MBoC | ARTICLE

Electric field–induced migration and intercellular

stress alignment in a collective epithelial
monolayer
Youngbin Cho, Minjeong Son, Hyuntae Jeong, and Jennifer H. Shin*
Department of Mechanical Engineering, Korea Advanced Institute of Science and Technology, Daejeon 34141,
Republic of Korea

ABSTRACT During wound healing, cells migrate with electrotactic bias as a collective entity. Monitoring Editor
Unlike the case of the electric field (EF)-induced single-cell migration, the sensitivity of electro- Yu-Li Wang
Carnegie Mellon University
tactic response of the monolayer depends primarily on the integrity of the cell–cell junctions.
Although there exist biochemical clues on how cells sense the EF, a well-defined physical por- Received: Feb 7, 2018
trait to illustrate how collective cells respond to directional EF remains elusive. Here, we de- Revised: Jun 14, 2018
veloped an EF stimulating system integrated with a hydrogel-based traction measurement Accepted: Jul 20, 2018
platform to quantify the EF-induced changes in cellular tractions, from which the complete
in-plane intercellular stress tensor can be calculated. We chose immortalized human keratino-
cytes, HaCaT, as our model cells to investigate the role of EF in epithelial migration during
wound healing. Immediately after the onset of EF (0.5 V/cm), the HaCaT monolayer migrated
toward anode with ordered directedness and enhanced speed as early as 15 min. Cellular trac-
tion and intercellular stresses were gradually aligned perpendicular to the direction of the EF
until 50 min. The EF-­induced reorientation of physical stresses was then followed by the de-
layed cell-body reorientation in the direction perpendicular to the EF. Once the intercellular
stresses were aligned, the reversal of the EF direction redirected the reversed migration of the
cells without any apparent disruption of the intercellular stresses. The results suggest that the
dislodging of the physical stress alignment along the adjacent cells should not be necessary
for changing the direction of the monolayer migration.

INTRODUCTION
Cells divide, differentiate, migrate or die in response to various phys- ble molecules. In addition to biochemical factors, all cells produce
iological cues from the microenvironment. Among many factors that membrane potential by segregating ions and charged molecules
trigger cellular responses, the most prevalent cues are biochemical between plasma membranes to generate endogenous electric fields
origins such as hormones, cytokines, growth factors, and other solu- (EFs) from the early embryonic development (Funk, 2015). Bioelec-
tricity, an endogenous electrical cue, can override most chemical
gradients to promote electrotactic response, termed electrotaxis.
This article was published online ahead of print in MBoC in Press (http://www Electrotaxis, the phenomenon by which cells migrate directionally to
.molbiolcell.org/cgi/doi/10.1091/mbc.E18-01-0077) on July 25, 2018.
electrical stimulation, affects a number of physiological processes
The authors declare that they have no competing financial interest.
Author contributions: Y.C. and H.J. performed the experiment. Y.C., M.S., and
such as embryonic development, directing nerve cell growth, angio-
J.H.S. analyzed and discussed the data. Y.C. and J.H.S. wrote the article. genesis, cancer metastasis, and wound healing (McCaig et al., 2004;
*Address correspondence to: Jennifer H. Shin ([email protected]). Zhao, 2009; Cortese et al., 2014). When exogenous EFs are applied
Abbreviations used: dcEF, direct current electric field; EF, electric field; MSM, to cells in culture to mimic the naturally occurring EF, they exert pro-
monolayer stress microscopy; PDMS, polydimethylsiloxane; TFM, traction force
microscopy. found polarization effects, directing the cellular migration.
© 2018 Cho et al. This article is distributed by The American Society for Cell Biol- Cell migration is constitutive for multiple physiological settings
ogy under license from the author(s). Two months after publication it is available to position the cells at appropriate places at a right timing during
to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported
Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).
biological processes. For example, during the process of wound
“ASCB®,” “The American Society for Cell Biology®,” and “Molecular Biology of healing, the cells in our body must know not only when but, very
the Cell®” are registered trademarks of The American Society for Cell Biology. importantly, in which direction to migrate, for effective healing of

2292 | C. M. Hickey et al. Molecular Biology of the Cell

the wounded tissue. Numerous in vitro experiments confirmed the changes in the physical state of junctional proteins, the quantifica-
EF-induced directional migration in many cell types such as cor- tion of physical stresses that govern the EF-induced collective mi-
neal epithelial cells (Zhao et al., 1996, 1999, 2006; Song et al., gration requires further investigation. More specifically, experimen-
2002), endothelial cells (Zhao et al., 2003), keratocytes (Cooper tal observations on the EF-induced changes in the underlying
and Schliwa, 1985; Sun et al., 2013), keratinocytes (Nakajima et al., tractions exerted by each cell on its substrate, and intercellular
2015), and breast cancer cells (Mycielska and Djamgoz, 2004; Pu stresses exerted between immediate neighbors, have been a signifi-
et al., 2007). Both the speed and direction of electrotaxis are cell- cant challenge. Consequently, a well-defined physical portrait to il-
type dependent. The typical range of physiologically relevant EF lustrate how collective cells respond to directional EF remains
has been reported to be 0.1–10 V/cm. The physiological range of elusive.
EF also induced significant morphological changes in many cell In this work, we developed an EF stimulating system integrated
types, including endothelial cells (Zhao et al., 2003), epithelial cells with a hydrogel-based traction measurement platform to quantita-
(Luther and Peng, 1983), neural crest cells (Cooper and Keller, tively measure the EF-induced changes in cellular tractions (Trepat
1984), and osteoblasts (Curtze et al., 2004). The EF-induced reori- et al., 2009), from which the complete in-plane intercellular stress
entation was accompanied by the asymmetric redistribution of cy- tensor can be obtained (Tambe et al., 2011). Our observations
toskeletal structures such as actin stress fiber (Luther and Peng, showed initially randomly migrating HaCaT cells immediately re-
1983) and microtubule (Song et al., 2002) as well as Golgi appara- sponded and rapidly synchronized their migration toward the anode
tus (Pu and Zhao, 2004). A number of researchers investigated the within 15–20 min after the onset of 0.5-V/cm EF stimulation. Cellular
biomolecular intracellular signaling pathways to reveal how the traction and intercellular stresses were gradually aligned perpen-
cells sense and control the polarity in response to the directional dicularly to the direction of the EF until 50 min after stimulation.
electric cue at a single-cell level (McCaig and Zhao, 1997; Furthermore, intercellular stress tensor perpendicular to the EF di-
Robinson, 1985). The intracellular “compass model” suggests a rection increased significantly while the stress tensor parallel to the
competition between the PI3K-dependent pathway at the front EF direction decreased during the EF application. The overall mag-
and the myosin-dependent pathway at the rear of the cell that nitude of average intercellular stress maintained or slightly increased
determines the direction of single-cell migration by the active for- during the EF stimulation experiment. This reorientation of physical
mation of lamellopodia in directional response to the applied EF stresses under EF stimulation preceded the morphological reorien-
(Sun et al., 2013). The dcEF was shown to induce a polarized acti- tation perpendicular to the EF direction. Once the intercellular
vation of several other signaling pathways such as phosphatase stresses were aligned, the disruption of the intercellular stress orien-
and tensin homologue (PTEN), epidermal growth factor (EGF) tation was not necessary for changing the direction of monolayer
receptors, mitogen-activated protein kinase (MAPK), extracellular- migration during the reversal of EF direction.
signal-regulated kinase (ERK), and Src (Fang et al., 1999; Zhao
et al., 1999, 2002, 2006; Pu et al., 2007). Furthermore, with the RESULTS
advancement of techniques to visualize cellular traction, few re- Establishment of the EF–TFM (traction force microscopy)
searchers observed the surprisingly immediate response of cellular chamber
traction to the applied EF, which preceded the polarized rear- To quantify the immediate electrotactic response of a collective
rangement of the intracellular cytoskeleton in the cells cultured in monolayer, we set up the EF stimulating system with the following
low density (Harris et al., 1990, Curtze et al., 2004). These findings specifications: 1) small size and transparency for real-time imaging
indicate that the physical traction may be the very early target of on the microscope, 2) stable EF stimulation without fluctuations of
the EF-induced polarized signaling pathway during the electrotac- temperature or pH, and 3) hydrogel-based traction measurement
tic response. platform. For the size and transparency constraints, 76 × 52-mm-
The limitation of current knowledge is that the studies on the sized glass slides were used for the base part with a cover made of
electrotactic response dealt with the cells that are in isolation with- transparent polydimethylsiloxane (PDMS) membrane. To warrant
out mature cell–cell adhesions. However, cellular motility in many stable EF stimulation without disturbing cell culture condition, we
physiological conditions concerns with the cluster of cells held by redesigned the EF stimulation platform by implementing several
intimate cell–cell contacts. Especially during embryonic develop- key geometrical features from the existing EF devices (Song et al.,
ment and wound healing, a sheet of cells expands with electrotactic 2007; Cohen et al., 2014). Most importantly, the specific geometry
bias as a collective pack. Each cell in a cellular cluster is physically and the medium reservoir height (Figure 1b) were carefully selected
coupled to neighboring cells confining the cells in the monolayer to minimize the undesirable effects of electrochemical byproducts
while suppressing active lamellipodia. Consequently, the cell-cell and pH change while guaranteeing the linear EF lines (simulated by
interactions may induce the differential impact of the EFs on cells in COMSOL Multiphysics Software in Figure 1c). Since the shallow
the confluent monolayer, compared with the isolated cells. Recent clearance in the cell observation region yields high electrical resis-
studies demonstrated the different electrotactic response between tance compared with the medium reservoir with higher depth, up
single cells and a collective monolayer. Li et al. showed that the epi- to 60% of total voltage drop can be concentrated at the cell obser-
thelial cells in a monolayer migrated far more efficiently with better vation region (Figure 1d) (Cohen et al., 2014). Also, a sufficient vol-
directional persistence compared with those in isolation or smaller ume of medium in the chamber reservoir helped to maintain a sta-
clusters (Li et al., 2012). Interestingly, even within the monolayer, ble pH and temperature within the chamber (Song et al., 2007).
cells near the free edge, which show the distinct behavior compared Finally, the most critical feature of our device is the hydrogel-based
with the cells in the bulk of the monolayer such as the emergence of traction measurement platform inside the EF stimulation system. To
leader cells with aligned actin stress fiber and weak E-cadherin, did measure the cellular forces, we cultured the HaCaT cell monolayer
not show the efficient electrotactic migration (Cohen et al., 2014). on a fluorescence-bead-embedded polyacrylamide (PA) gel within
Thus, one can speculate that the sensitivity of collective electrotactic the cell observation region (Figure 1b).
response depends primarily on the integrity of the cell–cell junc- A glass slide and the first PDMS membrane with the pattern were
tions. While there exist biochemical clues on the EF-induced first treated with O2 plasma for the permanent bonding between

Volume 29 September 15, 2018 Behavior of cell monolayer under EF | 2293

FIGURE 1: Establishment of EF stimulating chamber implemented with force measurement system. (a) Schematics of
the chamber components and fabrication process. Polyacrylamide gel is embedded in the chamber for the force
measurement. (b) Schematics of the experimental set-up for EF stimulation. Chamber is connected to the power supply
through agar bridge and Steinberg’s solution. Red dotted box in the middle of the chamber indicates the observation
region for imaging. In the observation region, fluorescent bead–embedded PA gel is fabricated on the slide-glass
surface. HaCaT monolayer was cultured on the PA gel to measure the traction force during the EF stimulation.
(c) COMSOL simulation of the linear EF line at the cell observing area. (d) Experimental measurement of the voltage
drop within the chamber. Owing to the high electrical resistance, up to 60% of total voltage drop was concentrated at
the cell observation region (blue shaded region), indicating the enhancement of the voltage drop efficiency of the
chamber.

two layers. After bonding two layers, the glass surface at the center sponse was so rapid that the directed migration became predomi-
of the observation region was pretreated with silane for the stable nant through the monolayer as early as 10 min after the onset of EF
linkage between the glass and PA gel. Then, 10 μl of PA gel (3 kPa; application. Continued exposure to the 0.5 V/cm EF for 30 min, the
5.5% acrylamide, 0.09% bisacrylamide, 0.5% ammonium persulfate) cell migration became almost completely ordered in the direction
mixed with 0.5-μm-diameter fluorescent beads was polymerized by parallel to the EF with the directedness value of 0.97 where the di-
pressing the 12-mm-diameter cover glass on the PA gel drop. Dur- rectedness value of 1 indicates a perfect migratory alignment with
ing the polymerization of PA gel, the whole base part was centri- the direction of the EF (Figure 2f; see Materials and Methods for
fuged upside down for 15 min at 1000 rpm to pull up the beads to details on the directedness). As such, the parallel component of the
the surface of the gel. After polymerization, the surface of the PA gel migration velocity (V‖) of the collective cells was greatly enhanced
was functionalized by Sulfo-SANPAH and then coated with 10 µg/ml by approximately fourfold compared with the case without EF ap-
collagen I for the cell attachment. To culture the HaCaT cells as a plication. However, the perpendicular component of the velocity
confluent monolayer slab, we prepared 250-μm-thick PDMS stencil did not show any significant change (Figure 2g).
with 1.8 mm2-sized rounded rectangular patterns and gently pressed
the stencil down on the gel surface. HaCaT cells were seeded within Perpendicular reorientation of cell body to the EF direction
the PDMS stencil and incubated for 12 h to establish the confluent lags electrotactic migration
monolayer with mature cell–cell junctions. Before the start of the After the onset of DC EF, initially randomly oriented HaCaT cells
experiment, we peeled off the PDMS stencil to prevent the distor- gradually changed their orientation in the direction perpendicular to
tion of the EF near the cell monolayer. We then finalized the assem- the EF (Figure 3, a, c–f). We compared the time-course electrotactic
bly of the chamber by placing a 6-mm-thick second PDMS layer, response of cell migration and cell-body reorientation using the rose
pretreated with O2 plasma, onto the first PDMS membrane to seal plot at discrete time points of pre-EF, and 30, 50, 100 min after 0.5
the observation region. Agar bridges were placed between the res- V/cm dcEF application (Figure 3, b and g–j). While the anodal elec-
ervoirs and the Steinberg’s solutions with an Ag/AgCl electrode trotactic migration response was immediate, the cellular reorienta-
connected to the voltage controlled power source. tion happened gradually over 100 min. We took the absolute values
for both velocity and cell-body orientations to plot the data within
Immediate electrotaxis of collective epithelial monolayer 0°–90° range. The cellular velocities were calculated by the PIV anal-
in the direction of EF ysis, and the changes in the cell-body orientation were obtained by
In the absence of EF, HaCaT cells within the confluent monolayer the ellipsoidal fit using the ImageJ (see Materials and Methods and
migrated in random directions (Figure 2a). At the onset of 0.5 V/cm Supplemental Figure S1). The red bars depict the velocity orienta-
direct current EF (dcEF), the cells began to migrate toward the an- tion, and the blue bars represent the cell-body orientation. Before
ode (upper direction in Figure 2, b–e). The anodal migration re- the EF stimulation, the velocity and cell-body orientation were both

2294 | Y. Cho et al. Molecular Biology of the Cell

FIGURE 2: Immediate electrotactic migration in a collective monolayer. (a) Migration of HaCaT monolayer in the
random direction at the pre-EF condition. (b–e) Time-course alignment of migration of HaCaT monolayer with the onset
of EF stimulation. (b) Five minutes after start of EF, the monolayer still migrated in the random direction. (c) Within
15 min, the monolayer initiated the anodal migration. (d, e) With time, migration of overall monolayer aligned toward
the anode. (f) Average migration directedness and (g) average migration velocity of HaCaT monolayer over time. Data
are presented in 10-min intervals. Dashed lines indicate the time point of EF initiation. Data are presented as mean ± SD
(n = 3 independent monolayers). Scale bars, 50 µm.

randomly distributed (Figure 3b). With the initiation of EF stimula- et al., 2009; Maruthamuthu et al., 2011; Tambe et al., 2011). Cells
tion, the cellular velocities were biased early in the direction parallel communicate intimately with the surroundings and neighboring
to the EF within 30 min (Figure 3g). However, the cell body showed cells through these cellular junctions (Gomez et al., 2011). Both junc-
the tendency of perpendicular reorientation from 70 min after the tion types are associated tightly with various structural molecules
EF stimulation (Figure 3i) and became apparent by 100 min (Figure and cytoskeletal networks intracellularly (Vasioukhin et al., 2000; le
3j). Our data suggest that in HaCaT monolayer, the collective cellu- Duc et al., 2010; Jang et al., 2017). Cohen et al. (2014) reported a
lar electrotactic migration happens immediately after the onset of detailed portrait of the electrotactic migratory response of collective
the EF, without having to require cellular reorientation or cytoskele- Madin–Darby canine kidney (MDCK) cells. Motivated by these find-
tal rearrangement. Cell-body orientation, in fact, seems to follow the ings, we became curious about the physical stress states of the EF-
electrotactic migratory response with a time delay. This time delay induced migration of the collective skin epithelial cell, HaCaT. To
may be due to the inherent property of the confluent monolayer investigate the state of cellular stresses in response to the EF, we
where the neighboring cells are tightly bound to one another, hin- first quantified local traction forces exerted by collective HaCaT
dering the morphological degree of freedom. monolayer using Fourier-transform traction microscopy (Trepat
et al., 2009). In both the pre- and post-EF stimulation, cellular trac-
EF reorients cellular traction forces and intercellular stresses tion showed substantial spatial heterogeneity with dynamic fluctua-
When the cells migrate as a collective in a monolayer, cells generate tions in the magnitude and direction, evidenced by a color change
both traction forces and intercellular stresses through the cell– in the locations marked by dotted circles (Figure 4, c–f). Tx repre-
substrate adhesions and cell–cell adhesions, respectively (Trepat sents the x component of traction, which is the axis perpendicular to

Volume 29 September 15, 2018 Behavior of cell monolayer under EF | 2295

FIGURE 3: Perpendicular alignment of cell body follows the electrotactic migration. Phase contrast images of HaCaT
monolayer under (a) pre-EF condition and (c–f) EF stimulation. Overlay of the angular distribution of cell velocity (red
bars) and cell body (blue bars) at (b) pre-EF condition and (g–j) EF stimulation. (b) At the pre-EF condition, both cell
velocity and cell body showed no preferred orientation. With the onset of EF stimulation, (g) cell velocity showed
apparent alignment in the direction parallel to the EF within 30 min, while (h) the cell body still exhibited no biased
alignment until 50 min. (i) After 70 min, the cell body showed the tendency of reorientation in the direction
perpendicular to the EF, and (j) after 100 min, the cell body now showed apparent alignment in the direction
perpendicular to the EF. Scale bars, 50 µm.

EF direction in our experimental setting (Figure 4, c and d), where Poisson’s ratio ν. Young’s modulus E is not relevant to the MSM solu-
the red indicates upward traction exerted on the substrate by the tion, but Poisson’s ratio ν can induce the inexact solution. Neverthe-
cells and the blue indicates downward traction exerted on the sub- less, the effect of Poisson’s ratio has been shown to be quite small;
strate by the cells. Ty represents the y component of traction, the axis therefore, the solution recovers a wide range of the cell monolayer
parallel to EF direction (Figure 4, e and f; the red indicates rightward property (Tambe et al., 2011, 2013). Recently, to exclude the effect
traction, and the blue indicates leftward traction). When the average of the cell monolayer rheology, an alternative method to measure
magnitudes of the traction components were plotted as a function of stresses has been suggested (Nier et al., 2016). On the basis of the
time, we noticed a gradual, persistent decrease in the parallel com- previous report by Li et al. (2012) on the role of EF on intercellular
ponent of traction with respect to the field direction (|T y |, the red line junctions, we hypothesized that the intercellular stress should play a
in Figure 4g). However, the average traction magnitude perpendicu- crucial role in the electrotactic response of the epithelial monolayer.
lar to the EF direction ( | T x |, the blue line in Figure 4g) showed neg- To validate this, we utilized the MSM to visualize the intercellular
ligible change. Thus, the | T x | |T y | ratio (gray line in Figure 4g) exhib- stresses within the monolayer in both pre- and post-EF stimulation.
ited significant, steady increase for first 50 min after the onset of The average normal intercellular stress of the HaCaT monolayer be-
the EF stimulation. Fifty minutes of EF stimulation was shown to be fore the EF stimulation was tensile throughout the monolayer (com-
insufficient for the completion of cell-body orientation. pressive normal stress would have negative values) with the hetero-
Next, local intercellular stresses exerted among neighboring geneous spatial distributions with varying magnitudes (Figure 5a).
cells were calculated using the monolayer stress microscopy (MSM) With the onset of EF, the overall magnitude of the average intercel-
technique (Tambe et al., 2011, 2013). The MSM method encom- lular stress slightly increased while the spatial heterogeneity was still
passes the principle that the local tractions must be balanced by maintained (Figure 5, b–d, gray line in q). Since the EF contains the
the intra- and intercellular forces according to Newton’s laws. This directional information, scalar value of intercellular stress alone
method rests on the assumption that the cell monolayer behaves as would be insufficient to describe the physical stresses. We decom-
a continuous, linear elastic material with Young’s modulus E and posed the local intercellular stress into x-y components where σxx

2296 | Y. Cho et al. Molecular Biology of the Cell

 stress anisotropy, and the orientation of each ellipse shows the local
principal stress orientation. Each ellipse was color coded to repre-
sent the degree of ellipse orientation departure from the y-axis,
which is the axis representing the EF direction. Before EF stimula-
tion, the orientation of ellipse distributed in all directions between
0° and 90° over the monolayer. Application of EF stimulation in-
duced remarkable reorientation of intercellular stress toward the x-
axis perpendicular to the EF direction (90° from the y-axis, red col-
ored ellipse in Figure 5, n–p).

Physical stress reorientation precedes cell-body

reorientation
From the observation of anisotropic change in the intercellular
stress, we then asked which of the stress rearrangement and cell-
body orientation comes first. To answer this question, we compared
the time-course rearrangement of cell migration velocity, cell-body
orientation, and intercellular stress orientation simultaneously by
overlapping all data on the rose plots at pre-EF, 30 min, and 100 min
after EF stimulation, respectively (Figure 5, s–u). At the pre-EF con-
dition, all of the cell migration (red bars), cell body (blue bars), and
intercellular stress (yellow bars) randomly oriented without any par-
ticular directedness (Figure 5s). Velocity field started to align in the
direction parallel to the EF direction as early as 10 min after the on-
set of EF stimulation and aligned almost fully by the 25-min time
point. However, intercellular stress exhibited no significant align-
ment until 20 min after the onset of EF and gradually aligned in the
direction perpendicular to the EF direction (Figure 5r). After 30 min
from the onset of the EF, nearly 70% of overall intercellular stress
showed the considerable reorientation toward the perpendicular
axis with respect to the EF direction, distributed between 60° and
90° from the EF direction. Between the migration velocity and inter-
cellular stress, there existed lagging of stress behind the migration.
However, cell-body orientation did not exhibit any preferred direc-
tion within 30 min (Figure 5t). At 100 min from the onset of EF stimu-
FIGURE 4: EF induces a polarized change of cellular traction force. lation, the cell migration was mostly synchronized along the EF
(a, b) Phase contrast images, (c, d) maps of x component of traction direction, and the intercellular stress dominantly well aligned per-
(perpendicular to EF direction), and (e, f) the y component of traction pendicular to the EF direction. At this time point, cell body now
(parallel to EF direction) at (a, c, e) pre-EF condition and (b, d, f) EF showed significantly biased orientation aligned with the intercellular
stimulating condition. White dotted circles indicate the locations of stress (perpendicular to the EF direction) (Figure 5u). Our results
dynamic traction fluctuations in direction and magnitude. (g) Time
confirmed that cell-body orientation happened at last, following
evolution graph of the average magnitude of Tx (blue line) and Ty (red
rapid electrotactic migration response and then physical stress
line) and the ratio of magnitude between Tx and Ty (gray line). Data
are presented in 10-min intervals. Dashed line indicates the timepoint alignment in the monolayer. Reorientation of intercellular tensile
of EF initiation. Data are presented as mean ± SD (n = 3 independent stress in the direction perpendicular to the EF must correlate closely
monolayers). Scale bars, 50 µm. with intracellular cytoskeletal rearrangement, leading to the cell-
body orientation in the same direction.
corresponds to the stress tensor perpendicular to the EF direction
and σyy corresponds to the stress tensor parallel to the EF direction. Reversed EF redirects migration of collective monolayer
Before the EF stimulation, σxx and σyy were similar in magnitude without rearrangement of intercellular stress
(Figure 5, e and i, blue and red line in q). After the onset of EF, During the abrupt change in the EF direction, cells in isolation could
however, σxx increased dramatically (Figure 5, f–h, blue line in q) change direction by either reversing their polarity or by making a
while σyy was only slightly affected (Figure 5, i–l, red line in q). This smooth U-turn in space in the absence of the physical constraints
prominent increase in perpendicular stress tensor indicates the ac- from neighboring cells or obstacles (Allen et al., 2013). What would
cumulation of tensile stress in tangential direction against the collec- happen if the EF direction were reversed in a monolayer of cells
tive migration in the EF direction. In particular, the time period of tightly held together by intercellular tension? A recent study by
0–50 min was when the cells are migrating without reorientation of Cohen et al. (2014) reported evidence that showed the local collec-
the cell body. The sudden drop in σxx at 80 min may correlate with tive “U-turn” redirection in an MDCK cell monolayer.
the loosening of intercellular tension as cells were allowed to reori- To clarify the physical basis for the redirection mechanism of the
ent in their ultimately preferred direction. We further investigated monolayer, we analyzed the intercellular stress orientation during
the intercellular stress anisotropy using the stress ellipse (Figure 5, the reversal of the EF. In our experiment, the monolayer was shown
m–p). The major axis of each ellipse indicates the maximum princi- to migrate upward in a synchronized manner under the continued
pal stress, and the minor axis indicates the minimum principal stress. exposure to EF for 100 min, the anode being on top of the page
The circularity of each ellipse indicates the degree of intercellular (Figure 6a). After a sudden reversal of the EF by exchanging the

Volume 29 September 15, 2018 Behavior of cell monolayer under EF | 2297

FIGURE 5: EF induces polarized intercellular stress that precedes the cell-body alignment. Maps of (a–d) average normal
intercellular stress), (e–h) σxx (stress component perpendicular to the EF direction), and (i–l) σyy (stress component parallel
to the EF direction) overlaid on phase contrast images at pre-EF and EF stimulating condition. With EF, σxx largely
increased while σyy slightly decreased. (m–p) Color-coded stress ellipses overlaid on phase contrast images at pre-EF and
EF stimulating condition. Color-code indicates the orientation of stress ellipse from the EF direction. (q) Time evolution
graph of intercellular stress magnitude in 5-min time intervals. The ray line shows average normal intercellular stress, the
blue line shows σxx, and the red line shows σyy. Lines indicate mean value (n = 3 independent monolayers). (r) Time
evolution graphs of migration directedness (black line) and stress orientation (gray line) in 5-min intervals. Degree
indicates departure of stress orientation from the y-axis (direction of EF). 0° indicates parallel direction to EF and 90°
indicates perpendicular direction to EF. Data are presented as mean ± SD (n = 3 independent monolayers). Migration
started to align already from 10 min after EF start and dominantly aligned around 25 min after EF stimulation. However,

2298 | Y. Cho et al. Molecular Biology of the Cell

anode and the cathode, HaCaT cells underwent a transient change was sensed by the cells. A number of studies identified the cellular
in their migration behavior (Figure 6b) and finally synchronized membrane as the primary sensor for the EF (Huang et al., 2009;
themselves to migrate toward the new anode (Figure 6c). During Minc and Chang, 2010; Allen et al., 2013; Sun et al., 2013). EF-in-
the transient repolarization of migration, the upward directedness duced asymmetry in the membrane characteristics was believed to
of migration diminished while either rightward or leftward move- be delivered through the polarized biochemical signaling pathways,
ments were observed for a short duration (Figure 6e). This orthogo- ultimately regulating the migratory machinery inside the cell to di-
nal migration with respect to the EF orientation spatiotemporally rect the migration. For a migratory response, the change in cellular
emerged as cooperative packs consisted of ∼10 cells, displaying traction and cytoskeletal rearrangements would be expected. In-
patches of swirl-like patterns within the monolayer. As shown in deed, when a single cell was stimulated by EF, the immediate
Figure 6, d–g, tracking the displacement of centroids of two se- change in the cellular traction was observed followed by the mor-
lected cells showed the local “U-turn” reorientation over the course phological rearrangement (Curtze et al., 2004). Li et al. visualized
of ∼60 min. This U-turn behavior has also been reported by Cohen the cellular traction force of the EF-stimulated epithelial monolayer
et al. (2014) in the MDCK monolayer. Time-course measurement of using the deflection of underlying microposts (Li et al., 2012), and
average migration directedness confirmed the approximately ∼60- the discontinuous distribution of microposts was unsuitable for the
min time delay between the EF reversal and actual cellular redirec- calculation of the intercellular stresses among neighboring cells.
tion response (Figure 6n). This time delay consisted of three phases. Polyacrylamide gel as the continuous underlying substrate for trac-
First, for ∼20 min after the EF reversal, the monolayer persisted in tion measurement was beneficial to quantify the EF-induced
the directional migration induced by the initial EF stimulation. Be- changes in cellular tractions and their correlations with the intimate
tween 20 and 40 min, the migration directedness was randomly neighbors within the collective monolayer.
oriented and, finally, by the 60-min time point, it rapidly recovered Using the MSM method, Tambe et al. (2011) observed the coin-
in the reversed direction toward the new anode. During the dy- cidence of the local direction of migration and the local orientation
namic alteration in migration, the average normal intercellular stress of maximal principal stress in collective cell migration and defined
showed insignificant change in magnitude (Figure 6, h–j). Consis- this phenomenon as “plithotaxis.” While the plithotaxis is physi-
tently, as evidenced by the stress ellipse, the stress orientation was cally intuitive and consistent with the tension-induced polarization
maintained without any transient changes throughout the entire of cellular migration, an exceptional case has been reported by Kim
redirection process of migration once the intercellular stress aligned et al. (2013). When the advancing cell monolayer was forced to
perpendicular to the EF field (Figure 6, k–m and o). Cell-body orien- encounter the nonadhesive vacant space, the local velocity vectors
tation also maintained its perpendicular orientation to the EF direc- veered systematically away from the orientations of the principal
tion during the redistribution of the velocity profile (Figure 6, p–r). stress by angles approaching 90° (Kim et al., 2013). What we ob-
These data suggest that the intercellular stress state within the served in our study with the EF is similar to what Kim et al. showed
monolayer is maintained during the EF-driven U-turn migration of with the frustrated edge where both cases consistently show the
the HaCaT monolayer. perpendicular alignment of the orientation of principal stress and
local velocity vectors rather than parallel one predicted by the
DISCUSSION plithotaxis.
The EF is a strong guidance cue that can override other coexisting Dynamic alterations in the physical stresses and the morphologi-
guidance cues from microenvironment (Zhao et al., 2009). Thus, cal reorientation during electrotactic responses shared some of the
the endogenous EF is expected to play a key role in essential common characteristics with the case of the shear flow induced re-
physiological events such as tissue regeneration and development alignment of endothelial cell monolayer reported by Steward et al.
by inducing the directional response of the cells. Especially, during (2015). Endothelial monolayer exhibited an early alignment of the
the wound healing of skin tissues, a sheet migration of epithelial traction forces and intercellular stresses along the direction of the
monolayer during the reepithelization process is known to be shear flow followed by the delayed cell-body alignment. This evi-
driven by endogenous EF. While the importance of such collective dence inferred that intercellular stress-guided morphological reor-
response to EF has been well accepted, the underlying physical ganization in the collective monolayer may be generalized to other
mechanism is yet to be elucidated. In this study, development of situations of exogenously applied stimulators such as chemical gra-
the integrated platform to simultaneously stimulate dcEF while dient or cyclic stretch. However, we identified the clear distinctions
observing cells and measuring cellular tractions enabled us to in- between electrotactic- and shear flow-induced responses. Unlike
vestigate the time evolution of collective electrotactic response of the gradual attenuation of intercellular stress triggered by the shear
a monolayer. flow on the endothelial monolayer (Steward et al., 2015), the EF
Since the discovery of EF-directed cell migration in the late 19th strengthened the intercellular stress and reoriented the principal
century, extensive studies investigated the role of EF in directing axes of stress ellipse in the direction perpendicular to the EF as
migration and cellular morphology across many cell types. In addi- shown in Figure 5. The reversal of the EF had little effect on both the
tion to the phenomenological observation of electrotactic cellular strength of the intercellular stresses and the orientation of the stress
response, researchers investigated the mechanism of how the EF ellipse of the intercellular stresses (Figure 6).

intercellular stress started to have alignment tendency not until 20 min after EF start and gradually aligned
perpendicularly from the EF direction until 50 min. (s–u) Overlay of angular distribution of cell velocity (red bars), cell body
(blue bars), and intercellular stress (yellow bars). (s) At pre-EF condition, velocity, body, and stress showed no preferred
alignment. (t) After 30 min from the EF initiation, dominant population of velocity already well oriented parallel to the EF
direction. Intercellular stress also showed considerable reorientation to perpendicular axis to the EF direction. Cell body,
however, showed no preferred orientation. (u) At 100 min after EF stimulation, velocity and intercellular stress were even
better aligned parallel and perpendicular to the EF direction, respectively. Now the cell body aligned perpendicular to the
EF direction (90°) with relatively weak tendency. Dashed lines indicate the EF initiation. Scale bars, 50 µm.

Volume 29 September 15, 2018 Behavior of cell monolayer under EF | 2299

 The adherens junction, where cadherins
are anchored to the cytoskeletal structure
through α- and β-catenin, mainly regulates
the force transmission and accumulation be-
tween neighboring cells (Maître and Heisen-
berg, 2013). These adherens junctions ex-
hibit plasticity, continually formed and
disassembled, to maintain the epithelial char-
acteristics (Baum and Georgiou, 2011).
Therefore, the polarized intercellular stress-
driven morphological alignment might origi-
nate from the polarized remodeling of the
adherens junction complex along the cell–
cell interface in the direction of the EF, induc-
ing a stress ellipse in the direction orthogonal
to the EF. In particular, given the importance
of α-catenin in the HaCaT monolayer for the
coordinated contraction, the anisotropic ac-
cumulation of α-catenin may be a promising
candidate responsible for the EF-induced
directional enhancement of intercellular
stress (Vedula et al., 2014, 2015).
In this study, we provided the evidence
suggesting that the EF-induced intercellular
stress reorientation drives the cell-body
alignment while the electrotatic migration
happens independently of the reorientation
of either intercellular stress or cellular mor-
phology. Once the intercellular stresses
were aligned, the reversal of the EF direc-
tion induced the reversed migration of the
cells without any apparent disruption of the
intercellular stress, suggesting that the dis-
lodging of the physical stress alignment
along the adjacent cells not be necessary for
changing the direction of monolayer migra-
tion. The reversal of EF also confirmed that
the orthogonal arrangement of intercellular
stress with respect to the EF direction is ir-
respective of the polarity of the EF. These
observations imply that the anodal/cathodal
exchange only directly influence the direc-
tion of migration, whereas the intercellular
stress orientation is aligned perpendicular
to the axis of EF itself rather than affected by
the position of anodal/cathodal polarity.

MATERIALS AND METHODS

Cell culture
HaCaT cells, derived from spontaneously
FIGURE 6: Maintenance of intercellular stress during the EF-induced reversal of collective immortalized human keratinocytes, were
migration. (a–c) Redirection process of HaCaT monolayer induced by the reversal of EF cultured in DMEM supplemented with 10%
direction. (d–f) Enlarged field of red dotted boxes in a–c. (g) Outlines of the representative cells FBS and 1% penicillin/streptomycin at 37°C
in d–f showing the time-course relative positions. Dots represent the position of centroids at with 5% CO2.
each time point. Black arrows represent the relative displacement of centroids. (h–j) Maps of
average normal intercellular stress overlaid on phase contrast images. (k–m) Color-coded stress
EF stimulating chamber
ellipses overlaid on phase-contrast images showed no transient change of stress alignment
The EF chamber consisted of three parts: the
during the reversal of EF direction. (n) Time evolution graph of average migration directedness
of the monolayer in 10-min intervals during the pre-EF, first EF stimulation with the anode on slide-glass base part, the first PDMS layer,
top, and sudden reversal of EF direction. (o) Time evolution graph of intercellular stress and the second PDMS layer (Figure 1). First,
orientation in 10-min intervals. (p–r) Overlay of angular distribution of cell velocity (red bars), cell the slide-glass base part (76 × 52 mm, height
body (blue bars), and intercellular stress (yellow bars) during the reversal of EF direction. Data of 1.3 mm; Matsunami) was cleaned with
are presented as mean ± SD (n = 3 independent monolayers). Scale bars, 50 µm. ethanol. For the curved chamber geometry,

2300 | Y. Cho et al. Molecular Biology of the Cell

the first PDMS layer was prepared by patterning the desired geome- PIV analysis
try on a 1 mm height of the PDMS sheet. Then both slide-glass base Acquired stack images of the experiment were stabilized using ei-
part and the first PDMS layer were O2 plasma-treated for the perma- ther ImageJ or MATLAB. Postprocessed images were then analyzed
nent bonding. After the attached parts were autoclaved and sterilized, by custom particle image velocimetry (PIV) software written in
PA gel (Young’s modulus = 3 kPa, thickness = 100 μm) was fabricated MATLAB for the measurement of the velocity field. We used cross-
on the surface of the linear chamber geometry, where becomes the correlation with a window size of 64 × 64 pixels allowing spatial reso-
cell observation area. Gel preparation followed the protocols reported lution of 16 µm.
in previous publications (Butler et al., 2002; Kandow et al., 2007). For
the stable bonding between the glass and PA gel, the surface of the Cell migration velocity and directedness
slide glass was treated with silane. During the gel polymerization, we Cell migration calculated by PIV analysis from 5-min interval images
mixed fluorescent beads (diameter = 0.5 μm; FluoSpheres; Life Tech- was analyzed to determine the migration velocity and directed-
nologies) in the gel, loaded a 24-µl drop of PA gel on the base part, ness. Cell migration velocity indicates the instantaneous velocity
and immediately centrifuged the attached base part to pull up every quantified from the displacement of the cell between two succes-
bead to the surface of the PA gel. For the cell attachment on the gel, sive images at every time points. Directedness indicates the direc-
the PA gel surface was functionalized with sulfosuccinimidyl-6-(4-az- tional information of migration shown as cos θ, where θ represents
ido-2-nitrophenylamino) hexanoate (Sulfo-SANPAH; Proteochem) and the angle between the axis parallel to EF direction and the cell
1 mg/ml in 50 mM HEPES buffer (Life Technologies) and coated with migration direction. Therefore, if the average cell direction was par-
10 μg/ml collagen type I (PureCol; Advanced BioMatrix). After HaCaT allel to the EF, with the average value of θ close to 0° or 180°, di-
cell monolayer was seeded on the PA gel (details described below), rectedness was close to 1(upward migration) or –1(downward mi-
the 6-mm height of the second PDMS layer was finally covered on the gration). Average value of directedness close to 0 represented the
first PDMS layer to form the channel at the observation region, and random migration.
media were filled in the chamber. The assembled chamber was
mounted on the microscope, and agar bridges were placed into each Quantification of cell-body orientation
media reservoir. The other ends of agar bridges were connected to Cell-body orientation was quantified by the angle between the long
the voltage-controlled power source via Ag/AgCl electrodes in bea- axis of the cell and the axis parallel to the EF direction. The long axis
kers filled with 5x Steinberg’s solution (Figure 1). The field strength at of the cell was defined as the major axis of the ellipsoidal fit of the
the cell observation window was measured at the beginning and end cell boundary. All of these steps were conducted using ImageJ (Sup-
of the experiment through Ag/AgCl monitoring electrodes placed at plemental Figure S1).
both ends of the enclosed chamber by voltage meter.
Fourier transform traction microscopy
Cell monolayer seeding Traction maps exerted by the cell monolayer were measured from
To seed the HaCaT cells in the observation region of the chamber, we the bead displacements in the gel using the unconstrained Fourier
prepared 10:1 PDMS stencil with rounded rectangular patterns and transform traction microscopy, which was well described in previ-
covered it on the gel substrate. Before seeding the cells, patterns in ously published references (Butler et al., 2002; Trepat et al., 2009).
the stencil were carefully filled with DMEM to avoid the generation of
air bubbles. Next, a 200-μl drop of cell suspension with 1 × 106 cells/ Monolayer stress microscopy
ml density was loaded into the PDMS stencil. After allowing cells to To calculate the intercellular stress, we used the monolayer stress
settle down and attach to the substrate for ∼1 h, the remaining cells microscopy (MSM) reported by Tambe et al. (2011, 2013). MSM
were flushed out by gentle pipetting. Then the cells were incubated calculated intercellular stress based on the measured traction by
for additional 12 h to form a confluent monolayer with mature cell– applying straightforward force balance according to Newton’s
cell junctions. Immediately before mounting the chamber on the mi- law; the two-dimensional stress tensor within the monolayer
croscope, the PDMS stencil was carefully removed, and the second must be balanced by the traction. At each point within the mono-
PDMS layer was placed to seal the observation region. layer, the stress tensor perpendicular to the EF direction (σxx) and
parallel to the EF direction (σyy) were presented in data. To ob-
Region of interest tain the principal stresses, σmax, and σmin, the coordinate was
During the assembly of the EF stimulation system, we remove the converted at each point within the monolayer by eigenvalue de-
PDMS stencil to prevent the undesirable distortion of the EF. Once composition. From the principal stresses, the local average nor-
the stencil was removed, cells at the edge were free to move out- mal stress was defined as (σmax + σmin)/2, which represents the
ward with active lamellipodia while the cells in the core, far enough scalar tension within the monolayer. To represent the intercellular
away from the edge, exhibited distinct phenotypes (Poujade stress orientation, we plotted stress ellipse with the σmax at the
et al., 2007). In this study, we aimed to focus on the effect of EF major axis and the σmin at the minor axis. The local principal
only within the bulk of the confluent monolayer. For this reason, stress orientation defined the local stress orientation, repre-
we eliminated any potential edge effects by confining our region sented by the ellipse orientation in this report (Kim et al., 2013;
of interest only within the interior location of the monolayer, at Steward et al., 2015).
least 12–15 rows of the cells (250 µm) from the nearest free edge.
ACKNOWLEDGMENTS
Time-lapse imaging The Matlab codes for TFM and MSM were generously provided
Phase-contrast and fluorescence bead images of the cell monolayer by J. J. Fredberg’s lab at the Harvard T. H. Chan School of Public
within the region of interest were taken every 5 min using the 5× Health. This work was supported by National Research Founda-
objective lens. All experiments were performed on the Axiovert tion of Korea (NRF) grants funded by the Korean Government
200M (Carl Zeiss) microscope with the maintenance of incubating (NRF-2013S1A2A2035518, NRF-2015M3A9B3028685, and NRF-
condition (37°C and 5% CO2). 2016K2A9A2A08003761).

Volume 29 September 15, 2018 Behavior of cell monolayer under EF | 2301

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