3 hrs and subsequently in stop-flow condition, showing actinin softening in flow and hardening in the no-flow condition.
The changes in flow are indicated by red lines. b: Fluorescence images (CFP channel) at times indicated in (a), showing a reversible F-actin
reorganization. The scale bar represents 10 mm.
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subcloned from non-erythrocytic spectrin isoform 1 , Fig. 1a.
To insert sstFRET into a-actinin, we subcloned a-actinin into
pEYFP-C1 vector (Clontech, Mountain View, CA, USA) by
replacing the original YFP gene. The new construct was called
PEG-actinin. The sstFRET was then inserted into PEG-actinin to
get PEG-actinin-sstFRET. For maximum sensitivity, sstFRET is
inserted into the middle of actinin, at amino acid 300; further
details of the construction are given elsewhere . The sstFRET
stress sensors have previously been characterized for their stress
sensitivity and it has also been shown that the sensors have no
adverse effect on the host protein .
Microfluidic chamber and environmental control
The microfluidic chip consists of a PDMS flow channel that is
500 mm wide, 100 mm high, and 10 mm long. The flow channel is
held between two parallel glass plates; a glass cover slip base and a
glass top. Holes through the PDMS form the inlet and outlet of the
channel. For long-term imaging under flow, the microfluidic chip
was placed in an incubator (INUB-ZILCSD-F1-LU, Tokai Hit
CO., Ltd, Japan) at 37uC and 5% CO2. Cell culture media was
perfused through the chamber using a syringe pump (Harvard
Apparatus PHD2000). The fluid shear stress (t) was calculated
using the relationship t=6Qm/wh2, where Q is the flow rate, m is
the dynamic viscosity of the perfusing media, w and h are the width
and the height of the channel, respectively; a viscosity value of
0.861023Pa?s was used .
MDCK cells (ATCC) were cultured directly in the microfluidic
chamber. Cells were cultured in Dulbecco’s Modified Eagle
Medium (DMEM) having 10% fetal bovine serum, with 1%
penicillin and streptomycin. To seed the cells in the flow chamber,
the microfluidic chamber were first coated with fibronectin and
then the media with suspended cells was perfused into the
chamber using a pipette. The microfluidic chip was placed in the
incubator and culture media was changed every 24 hours.
Experiments were conducted after culturing the cells for 3 days
with typical confluence of 80–90%. Cells were transfected with
sstFRET a-actinin 24 hours before the experiments using
FuGENE 6 transfection reagent (3.3:1, FuGENE 6 volume to
DNA weight ratio). Average transfection efficiency was 20–30%.
During the experiments, the chamber was perfused with Phenol-
red free DMEM (InvitrogenTM) in order to eliminate background
fluorescence from the media.
FRET imaging and analysis
Fluorescence imaging was done using a Zeiss inverted
microscope with 636 oil immersion objective (Axiovert 200 M,
Figure 4. Fluid shear stress-induced cytoskeletal stresses in the presence of MSC inhibitors Gd3+and GsMTx4. a,b: Application of
100 mM Gd3+(solid squares) or 5 mM GsMTx4 (unfilled squares) inhibited the dynamic changes in actinin stresses under flow, showing only small
variations. c,d: Fluorescence images (CFP channel) and FRET ratio at various times in (a) (solid square), showing that shear stress did not cause
significant redistribution of cytoskeleton, and cell showed active locomotion. The scale bar represents 10 mm.
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Zeiss). Images were acquired using Hamamatsu EM-CCD camera
(ImagEM C9100-13, Hamamatsu, Japan). Both emission wave-
lengths (CFP and YFP) were acquired simultaneously using a dual-
view optical system (Optical Insights, USA) having an excitation
filter set that includes a bandpass filter (436620 nm) and a
dichroic mirror (455DCLP), and an emission filter set that includes
Figure 5. Effect of mechanosensitive channel inhibitors on flow-induced cytoskeleton reorganization. Immunostained F-actin in MDCK
cells under a: static condition and b: shear stress of 0.74 dyn/cm2for 3 hrs. c: Statistics of actin arrangements under static (red) and shear stress
(green). The percentage was estimated for each experiment, and the error bars show standard errors of ‘‘n’’ experiments (static, n=7; shear flow,
n=10). d: F-actin staining in the presence of 100 mM Gd3+in cells in static condition and e: under shear flow of 0.74 dyn/cm2for 3 hrs, showing Gd3+
inhibits flow induced cytoskeleton reorganization. f: Statistics of actin arrangements of (d) vs (e) (static+Gd3+: n=4; flow+Gd3+: n=8). The scale bar is
Figure 6. Effect of shear stress on cell motility. a–c: Trace of single cell movement tracked by centroid of the nucleus for 2.5 hrs under a: static
condition; b: shear stress of 0.74 dyn/cm2; and c: same flow condition with addition of 100 mM Gd3+. The cell’s original location (defined by the center
of nucleus) is at (0,0) and the motion is tracked along the x and y axis every 16 min for a total ,160 min. The arrows in b and c indicate flow
directions. d: CFP images showing the typical movement of a cell under static condition. The yellow stars in (d) indicate the center position of nucleus
that was traced and plotted in (a). The scale bar represents 10 mm.
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two emission filters (480630 nm and 535640 nm) and a dichroic
mirror (505DCXR). Time-lapse images were obtained using Zeiss
software (Axiovision, Zeiss). The images from CFP and YFP
channels were aligned and processed to obtain the ratio of
acceptor to donor (FRET A/D ratio) using Image-J (NIH)
software following previously published methods [28,46]. The
bleedthrough of CFP into YFP was measured prior to the
experiments and subtracted from YFP images to obtain the true
FRET signal. The spatiotemporal variation of the FRET ratio is
shown in a 16 color map. The warm colors (red) indicate a higher
ratio, i.e., a lower a-actinin stress, while cool colors (blue) indicate
a lower FRET ratio (higher a-actinin stress).
Cells were treated with 4% paraformaldehyde at room
temperature for 15 minutes and washed three times with
phosphate buffered saline (PBS). Cells were then incubated in
0.5% Triton X-100 solution for 15 min and washed three times
with PBS. Cells were stained with 0.16 mM phalloidin-Alexa Fluor
568 (Invitrogen) for 20 minutes, washed three times with PBS and
were then observed under the microscope.
Ca2+response to flow shear stress. Time course
of intracellular Ca2+response to a stepwise increase in shear stress
from 0.03 to 1 dyn/cm2in control (red dots), 100 mM Gd3+(solid
squares), and 5 mM GsMTx4 (unfilled squares), showing that Ca+2
influx was blocked by MSC inhibitors.
formation of actin fibers and cytoskeletal stress. a: Live
cell imaging of actinin-sstFRET (CFP channel) during the
application of Y27632 at times indicated in (b), showing that
blockage of Rho-ROCK reversibly disassociates actin stress fibers.
The scale bar represents 10 mm. b: Average FRET ratio over the
two cells in (a). c: FRET ratio measured in a cell subjected to shear
stress of 0.74 dyn/cm2in the presence of 20 mM Y27632, showing
Effect of Rho-ROCK inhibitor Y27632 on the
that the inhibitor blocked the flow induced changes in cytoskeleton
ing MDCK cells in response to a shear stress of 0.74 dyn/cm2,
showing the shear induced actin reorganization. The arrow (top
left) indicates the start of flow. The nucleus ‘ring formation’, few
minutes after application of shear, and later peripheral actin
arrangement is readily observable. The images were captured
every 2 min for a total of 3 hours.
Time-lapse CFP images of actinin-sstFRET express-
0.74 dyn/cm2, showing shear induced FRET changes and a net
increase in FRET ratio upon the application of shear. The arrow
indicates the start of flow. The images were captured every 2 min
for 3 hours.
FRET response of MDCK cells to a shear stress of
ing MDCK cell in static environment. Actin dynamics that were
observed in cells under shear are absent and the cell is mobile. The
images were taken every 3 min for 3 hours.
Time-lapse CFP images of actinin-sstFRET express-
condition showing only slight variations with time. The images
were captured every 3 min for a total of 3 hours.
Typical FRET changes in MDCK cell under static
ed MDCK cell in response to a shear stress of 0.74 dyn/cm2in the
presence of 100 mM Gd3+, showing no significant redistribution of
actin due to shear. The arrow (top left) indicates the onset of flow.
The images were taken every 2 min for a total of 3 hours.
Time-lapse CFP images of actinin-sstFRET transfect-
0.74 dyn/cm2in presence of 100 mM Gd3+, showing only small
variations and no apparent change in FRET. The arrow indicates
FRET response of MDCK cell to a shear stress of
Figure 7. Schematic of proposed multiphase-adaptation to fluid shear stress in epithelial cells.
Cytoskeletal Stress and Cell Adaptation
PLOS ONE | www.plosone.org8September 2012 | Volume 7 | Issue 9 | e44167
the start of flow. The images were taken every 2 min for a total of
Conceived and designed the experiments: DV SZH. Performed the
experiments: DV NY FM JR. Analyzed the data: DV FS SZH.
Contributed reagents/materials/analysis tools: DV FM. Wrote the paper:
DV FS SZH.
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