© 2017. Published by The Company of Biologists Ltd | Journal of Cell Science (2017) 130, 2459-2467 doi:10.1242/jcs.
203752
RESEARCH ARTICLE
Electrophoresis of cell membrane heparan sulfate regulates
galvanotaxis in glial cells
Yu-Ja Huang1,2, Paula Schiapparelli3, Kristen Kozielski1,4, Jordan Green1,4, Emily Lavell3,
Hugo Guerrero-Cazares1,3, Alfredo Quinones-Hinojosa3 and Peter Searson1,2, *
ABSTRACT
Endogenous electric fields modulate many physiological processes
by promoting directional migration, a process known as
galvanotaxis. Despite the importance of galvanotaxis in
development and disease, the mechanism by which cells sense
and migrate directionally in an electric field remains unknown. Here,
we show that electrophoresis of cell surface heparan sulfate (HS)
critically regulates this process. HS was found to be localized at the
anode-facing side in fetal neural progenitor cells (fNPCs), fNPC-
derived astrocytes and brain tumor-initiating cells (BTICs),
regardless of their direction of galvanotaxis. Enzymatic removal of
HS and other sulfated glycosaminoglycans significantly abolished
or reversed the cathodic response seen in fNPCs and BTICs.
Furthermore, Slit2, a chemorepulsive ligand, was identified to be
colocalized with HS in forming a ligand gradient across cellular
membranes. Using both imaging and genetic modification, we
propose a novel mechanism for galvanotaxis in which
electrophoretic localization of HS establishes cell polarity by
functioning as a co-receptor and provides repulsive guidance
through Slit-Robo signaling.
KEY WORDS: Galvanotaxis, Brain tumor-initiating cells, Heparan
sulfate, Electrophoresis
INTRODUCTION
Endogenous electric fields (EFs) are known to drive many
physiological processes including embryo development, wound
healing and immune responses by promoting directional migration,
a process known as galvanotaxis (Mycielska and Djamgoz,
2004; Lin et al., 2008). Disruption of endogenous EFs with
pharmacological agents or externally applied EFs of opposite
polarity disturbs these processes, whereas enhancement of
endogenous EFs (same polarity, increased magnitude) increases
the rate of regeneration by promoting the extent of nerve sprouting
and the rate of wound healing in vivo (Song et al., 2004; Messerli
and Graham, 2011). The brain exhibits one of the highest electrical
activities amongst all organs in the body; electric fields in the brain
are not an epiphenomenon but actively regulate cellular functions.
For example, the endogenous electric field between the
1
Institute for Nanobiotechnology, Johns Hopkins University, Baltimore, MD 21218,
USA. 2Department of Materials Science and Engineering, Johns Hopkins
University, Baltimore, MD 21218, USA. 3Department of Neurosurgery and
Oncology, Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins
University, Baltimore, MD 21231, USA. 4Department of Biomedical Engineering,
Johns Hopkins University, Baltimore, MD 21231, USA.
*Author for correspondence (searson@jhu.edu)
P.S., 0000-0002-5417-0828
Received 15 March 2017; Accepted 2 June 2017
subventricular zone and olfactory bulb was found to direct the
migration of neuroblasts and guide the migration of neural
precursor cells along the rostral migratory stream (Cao et al.,
2013). Furthermore, increased electrical activity stimulated by
optogenetics accelerates glioma growth in vivo (Venkatesh et al.,
2015). Taken together, these results suggest that endogenous
electric fields modulate neural regeneration and glioma infiltration
by regulating galvanotaxis; however, the mechanism by which brain
cells sense and migrate directionally in an electric field remains
unknown. Therefore, elucidating the mechanism of galvanotaxis
can provide new insight into brain development and the progression
of diseases such as glioma, and provide the foundations for new
clinical interventions.
Proposed explanations for galvanotaxis include electrophoretic
distribution of charged membrane components (Jaffe, 1977; Poo
and Robinson, 1977; Allen et al., 2013), asymmetric activations
of ion channels (Yang et al., 2013; Nakajima et al., 2015), and
membrane-associated electro-osmotic forces (McLaughlin
and Poo, 1981). Interestingly, while most cell types exhibit
galvanotaxis, the response can be either cathodic or anodic,
suggesting that there may be competing mechanisms (Mycielska
and Djamgoz, 2004; Sato et al., 2009; Sun et al., 2013). Here, we
investigate the galvanotaxis in three different types of glial cells
including primary neural progenitor cells (fNPCs), fNPC-derived
astrocytes, and malignant brain tumor-initiating cells (BTICs). We
show that all three cell types exhibit a directional response to an
external EF. More importantly, we identify the novel role of
surface heparan sulfate (HS), a highly negatively charged
sulfated glycosaminoglycan (GAG), in sensing and mediating
galvanotaxis. HS was found to be highly localized towards the
positive electrode (anode) of the cells in the presence of an EF in
all cell types due to electrophoretic interactions. Enzymatic
digestion of HS significantly abolished the cathodic response in
cells. Furthermore, using non-viral siRNA knockdown, we
showed that galvanotaxis is unlikely to be due to any single
heparan sulfate proteoglycan, but is rather a collective outcome
due to the localization of HS chains. HS was identified as a
co-receptor, establishing a Slit2 gradient across cellular
membranes as a consequence of electrophoretic localization.
Journal of Cell Science
Slit2, a chemorepulsive ligand critical for central nervous system
development (Shi and Borgens, 1994; Ba-Charvet et al., 1999;
Kaneko et al., 2010), subsequently provides a repulsive guidance
through Slit-Robo signaling as indicated by the attenuation of
galvanotaxis in response to downregulation of Robo1. We propose
that HS is a novel EF sensor that regulates galvanotaxis through
electrophoretic interactions and its function as a co-receptor, to
establish a ligand gradient. Our findings provide direct evidence in
support of electrophoretic interactions in regulating galvanotaxis,
and highlight the possibility of an EF in promoting autologous
chemotaxis.
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RESULTS
fNPCs, astrocytes and BTICs exhibit galvanotaxis with
different characteristics
To understand the mechanisms regulating the galvanotaxis of brain
cells, we first characterized the responses of fNPCs, astrocytes and
BTICs using a custom galvanotaxis chip (Huang et al., 2013)
(Fig. 1A). All experiments were conducted under the same culture
conditions (see Materials and Methods) to avoid any bias. The
trajectories of the cells in the presence of an EF were tracked and
analyzed to characterize the cellular response. We showed that
Fig. 1. Galvanotaxis of fetal neural progenitor cells (fNPCs), astrocytes
and brain tumor-initiating cells (BTICs). (A) Each galvanotaxis chip contains
two symmetrical devices on a 35 mm×50 mm glass coverslip. Each device
features two coiled Ag/AgCl electrodes embedded in agarose reservoirs
located at each end of the cell culture channel, along with two media reservoirs
also located at each end of the channels. The dimensions of the cell culture
channel are 10 mm×1 mm×250 μm (length×width×height). A cell injection port
located in the middle of the cell culture channel is used to introduce cells into
the device and is clamped with an alligator clip afterwards to prevent
evaporation. Trajectories of fNPCs (B), astrocytes (C) and BTICs (D) in the
presence of an EF are analyzed and overlaid at the origin to characterize the
galvanotaxis of each cell line. Each trajectory represents the actual path
traveled by a cell in 3 h either to the cathode (left, black) or anode (right, red).
(E) Quantitative analysis of cell motility and directedness in the presence of a
1 V cm−1 EF.
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galvanotaxis is highly dependent on cell type: while 100% of fNPCs
exhibited strong directional response towards the cathode (Movie 1
and Fig. 1B), astrocytes derived from fNPCs showed an anodic
directional response opposite to fNPCs (Movie 2, Fig. 1C).
Meanwhile, the majority of BTICs (73%) migrated towards the
cathode in the presence of a 1 V cm−1 EF (Movie 3 and Fig. 1D).
Further quantifying cell motility and directedness in the presence
of an EF (Fig. 1E) showed that fNPCs exhibited the highest motility
on a laminin-coated surface in the presence of an EF (0.87±
0.08 μm min−1) followed by BTICs (0.75±0.15 μm min−1) and
astrocytes (0.56±0.03 μm min−1). fNPCs also exhibited the highest
directedness (d) towards the cathode (d=0.85±0.09) followed by
BTICs (d=0.44±0.01), whereas astrocytes migrated strongly
towards the anode (d=−0.85±0.06).
HS is localized towards the anode regardless of cell type and
the direction of galvanotaxis
We next investigated the involvement of membrane components to
understand how cells were able to sense and respond to an electric
field. Heparan sulfate (HS) is a candidate as an EF sensor as it is not
only ubiquitously expressed on all cellular membranes, but also
highly negatively charged. To understand the involvement of HS,
fNPCs, astrocytes and BTICs were stained with antibodies against
HS both in the absence of an EF and after being stimulated with a
1 V cm−1 EF for 3 h. We showed that HS was indeed abundantly
expressed in each cell type, as indicated by the punctate aggregates
throughout the cellular membranes (Fig. 2A–F). In the absence of
an EF, HS distributed uniformly across cellular membranes in each
cell type (Fig. 2A,C,E). However, in the presence of an EF, where
fNPCs and BTICs migrated towards the cathode and astrocytes
migrated towards the anode, HS was found to be highly localized
towards the anode ( positive electrode) regardless of cell type and the
direction of migration (Fig. 2B,D,F). The localization of HS is
particularly obvious in astrocytes where the average size of the cells
is much larger than fNPCs and BTICs. In both the absence and
presence of an EF, astrocytes secreted and left behind trails of
extracellular matrix (ECM) abundant with HS during migration
(Fig. S1A,B). However, localization of HS was only observed in the
presence of an EF (Fig. 2D), not in the absence of an external field
(Fig. 2C). Further quantifying the localization of HS from analysis
of processed images (Fig. 2G), we showed that an EF resulted in a
substantial increase in the percentage of cells with anodic
localization of HS (Fig. 2H). In the absence of an EF, the anodic
localization of HS was close to 50%, indicating no polarization:
48% of fNPCs, 37.5% of astrocytes and 43.3% of BTICs. In
contrast, in the presence of a 1 V cm−1 EF, the localization increased
to 65.2, 86.1 and 77.8%, respectively. The distribution of HS in a
smaller EF (0.5 V cm−1) was further examined in BTICs; however,
there was no evidence for significant HS polarization (47.7%)
(Fig. S1C,D).
Journal of Cell Science
HS regulates the cathodic response in fNPCs and BTICs
To establish the relevance of anodic localization of HS in cellular
galvanotaxis, we next used heparinase (HPase) to digest surface
HS. The effectiveness of HPase treatments was verified by
immunostaining of HS, where cells treated with HPase were
devoid of any HS signal except at the periphery near focal adhesions
(Fig. 3A, right, B, right). fNPCs, astrocytes and BTICs were each
treated with HPase and stimulated with an EF to compare their
responses to the corresponding wild-type cells (Fig. 3C-E). We
showed that while HPase treatment had no significant effect on cell
motility in either cell type (Fig. 3F), it significantly influenced the
RESEARCH ARTICLE
cathodic responses of fNPCs and BTICs (Fig. 3G). The directedness
of fNPCs significantly decreased from 0.85 to 0.38 (P=0.038)
after treatment with HPase, whereas the directedness of BTICs
significantly decreased from 0.48 to −0.12 (P<0.01). The anodic
directional response of astrocytes, however, remained unaffected
even after treatment with HPase (Fig. 3G).
Chondroitin sulfate (CS) and dermatan sulfate are two other
main types of GAG that bear a negative charge due to sulfation
modification. To investigate its involvement in galvanotaxis, we
treated cells with both HPase and chondroitinase ABC (chABC),
enzymes that catalyze the removal of chondroitin sulfate and
dermatan sulfate GAG chains. Addition of chABC did not affect the
motility of either cell type (Fig. 3F), nor did it further affect the
directedness of fNPCs (Fig. 3G). The directedness of astrocytes
remained comparable to the wild-type cells despite the removal of
HS, CS and dermatan sulfate (Fig. 3G). However, treatments with
both HPase and chABC completely reversed the direction of
galvanotaxis in BTICs from cathodic to anodic (Fig. 3E, right;
Movie 4). BTICs treated with HPase and chABC migrated towards
the anode with a mean directedness of −0.40±0.12, significantly
different from wild-type BTICs (d=0.48±0.07, P<0.01).
HS-mediated directional response is unlikely to be due to any
specific HSPG
HS exists as a heparan sulfate proteoglycan (HSPG), where a core
protein is covalently attached to several sulfated HS chains. To
understand how the anodic localization of HS mediates the cathodic
directional migration of fNPCs and BTICs, we considered two
possible explanations involving either HSPG core proteins or HS
GAGs, and investigated them accordingly.
Journal of Cell Science (2017) 130, 2459-2467 doi:10.1242/jcs.203752
Fig. 2. Anodic localization of HS in the presence of
an EF. The distribution of HS was measured by
performing a maximum intensity projection of confocal
z-stacks of individual cells to minimize any error
associated with out-of-focus pixels. In the absence of
an EF, HS is distributed uniformly across the
membranes of fNPCs (A), astrocytes (C) and BTICs (E).
However, strong anodic localization of HS was
observed across all three cell lines (B,D,F) in the
presence of an EF despite fNPCs and BTICs migrating
towards the cathode and astrocytes migrating towards
the anode. (G) Distribution of HS was analyzed by
performing a signed rank test among all the pixels in the
cathode face (c) versus pixels in the anode face (a).
(H) Percentage of cells with anodic localization of HS is
higher in the presence of an EF than no EF among all
three cell types. Each condition represents more than
50 cells. White arrows in panels C and D indicate the
ECM left behind by cells during migration.
We first hypothesized that cathodic galvanotaxis is due to the
asymmetric distribution of one of the HSPG core proteins that interacts
with its downstream effectors and establishes cell polarity. To examine
this hypothesis, the expression levels of selected individual HSPGs
were systematically downregulated using siRNA. Two main types of
HSPG are expressed on the membranes of mammalian cells: syndecan
(SDC1–SDC4) and glypican (GPC1–GPC6) (Sarrazin et al., 2011).
Using polymeric nanoparticles containing optimized siRNA
sequences, we downregulated SDC1, SDC2, SDC3, SDC4 and
GPC1; downregulation of the corresponding protein expression levels
was confirmed by western blot and further validated by qRT-PCR
(Fig. S2A–F). While the expression levels of selected HSPGs were
downregulated by 40–60% based on western blots and the mRNA
levels were downregulated by >80% (Fig. S2), the motility of BTICs
remained unaffected by the transfection (Fig. S2G). Furthermore,
downregulation of SDC1, SDC2, SDC3, SDC4 or GPC1 alone had no
significant effect on the directedness of BTICs in the presence of an
EF when compared with either wild-type cells or cells treated with a
scrambled sequence (Fig. S2H).
Journal of Cell Science
Disrupting the sulfation of HS abolishes the galvanotaxis
of fNPCs and BTICs
As cell polarity during galvanotaxis is unlikely to be due to any
single HSPG, we considered the possibility that galvanotaxis is a
collective outcome of the localization of sulfated GAG chains. We
hypothesized that as a known co-receptor for many ligands
(Sarrazin et al., 2011), HS is capable of binding and forming a
ligand gradient across cellular membranes in the presence of an EF,
and promotes directional migration in a way similar to chemotaxis.
To test this hypothesis, each cell type was treated with NaClO3 to
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Fig. 3. Enzymatic digestion of HS significantly attenuates cathodic
galvanotaxis in fNPCs and BTICs. Heparinase (HPase) significantly reduced
the amount of surface HS GAGs in astrocytes (A) and BTICs (B), as shown by the
absence of fluorescence except near cell peripheries close to focal adhesions
after enzymatic treatment. (C–E) Trajectories of wild-type cells (WT) and cells
treated with both HPase and chondroitinase ABC (chABC) are shown side by
side to highlight the effect of sulfated GAGs on cellular galvanotaxis. Each
trajectory represents the actual path traveled by a cell in 3 h either to the cathode
(left, black) or anode (right, red). (F) Enzymatic treatment with either HPase alone
or a combination of HPase and chABC had no significant effect on cell motility
when compared with the corresponding WT. (G) HPase significantly attenuated
the cathodic response of fNPCs and completely abolished the galvanotaxis of
BTICs, but had no effect on astrocytes. The combination of HPase and chABC
did not further reduce the directedness of fNPCs but completely reversed the
directional response in BTICs. *P<0.05; **P<0.01; Student’s t-test. Statistics were
obtained from at least three independent experiments with at least 60 cells in
each experiment. Error bars represent standard deviation.
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disrupt the sulfation of HS, as the ability of HS to bind to different
ligands relies heavily on the degree of sulfation (Shipp and Hsieh-
Wilson, 2007). Chlorate competitively inhibits the formation of
high-energy sulfate donors required for sulfation reactions and thus
substantially undermines the capability of HS to bind ligands such
as fibroblast growth factor (FGF) and Slit (Safaiyan et al., 1999;
Shipp and Hsieh-Wilson, 2007). fNPCs, astrocytes and BTICs were
treated with 50 mM NaClO3 for 48 h before being subjected to an
applied EF. While the motility of fNPCs and BTICs remained
unaffected by the treatment (Fig. 4C), chlorate treatment completely
abolished the cathodic galvanotaxis in both fNPCs and BTICs
(Fig. 4A and B); the directedness of fNPCs decreased from 0.85 to
−0.03 (P<0.01), whereas the directedness of BTICs decreased from
0.44 to 0.00 (P<0.01) (Fig. 4D). Astrocytes, however, remained
unaffected by the chlorate treatment both in terms of motility and
directedness (Fig. S3A).
Localization of HS establishes a ligand gradient across
cellular membranes
As NaClO3 completely abolished the cathodic response in fNPCs
and BTICs, we continued to investigate the capability of HS in
forming a gradient of ligands across a cellular membrane. Cells in
the presence of an EF were stained with both HS and Slit2, a ligand
of the Robo family of receptors essential for development in the
central nervous system (Brose et al., 1999). Slit2 was chosen not
only for its well-characterized affinity to HS (Hu, 2001), but also for
its role in providing a repulsive migration cue in various types of
brain cells (Shi and Borgens, 1994; Ba-Charvet et al., 1999; Kaneko
et al., 2010). We have previously shown that Slit2-Robo1 signaling
greatly enhanced the invasion of BTICs by providing a
chemorepulsive signal in vitro (Guerrero-Cazares et al., 2015).
The observation that HS localized towards the back of fNPCs and
Journal of Cell Science
Fig. 4. NaClO3 abolishes cathodic response in fNPCs and BTICs. fNPCs
(A) and BTICs (B) were treated with 50 mM sodium chlorate for 48 h to
investigate how the sulfation of HS influences cathodic galvanotaxis.
Treatment with sodium chlorate had no effect on cell motility (C) but abolished
the directional response of fNPCs and BTICs in the presence of an electric
field (D). **P<0.01 (Student’s t-test). Statistics were obtained from at least
two independent experiments with at least 60 cells in each experiment. Error
bars represent standard deviation.
RESEARCH ARTICLE
BTICs (anode) while cells migrated towards the cathode supports a
mechanism involving Slit-mediated repulsive guidance. From
immunofluorescence staining, we discovered that Slit2 was
colocalized with HS towards the anode in the presence of an EF
in all three cell types, demonstrating the capability of HS in forming
a ligand gradient across a cellular membrane in an EF (Fig. 5A–C).
Furthermore, we also showed that the ability of HS to bind Slit was
significantly attenuated in cells treated with chlorate, as shown by a
significant decrease in the intensity of Slit but not HS (Fig. 5D).
Downregulation of Robo1 attenuates galvanotaxis
To probe the involvement of Slit-Robo signaling in promoting
galvanotaxis, BTICs were transduced with lentiviral particles
containing a shRNA sequence against Robo1 receptor (Fig. 6A
and B) and subjected to an EF. We showed that downregulation of
Robo1 attenuated the galvanotaxis of cells as both cell motility and
directedness decreased compared with the control group transduced
with an empty virus (EV) (Fig. 6C,D). Downregulation of Robo1
decreased the motility of BTICs from 0.9 to 0.58 μm min−1
(P=0.039), whereas the directedness decreased from 0.72 to 0.36
(P=0.031) (Fig. 6E,F).
DISCUSSION
Gradients of molecular cues along the cell migration path is widely
believed to be the driving mechanism for brain cell migration during
development and pathogenesis. Our findings suggest that an
endogenous or an applied EF can also establish a gradient of
molecular cues at the cellular level through electrophoresis of HS,
and resulted in directional migration. These findings have
implications in understanding brain homeostasis and may be
utilized for disease treatments.
Electrophoresis of cellular membrane components has long been
hypothesized to be the driving mechanism for galvanotaxis (Jaffe,
1977). Results from galvanotaxis experiments in response to changes
in media pH and viscosity also provide support for this mechanism
(Allen et al., 2013). Membrane components such as ConA receptors,
ricin receptors, sialic acids and EGF receptor (EGFR) have been
shown to be polarized in an electric field (Poo et al., 1979; Zagyansky
Journal of Cell Science (2017) 130, 2459-2467 doi:10.1242/jcs.203752
and Jard, 1979; Fang et al., 1999; Finkelstein et al., 2007; Nakajima
et al., 2015). However, the identity of a macromolecule that exhibits
both electrophoretic polarization and is also necessary for
galvanotaxis has not been reported. For example, the ConA
receptor has been shown to be polarized in an EF and its direction
of polarization reversed when cells were treated with neuraminidase,
but the effects of its polarization on galvanotaxis is unclear
(McLaughlin and Poo, 1981). EGFR, however, has been shown to
be important for galvanotaxis as pharmacological inhibition
abolished galvanotaxis in keratinocytes, although it is unclear
whether the polarization of EGFR towards the cathode is due to an
electrophoretic force. Here, we present direct evidence in support of
the electrophoresis in regulating galvanotaxis. As HS and other
sulfated GAGs are amongst the most highly negative charged
biopolymers in nature (Sarrazin et al., 2011) and HS is polarized
towards the anode-facing side of glial cells regardless of their
direction of galvanotaxis, localization of HS is probably a physical
process involving an electrophoretic force. Furthermore, HS is also
critical for the cathodic response observed in fNPCs and astrocytes.
Taken together, we conclude that HS is a novel EF sensor that relays
electrical signals to directional migration cues through electrophoretic
polarization.
However, since the anodic directional response of astrocytes was
not affected upon removal of HS, other mechanisms, perhaps in
competition with HS, probably exist and collectively determine
the direction of galvanotaxis. This could explain the surprising
observation that the directional response of BTICs was reversed
from cathodic to anodic upon removal of different types of sulfated
GAGs (Fig. 3E); as we weakened the cathodic response mediated
by sulfated GAGs, the mechanisms governing an anodic response
dominated and cells migrated towards the anode. Interestingly, we
have also previously observed that the galvanotaxis of BTICs can
also be reversed by their surrounding microenvironment (Huang
et al., 2016); galvanotaxis of BTICs switched from cathodic to
anodic upon addition of poly-L-ornithine on top of a laminin-
coated substrate, similar to the galvanotropism of neurons reported
previously (Movie 5 and Fig. S4) (Rajnicek et al., 1998). These
results highlight the existence of potential competing mechanisms.
Fig. 5. Slit2 colocalizes with HS and forms a
gradient across cellular membranes in the
presence of an EF. fNPCs (A), BTICs (B) and
astrocytes (C) were stained for both HS (red) and Slit2
(green) to investigate whether localization of HS is
capable of forming a ligand gradient across cellular
membranes. Antibodies raised in different species
were selected to avoid cross-reaction. Slit2 colocalized
with HS as indicated in the overlays (yellow) and
formed a gradient across cellular membranes in all
three cell types. (D) Chlorate treatments significantly
hindered the affinity of HS to Slit2, as indicated by the
decrease in Slit2 fluorescence signal.
Journal of Cell Science
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Fig. 6. Downregulation of Robo1 attenuates the galvanotaxis of BTICs.
BTICs were transduced with shRNA against Robo1 (sh-Robo1) to evaluate the
contribution of Slit-Robo signaling in galvanotaxis. (A,B) The efficiency of the
knockdown against cells transduced with an empty virus (EV) was evaluated
using western blot, where the transduction resulted in a 47±9.7%
downregulation of Robo1. Trajectories of EV cells (C) and sh-Robo1 cells (D) in
the presence of an EF were analyzed to examine the involvement of Slit-Robo
signaling in galvanotaxis. Robo1 knockdown resulted in a decrease in both cell
motility (E) and directedness (F). *P<0.05; Student’s t-test. Statistics were
obtained from two independent experiments with at least 60 cells in each
experiment. Error bars represent standard deviation.
We hypothesize that one of the contributing factors in mediating
the competing mechanisms may be the cellular level of cAMP, as
poly- L-lysine was shown to elevate cellular cAMP levels more
than twofold in oligodendrocytes (Vartanian et al., 1988). Cellular
levels of cAMP have been shown to modulate the repulsive versus
attractive response towards netrin-1 in Xenopus spinal neurons
(Ming et al., 1997) and elevated cAMP levels abolished the
galvanotaxis of keratinocytes and keratocyte fragments (Pullar and
Isseroff, 2005; Zhu et al., 2015). Another candidate in regulating
the anodic galvanotaxis is sialic acid, a negatively charged
sugar molecule that contributes to the overall charge of the
membrane. Removal of sialic acid has been shown to reverse
the polarization of ConA receptors and impair the cathodic
response in HeLa and 3T3 cells (McLaughlin and Poo, 1981;
Finkelstein et al., 2007).
In an attempt to find out how the anodic localization of HS leads
to a directional response towards the cathode, we tested two
hypotheses; one involved the function of the core proteins of
HSPGs and the other involved sulfated GAGs. We examined the
first hypothesis by selectively knocking down the expression level
of five individual syndecans and glypicans, as both types of surface
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HSPG have been shown to influence cell migration (Wight et al.,
1992). For example, syndecan-1 has been shown to localize at the
uropods (trailing edge) of polarized myeloma cells and promote cell
adhesion (Børset et al., 2000). Similarly, syndecan-4 has also been
observed to inhibit Rac at the back of neural crest cells in vivo during
development (Matthews et al., 2008) and mediate the persistent
migration of fibroblasts by locally regulating Rac activity (Bass
et al., 2007). These results support our observations and hypothesis,
where HSPGs were found to be localized at the anodal face (trailing
end) while cells migrated towards the cathode. However, knocking
down any individual HSPG alone did not have any significant effect
on the cathodic response of BTICs, suggesting that galvanotaxis is
unlikely to be dependent on the function of any individual HSPG
core protein. Given that mammalian cells have four syndecan
and six glypican genes, and our knock-downs yield 40–60%
downregulation at the protein level, the possibility of redundancy/
compensation between different family members remains.
We tested the possible role of sulfated HS GAG, as HS is known
to serve as a co-receptor for many different ligands based on various
sulfation modifications (Sarrazin et al., 2011). Treatment with
sodium chlorate completely abolished the cathodic response in
fNPCs and BTICs (Fig. 4D), suggesting the important role of
sulfated HS in sensing and migrating during galvanotaxis. In
addition, as Slit2 was found to be colocalized with HS in forming a
positive surface gradient towards the anode (Fig. 5A–C) and
downregulation of Robo1 attenuated galvanotaxis (Fig. 6), we
propose a model where galvanotaxis is mediated by the
electrophoresis of HS and its function to serve as a co-receptor for
ligands such as Slit2 (Fig. 7). However, it is likely that other
mechanisms and ligands, such as other family members of Slit and
Robo as well as FGF, may have also contributed to the process as
downregulating Robo1 only partially attenuated galvanotaxis.
Nevertheless, our results highlight the novel role of HS as an EF
sensor during galvanotaxis and provide direct evidence in support
of the electrophoretic galvanotaxis model. These findings could
have broad implications in many physiological events as cells
ubiquitously express HS and small EFs have been associated with
neural development (Burr, 1941; Cao et al., 2013), wound healing
(Zhao et al., 2006) and metastatic disease (Mycielska and Djamgoz,
2004). Understanding how endogenous EFs could guide cell
migration and how an applied EF could potentially be leveraged to
modulate this process provide a rationale for new therapeutics. For
example, transcranial direct current stimulation (tDCS) has shown
clinical benefits for central nervous system diseases (Fregni et al.,
2015) and was able to promote cathodal accumulation of
endogenous neural stem cells (Rueger et al., 2012). As more is
learned about the role of bioelectricity on cell function it is likely
that new opportunities for interventions will emerge.
MATERIALS AND METHODS
Cell lines
Journal of Cell Science
All cell cultures were established with institutional approval by the Johns
Hopkins University Internal Review Board.
fNPCs: F54 cells were derived after 17 weeks of gestation, obtained from
elective abortion (Tzeng et al., 2011) and were maintained in 2:1 high-
glucose Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen)/Ham’s
F12 (Cellgro) mixture supplemented with 2% B-27, 1% antibiotic–
antimyotic, 20 ng ml−1 bFGF, 20 ng ml−1 EGF, 20 ng ml−1 leukemia
inhibitory factor (LIF; Millipore, Billerica, MA) and 5 μg ml−1 heparin
(Sigma).
Astrocytes: Astrocytes were derived by plating fNPCs in a tissue culture flask
in DMEM/F12 medium (Sigma) supplemented with 10% fetal bovine serum
(Sigma) and 1% penicillin-streptomycin (Invitrogen) (Placone et al., 2015).
RESEARCH ARTICLE
BTICs: GBM612 cells were used and previously validated by Johns
Hopkins Genetic Resources Core Facility (Li et al., 2014). GBM612 cells
isolated from intra-operative tissue are multipotent and are able to form
diffuse tumors when implanted into animal models (Guerrero-Cázares et al.,
2009; Li et al., 2014; Kondapalli et al., 2015). BTICs were grown in
culture flasks coated with laminin and cultured in DMEM/F12 media
supplemented with 2% B-27, 1% antibiotic–antimyotic, 20 ng ml−1 FGF
and 20 ng ml−1 EGF.
Two-dimensional galvanotaxis and cell tracking
Galvanotaxis experiments were carried out using a customized galvanotaxis
device reported previously (Huang et al., 2013), with standard
microfabrication techniques (Fig. 1A). Briefly, a galvanotaxis chip is
fabricated from polydimethylsiloxane (PDMS) and oxygen plasma bonded
onto a glass coverslip. Ag/AgCl electrodes embedded in agarose are inserted
into the reservoirs, and the exposed glass slide in the channel is coated with
laminin. Before each experiment, different cell media were replaced with a
standard medium for 6 h to ensure that the reported phenotypes are not
medium dependent. The standard medium was DMEM/F12 supplemented
with 2% B-27, 1% antibiotic–antimycotic, 20 ng ml−1 bFGF and
20 ng ml−1 EGF. The galvanotaxis device was mounted onto an inverted
microscope (Nikon Ti-E 2000) equipped with a live-cell chamber at 37°C
and 5% CO2. The cells were imaged through the glass slide. Cells were
stimulated in a 1 V cm−1 DC EF for 3–9 h before being fixed for
immunofluorescence studies.
The trajectories of cells from time-lapse images were automatically
tracked using MetaMorph software (Molecular Devices, Sunnyvale, CA,
USA) to minimize any tracking biases. Only isolated cells that remained
in the field of view and did not undergo mitosis were selected for analysis.
Cell trajectories were further analyzed using a customized MATLAB
(MathWorks, Natick, MA, USA) script to characterize physical parameters
including cell motility and directedness. Here, we define cell motility as the
total path length traveled by a cell divided by the elapsed time. The
directedness is defined as Σcosθi/n and ranges between −1 and +1, where n
is the total number of cells and θi is the angle between the vector of cell
displacement and electric field vector (Huang et al., 2013). A directedness
close to zero indicates random motion, whereas positive and negative values
indicate cathodic and anodic responses, respectively. To determine
statistical significance, we use a two-tailed Student’s t-test (*P≤0.05;
**P≤0.01; ***P≤0.001).
Confocal immunofluorescence imaging
For immunofluorescence imaging, cells were fixed with 3.7% formaldehyde
and stained with selected antibodies without a permeabilization step to
minimize any fluorescence background arising from inside the cells. Cells
were then blocked with tris-buffered saline with 0.1% Tween 20 (TBST)
containing 5% bovine serum albumin (BSA) for 1 h and incubated with
mouse anti-HS (1:100, US Biological, 10E4 epitope) or rabbit anti-Slit2
Journal of Cell Science (2017) 130, 2459-2467 doi:10.1242/jcs.203752
Fig. 7. Proposed mechanism for cathodic
galvanotaxis mediated by electrophoretic
localization of HSPG. Electrophoresis of HS in the
presence of an EF redistributes HSPGs towards the
anode and consequently establishes a ligand gradient
(for example, Slit2) across a cellular membrane, as HS
is a co-receptor for many ligands. A ligand gradient
across a cellular membrane asymmetrically activates
downstream signaling, in this case Slit-Robo repulsive
guidance, and promotes cell migration towards the
cathode possibly by locally suppressing the activation
of Cdc42 and Rac1 at the anode.
(1:100, Abcam, ab7665) overnight at 4°C. After incubation, cells were
extensively washed with TBST and stained with a secondary antibody
conjugated to a fluorophore (1:200, Invitrogen).
To quantify the localization of membrane proteins, confocal fluorescence
images were collected to minimize any out-of-focus pixel due to any
variation throughout the cell surface (TiE, Nikon). Confocal z-stacks at 1 μm
per step were collected for each cell at the excitation wavelength
corresponding to the fluorophore used.
Quantification of protein localization
For each confocal z-stack, a maximum intensity projection was created using
NIS-Elements software to generate a 2D image and converted to an 8-bit
grayscale to quantify the localization of heparan sulfate. The contour of a 2D
cell image was identified and vertically divided along its geometric center
into a cathode- and an anode-facing side using ImageJ (Fig. 2G).
Subsequently, a Wilcoxon signed rank test was performed among all the
pixels in the cathode face to pixels in the anode face using R software, where
P<0.05 is defined as a cell with anodic localization of HS.
Enzymatic removal of surface glycosaminoglycans
Cells were treated with 12.5 U ml−1 of HPase I and III blend (Sigma) at
37°C for 6 h before being subjected to an EF for galvanotaxis experiments.
To remove CS and dermatan sulfate, cells were incubated with
chondroitinase ABC (chABC) (2.5 U ml−1; Sigma) at 37°C for 6 h.
siRNA transfection using polymeric nanoparticles
Polymers (R646) designed to condense siRNA into bioreducible
nanoparticles were made of poly(β-amino ester) as described previously
(Kozielski et al., 2014). The particles have been shown to be capable of
efficient gene knock-down in primary human glioblastoma cells without
significant cytotoxicity (Tzeng et al., 2011; Guerrero-Cázares et al., 2014).
BTICs plated in a 12-well plate at a density of 200,000 cells/well were
allowed to adhere overnight before transfection. Cells were then transfected
with siRNAs (OriGene Technologies, Rockville, MD, USA) for SDC1,
SDC2, SDC3, SDC4 and GPC1 genes or a scrambled siRNA (SC) at a final
concentration of 120 nM using polymeric nanoparticles (R646) following
Journal of Cell Science
procedures reported previously (Kozielski et al., 2014). Transfected cells
were collected for western blot and qRT-PCR analysis after 72 h.
Western blot and qRT-PCR
Cells in culture were lysed with RIPA buffer (Santa Cruz Biotechnology,
Dallas, TX, USA) on ice for 30 min before collection with a cell scraper.
Lysates were centrifuged at 10,000 g at 4°C for 15 min, and the supernatants
were removed from the pellets and collected. Protein collected from each
lysate was measured with a Pierce BCA protein assay kit (ThermoFisher
Scientific) to ensure equal loading. Denatured lysates were loaded onto
4–15% gradient SDS-polyacrylaminde gels (Bio-Rad) and transferred to a
nitrocellulose membrane (Bio-Rad). Membranes were blocked with TBST
2465
RESEARCH ARTICLE
containing 5% milk at room temperature for 1 h and incubated with primary
antibodies at 4°C overnight. For each experiment, GAPDH-HRP (Santa Cruz
Biotechnology, FL-335, 1:400) was used as a loading control. The following
antibodies were used at the specified concentrations: rabbit anti-syndecan 1
(Santa Cruz Biotechnology, H-174, 1:200), rabbit anti-syndecan 2 (Abcam,
ab79978, 1:200), rabbit anti-syndecan 3 (Proteintech, 10886-1-AP, 1:500),
rabbit anti-syndecan 4 (Abcam, ab24511, 1:200), mouse anti-glypican 1
(Santa Cruz Biotechnology, A-10, 1:200) and rabbit anti-Robo1 (Abcam,
ab85312, 1:800). Secondary antibodies conjugated to HRP (Bio-Rad) were
used at 1:2000. Immunoactive bands were detected using enhanced
chemiluminescence and quantified using Image Lab software (Bio-Rad).
Samples for qRT-PCR were prepared using a Taqman cells-to-CT kit
(ThermoFisher Scientific) following the manufacturer’s recommended
protocol and measured using a StepOnePlus system (ThermoFisher
Scientific). mRNA expression level was calculated using [Δ(ΔCt)]
relative to scrambled control, with GAPDH being used as a
housekeeping gene.
Stable transduction of cells with shRNA
To induce the knockdown of Robo1 expression, we used pLKO.1 lentiviral
particles containing shRNA sequences specific for human Robo1 transcripts
(Mission shRNA TRCN00000414 and TRCN00000417; Sigma Aldrich).
An empty vector was used as control (SHC001; Sigma Aldrich). Human
fetal neural progenitor cells were treated with one of the three viruses at 1 μl
per 2 ml with polybrene (8 μl ml−1) overnight. Virus-containing media were
replaced the next day with complete media with 0.25 μg ml−1 puromyocin as
selection agent. After one week, the concentration of puromycin was
reduced to 0.125 μg ml−1 for maintenance. Knockdown was confirmed
using western blot.
Acknowledgements
We thank Dr Cheng Ran (Lisa) Huang for scientific discussions.
Competing interests
The authors declare no competing or financial interests.
Author contributions
Conceptualization: Y.-J.H., P. Searson; Methodology: Y.-J.H.; Formal analysis:
Y.-J.H., P. Searson; Investigation: Y.-J.H.; Resources: P. Schiapparelli, K.K., J.G.,
E.L.; Writing – original draft: Y.-J.H.; Writing – review and editing: Y.-J.H., H.G.-C.,
A.Q., P. Searson; Supervision: P. Searson.
Funding
The work was supported by the National Institutes of Health (grant numbers
R01CA170629, R01NS070024). Deposited in PMC for release after 12 months.
Supplementary information
Supplementary information available online at
http://jcs.biologists.org/lookup/doi/10.1242/jcs.203752.supplemental
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J. Cell Sci. 130: doi:10.1242/jcs.203752: Supplementary information

Suplementary Information

Journal of Cell Science • Supplementary information

Supplementary Figure S1. Localization of HS in the presence of an EF (see Figure 2) .
Astrocytes left behind trails of ECM abundant with HS during migration (A and B, white arrows).
In the absence of an EF (A), the distribution of HS was uniform. Only in the presence of an EF
(B) was HS localized toward the anode. (C) The galvanotaxis of fNPCs and BTICs is dependent
on the magnitude of the EF, where a small but significant directional response was observed in a
0.5 V cm-1 EF. (D) The percentage of cells with anodic polarization of HS is significantly higher
in a 1 V cm-1 EF (77.8%) comparing to an EF of 0.5 V cm-1 (47.7%) and no EF (43.3%).
J. Cell Sci. 130: doi:10.1242/jcs.203752: Supplementary information

Supplementary Figure S2. Down-regulation of selected HSPGs did not influence the
galvanotaxis of BTICs (see Results sub-section HS-mediated directional response is unlikely
due to any specific HSPG).

(A-E) Five HSPGs (SDC1, SDC2, SDC3, SDC4, GPC1) were selected to be down-regulated using
polymeric nanoparticles containing optimized siRNA sequences. The efficiency of each

Journal of Cell Science • Supplementary information

knockdown on protein expression level was evaluated by Western blots and compared with cells
transfected with a scrambled (S.C.) sequence. (F) qRT-PCR result confirmed the downregulation
of selected HSPGs. Transfection with siRNA nanoparticles resulted in >80% downregulation in
gene expression among all targets. (G-H) Transfected cells were seeded in a device to evaluate
their galvanotaxis response, where we found that downregulating selected individual HSPGs had
no significant effect on cell motility (G) and directedness (H) in the presence of an EF. Statistics
were obtained from at least three independent experiments with at least 60 cells in each experiment.
Error bars represent mean ± standard deviation.
J. Cell Sci. 130: doi:10.1242/jcs.203752: Supplementary information

Supplementary Figure S3. Effects of sodium chlorate treatment on astrocyte motility and
directedness (see Figure 5).
(A) Chlorate treatment had no effect on both the motility and directedness of astrocytes.

Journal of Cell Science • Supplementary information

J. Cell Sci. 130: doi:10.1242/jcs.203752: Supplementary information

Supplementary Figure S4. Addition of a coating of positively charged poly-L-ornithine

reversed the direction of galvanotaxis in BTICs (see Discussion). BTICs were seeded on an
array of surfaces coated with different combinations of extracellular matrix (ECM) and charged
molecule. Regardless of ECM type, in the absence of poly-L-ornithine (PLO), a strongly
positively charged molecule, BITCs migrated toward the cathode as indicated by the positive
directedness. However, in the presence of PLO, cells migrated toward the anode.

Journal of Cell Science • Supplementary information

J. Cell Sci. 130: doi:10.1242/jcs.203752: Supplementary information

Movie 1. Galvnaotaxis of fNPCs. fNPCs in the preserce of a 1 V cm-1 EF migrated toward the
cathode with broad lamellpodium. Cells rapidly reversed their directional bias upon revesral of the
polarity of the applied EF.

Journal of Cell Science • Supplementary information

Movie 2. Galvanotaxis of astrocytes. In contrast to fNPCs, astrocytes differentiated from fNPCs

migrated toward the anode in the presence of a 1 V cm-1 EF.
J. Cell Sci. 130: doi:10.1242/jcs.203752: Supplementary information

Movie 3. Galvanotaxis of BTICs. BTICs migrated toward the cathode in the presence of an EF
similar to fNPCs.

Journal of Cell Science • Supplementary information

Movie 4. Galvanotaxis of BTICs with HPase and chABC. BTICs treated with 12.5 U mL-1 HPase
and 2.5 U mL-1 chABC for six hours were stimulated with an EF for galvanotaxis study. Cells
treated with HPase and chABC reversed their directional response and migrated toward the anode.
J. Cell Sci. 130: doi:10.1242/jcs.203752: Supplementary information

Movie 5. Galvanotaxis of BTICs on a PLO/LN-coated surface. Addition of PLO on a laminin-

coated surface reversed the directional response of BTICs toward the anode.

Journal of Cell Science • Supplementary information