RESEARCH ARTICLE

Electrophoresis of cell membrane heparan sulfate regulatesgalvanotaxis in glial cellsYu-Ja Huang1,2, Paula Schiapparelli3, Kristen Kozielski1,4, Jordan Green1,4, Emily Lavell3,Hugo Guerrero-Cazares1,3, Alfredo Quinones-Hinojosa3 and Peter Searson1,2,*

ABSTRACTEndogenous electric fields modulate many physiological processesby promoting directional migration, a process known asgalvanotaxis. Despite the importance of galvanotaxis indevelopment and disease, the mechanism by which cells senseand 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 theanode-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 ofHS and other sulfated glycosaminoglycans significantly abolishedor reversed the cathodic response seen in fNPCs and BTICs.Furthermore, Slit2, a chemorepulsive ligand, was identified to becolocalized with HS in forming a ligand gradient across cellularmembranes. Using both imaging and genetic modification, wepropose a novel mechanism for galvanotaxis in whichelectrophoretic localization of HS establishes cell polarity byfunctioning as a co-receptor and provides repulsive guidancethrough Slit-Robo signaling.

KEY WORDS: Galvanotaxis, Brain tumor-initiating cells, Heparansulfate, Electrophoresis

INTRODUCTIONEndogenous electric fields (EFs) are known to drive manyphysiological processes including embryo development, woundhealing and immune responses by promoting directional migration,a process known as galvanotaxis (Mycielska and Djamgoz,2004; Lin et al., 2008). Disruption of endogenous EFs withpharmacological agents or externally applied EFs of oppositepolarity disturbs these processes, whereas enhancement ofendogenous EFs (same polarity, increased magnitude) increasesthe rate of regeneration by promoting the extent of nerve sproutingand the rate of wound healing in vivo (Song et al., 2004; Messerliand Graham, 2011). The brain exhibits one of the highest electricalactivities amongst all organs in the body; electric fields in the brainare not an epiphenomenon but actively regulate cellular functions.For example, the endogenous electric field between the

subventricular zone and olfactory bulb was found to direct themigration of neuroblasts and guide the migration of neuralprecursor cells along the rostral migratory stream (Cao et al.,2013). Furthermore, increased electrical activity stimulated byoptogenetics accelerates glioma growth in vivo (Venkatesh et al.,2015). Taken together, these results suggest that endogenouselectric fields modulate neural regeneration and glioma infiltrationby regulating galvanotaxis; however, the mechanism by which braincells sense and migrate directionally in an electric field remainsunknown. Therefore, elucidating the mechanism of galvanotaxiscan provide new insight into brain development and the progressionof diseases such as glioma, and provide the foundations for newclinical interventions.

Proposed explanations for galvanotaxis include electrophoreticdistribution of charged membrane components (Jaffe, 1977; Pooand Robinson, 1977; Allen et al., 2013), asymmetric activationsof ion channels (Yang et al., 2013; Nakajima et al., 2015), andmembrane-associated electro-osmotic forces (McLaughlinand Poo, 1981). Interestingly, while most cell types exhibitgalvanotaxis, the response can be either cathodic or anodic,suggesting that there may be competing mechanisms (Mycielskaand Djamgoz, 2004; Sato et al., 2009; Sun et al., 2013). Here, weinvestigate the galvanotaxis in three different types of glial cellsincluding primary neural progenitor cells (fNPCs), fNPC-derivedastrocytes, and malignant brain tumor-initiating cells (BTICs). Weshow that all three cell types exhibit a directional response to anexternal EF. More importantly, we identify the novel role ofsurface heparan sulfate (HS), a highly negatively chargedsulfated glycosaminoglycan (GAG), in sensing and mediatinggalvanotaxis. HS was found to be highly localized towards thepositive electrode (anode) of the cells in the presence of an EF inall cell types due to electrophoretic interactions. Enzymaticdigestion of HS significantly abolished the cathodic response incells. Furthermore, using non-viral siRNA knockdown, weshowed that galvanotaxis is unlikely to be due to any singleheparan sulfate proteoglycan, but is rather a collective outcomedue to the localization of HS chains. HS was identified as aco-receptor, establishing a Slit2 gradient across cellularmembranes as a consequence of electrophoretic localization.Slit2, a chemorepulsive ligand critical for central nervous systemdevelopment (Shi and Borgens, 1994; Ba-Charvet et al., 1999;Kaneko et al., 2010), subsequently provides a repulsive guidancethrough Slit-Robo signaling as indicated by the attenuation ofgalvanotaxis in response to downregulation of Robo1. We proposethat HS is a novel EF sensor that regulates galvanotaxis throughelectrophoretic interactions and its function as a co-receptor, toestablish a ligand gradient. Our findings provide direct evidence insupport of electrophoretic interactions in regulating galvanotaxis,and highlight the possibility of an EF in promoting autologouschemotaxis.Received 15 March 2017; Accepted 2 June 2017

1Institute for Nanobiotechnology, Johns Hopkins University, Baltimore, MD 21218,USA. 2Department of Materials Science and Engineering, Johns HopkinsUniversity, Baltimore, MD 21218, USA. 3Department of Neurosurgery andOncology, Sidney Kimmel Comprehensive Cancer Center, Johns HopkinsUniversity, 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

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RESULTSfNPCs, astrocytes and BTICs exhibit galvanotaxis withdifferent characteristicsTo understand the mechanisms regulating the galvanotaxis of braincells, we first characterized the responses of fNPCs, astrocytes andBTICs using a custom galvanotaxis chip (Huang et al., 2013)(Fig. 1A). All experiments were conducted under the same cultureconditions (see Materials and Methods) to avoid any bias. Thetrajectories of the cells in the presence of an EF were tracked andanalyzed to characterize the cellular response. We showed that

galvanotaxis is highly dependent on cell type: while 100% of fNPCsexhibited strong directional response towards the cathode (Movie 1and Fig. 1B), astrocytes derived from fNPCs showed an anodicdirectional response opposite to fNPCs (Movie 2, Fig. 1C).Meanwhile, the majority of BTICs (73%) migrated towards thecathode in the presence of a 1 V cm−1 EF (Movie 3 and Fig. 1D).Further quantifying cell motility and directedness in the presenceof an EF (Fig. 1E) showed that fNPCs exhibited the highest motilityon 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) andastrocytes (0.56±0.03 μm min−1). fNPCs also exhibited the highestdirectedness (d) towards the cathode (d=0.85±0.09) followed byBTICs (d=0.44±0.01), whereas astrocytes migrated stronglytowards the anode (d=−0.85±0.06).

HS is localized towards the anode regardless of cell type andthe direction of galvanotaxisWe next investigated the involvement of membrane components tounderstand how cells were able to sense and respond to an electricfield. Heparan sulfate (HS) is a candidate as an EF sensor as it is notonly ubiquitously expressed on all cellular membranes, but alsohighly negatively charged. To understand the involvement of HS,fNPCs, astrocytes and BTICs were stained with antibodies againstHS both in the absence of an EF and after being stimulated with a1 V cm−1 EF for 3 h. We showed that HS was indeed abundantlyexpressed in each cell type, as indicated by the punctate aggregatesthroughout the cellular membranes (Fig. 2A–F). In the absence ofan EF, HS distributed uniformly across cellular membranes in eachcell type (Fig. 2A,C,E). However, in the presence of an EF, wherefNPCs and BTICs migrated towards the cathode and astrocytesmigrated towards the anode, HS was found to be highly localizedtowards the anode (positive electrode) regardless of cell type and thedirection of migration (Fig. 2B,D,F). The localization of HS isparticularly obvious in astrocytes where the average size of the cellsis much larger than fNPCs and BTICs. In both the absence andpresence of an EF, astrocytes secreted and left behind trails ofextracellular matrix (ECM) abundant with HS during migration(Fig. S1A,B). However, localization of HS was only observed in thepresence of an EF (Fig. 2D), not in the absence of an external field(Fig. 2C). Further quantifying the localization of HS from analysisof processed images (Fig. 2G), we showed that an EF resulted in asubstantial increase in the percentage of cells with anodiclocalization of HS (Fig. 2H). In the absence of an EF, the anodiclocalization of HS was close to 50%, indicating no polarization:48% of fNPCs, 37.5% of astrocytes and 43.3% of BTICs. Incontrast, in the presence of a 1 V cm−1 EF, the localization increasedto 65.2, 86.1 and 77.8%, respectively. The distribution of HS in asmaller 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).

HS regulates the cathodic response in fNPCs and BTICsTo establish the relevance of anodic localization of HS in cellulargalvanotaxis, we next used heparinase (HPase) to digest surfaceHS. The effectiveness of HPase treatments was verified byimmunostaining of HS, where cells treated with HPase weredevoid of any HS signal except at the periphery near focal adhesions(Fig. 3A, right, B, right). fNPCs, astrocytes and BTICs were eachtreated with HPase and stimulated with an EF to compare theirresponses to the corresponding wild-type cells (Fig. 3C-E). Weshowed that while HPase treatment had no significant effect on cellmotility in either cell type (Fig. 3F), it significantly influenced the

Fig. 1. Galvanotaxis of fetal neural progenitor cells (fNPCs), astrocytesand brain tumor-initiating cells (BTICs). (A) Each galvanotaxis chip containstwo symmetrical devices on a 35 mm×50 mm glass coverslip. Each devicefeatures two coiled Ag/AgCl electrodes embedded in agarose reservoirslocated at each end of the cell culture channel, along with two media reservoirsalso located at each end of the channels. The dimensions of the cell culturechannel are 10 mm×1 mm×250 μm (length×width×height). A cell injection portlocated in the middle of the cell culture channel is used to introduce cells intothe device and is clamped with an alligator clip afterwards to preventevaporation. Trajectories of fNPCs (B), astrocytes (C) and BTICs (D) in thepresence of an EF are analyzed and overlaid at the origin to characterize thegalvanotaxis of each cell line. Each trajectory represents the actual pathtraveled 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 a1 V cm−1 EF.

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cathodic responses of fNPCs and BTICs (Fig. 3G). The directednessof fNPCs significantly decreased from 0.85 to 0.38 (P=0.038)after treatment with HPase, whereas the directedness of BTICssignificantly decreased from 0.48 to −0.12 (P<0.01). The anodicdirectional response of astrocytes, however, remained unaffectedeven 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 sulfationmodification. To investigate its involvement in galvanotaxis, wetreated cells with both HPase and chondroitinase ABC (chABC),enzymes that catalyze the removal of chondroitin sulfate anddermatan sulfate GAG chains. Addition of chABC did not affect themotility of either cell type (Fig. 3F), nor did it further affect thedirectedness of fNPCs (Fig. 3G). The directedness of astrocytesremained comparable to the wild-type cells despite the removal ofHS, CS and dermatan sulfate (Fig. 3G). However, treatments withboth HPase and chABC completely reversed the direction ofgalvanotaxis in BTICs from cathodic to anodic (Fig. 3E, right;Movie 4). BTICs treated with HPase and chABC migrated towardsthe anode with a mean directedness of −0.40±0.12, significantlydifferent from wild-type BTICs (d=0.48±0.07, P<0.01).

HS-mediated directional response is unlikely to bedue to anyspecific HSPGHS exists as a heparan sulfate proteoglycan (HSPG), where a coreprotein is covalently attached to several sulfated HS chains. Tounderstand how the anodic localization of HS mediates the cathodicdirectional migration of fNPCs and BTICs, we considered twopossible explanations involving either HSPG core proteins or HSGAGs, and investigated them accordingly.

We first hypothesized that cathodic galvanotaxis is due to theasymmetric distribution of one of theHSPG core proteins that interactswith its downstream effectors and establishes cell polarity. To examinethis hypothesis, the expression levels of selected individual HSPGswere systematically downregulated using siRNA. Two main types ofHSPG are expressed on themembranes of mammalian cells: syndecan(SDC1–SDC4) and glypican (GPC1–GPC6) (Sarrazin et al., 2011).Using polymeric nanoparticles containing optimized siRNAsequences, we downregulated SDC1, SDC2, SDC3, SDC4 andGPC1; downregulation of the corresponding protein expression levelswas confirmed by western blot and further validated by qRT-PCR(Fig. S2A–F). While the expression levels of selected HSPGs weredownregulated by 40–60% based on western blots and the mRNAlevels were downregulated by >80% (Fig. S2), the motility of BTICsremained unaffected by the transfection (Fig. S2G). Furthermore,downregulation of SDC1, SDC2, SDC3, SDC4 or GPC1 alone had nosignificant effect on the directedness of BTICs in the presence of anEF when compared with either wild-type cells or cells treated with ascrambled sequence (Fig. S2H).

Disrupting the sulfation of HS abolishes the galvanotaxisof fNPCs and BTICsAs cell polarity during galvanotaxis is unlikely to be due to anysingle HSPG, we considered the possibility that galvanotaxis is acollective outcome of the localization of sulfated GAG chains. Wehypothesized that as a known co-receptor for many ligands(Sarrazin et al., 2011), HS is capable of binding and forming aligand 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

Fig. 2. Anodic localization of HS in the presence ofan EF. The distribution of HS was measured byperforming a maximum intensity projection of confocalz-stacks of individual cells to minimize any errorassociated with out-of-focus pixels. In the absence ofan EF, HS is distributed uniformly across themembranes of fNPCs (A), astrocytes (C) and BTICs (E).However, strong anodic localization of HS wasobserved across all three cell lines (B,D,F) in thepresence of an EF despite fNPCs and BTICs migratingtowards the cathode and astrocytes migrating towardsthe anode. (G) Distribution of HS was analyzed byperforming a signed rank test among all the pixels in thecathode face (c) versus pixels in the anode face (a).(H) Percentage of cells with anodic localization of HS ishigher in the presence of an EF than no EF among allthree cell types. Each condition represents more than50 cells. White arrows in panels C and D indicate theECM left behind by cells during migration.

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disrupt the sulfation of HS, as the ability of HS to bind to differentligands relies heavily on the degree of sulfation (Shipp and Hsieh-Wilson, 2007). Chlorate competitively inhibits the formation ofhigh-energy sulfate donors required for sulfation reactions and thussubstantially undermines the capability of HS to bind ligands suchas fibroblast growth factor (FGF) and Slit (Safaiyan et al., 1999;Shipp and Hsieh-Wilson, 2007). fNPCs, astrocytes and BTICs weretreated with 50 mM NaClO3 for 48 h before being subjected to anapplied EF. While the motility of fNPCs and BTICs remainedunaffected by the treatment (Fig. 4C), chlorate treatment completelyabolished 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 from0.44 to 0.00 (P<0.01) (Fig. 4D). Astrocytes, however, remainedunaffected by the chlorate treatment both in terms of motility anddirectedness (Fig. S3A).

Localization of HS establishes a ligand gradient acrosscellular membranesAs NaClO3 completely abolished the cathodic response in fNPCsand BTICs, we continued to investigate the capability of HS informing a gradient of ligands across a cellular membrane. Cells inthe presence of an EF were stained with both HS and Slit2, a ligandof the Robo family of receptors essential for development in thecentral nervous system (Brose et al., 1999). Slit2 was chosen notonly for its well-characterized affinity to HS (Hu, 2001), but also forits role in providing a repulsive migration cue in various types ofbrain cells (Shi and Borgens, 1994; Ba-Charvet et al., 1999; Kanekoet al., 2010). We have previously shown that Slit2-Robo1 signalinggreatly enhanced the invasion of BTICs by providing achemorepulsive signal in vitro (Guerrero-Cazares et al., 2015).The observation that HS localized towards the back of fNPCs and

Fig. 3. Enzymatic digestion of HS significantly attenuates cathodicgalvanotaxis in fNPCs and BTICs. Heparinase (HPase) significantly reducedthe amount of surfaceHSGAGs in astrocytes (A) andBTICs (B), as shown by theabsence of fluorescence except near cell peripheries close to focal adhesionsafter enzymatic treatment. (C–E) Trajectories of wild-type cells (WT) and cellstreated with both HPase and chondroitinase ABC (chABC) are shown side byside to highlight the effect of sulfated GAGs on cellular galvanotaxis. Eachtrajectory 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 aloneor a combination of HPase and chABC had no significant effect on cell motilitywhen compared with the corresponding WT. (G) HPase significantly attenuatedthe cathodic response of fNPCs and completely abolished the galvanotaxis ofBTICs, but had no effect on astrocytes. The combination of HPase and chABCdid not further reduce the directedness of fNPCs but completely reversed thedirectional response inBTICs. *P<0.05; **P<0.01; Student’s t-test. Statisticswereobtained from at least three independent experiments with at least 60 cells ineach experiment. Error bars represent standard deviation.

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 toinvestigate how the sulfation of HS influences cathodic galvanotaxis.Treatment with sodium chlorate had no effect on cell motility (C) but abolishedthe directional response of fNPCs and BTICs in the presence of an electricfield (D). **P<0.01 (Student’s t-test). Statistics were obtained from at leasttwo independent experiments with at least 60 cells in each experiment. Errorbars represent standard deviation.

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BTICs (anode) while cells migrated towards the cathode supports amechanism involving Slit-mediated repulsive guidance. Fromimmunofluorescence staining, we discovered that Slit2 wascolocalized with HS towards the anode in the presence of an EFin all three cell types, demonstrating the capability of HS in forminga ligand gradient across a cellular membrane in an EF (Fig. 5A–C).Furthermore, we also showed that the ability of HS to bind Slit wassignificantly attenuated in cells treated with chlorate, as shown by asignificant decrease in the intensity of Slit but not HS (Fig. 5D).

Downregulation of Robo1 attenuates galvanotaxisTo probe the involvement of Slit-Robo signaling in promotinggalvanotaxis, BTICs were transduced with lentiviral particlescontaining a shRNA sequence against Robo1 receptor (Fig. 6Aand B) and subjected to an EF. We showed that downregulation ofRobo1 attenuated the galvanotaxis of cells as both cell motility anddirectedness decreased compared with the control group transducedwith an empty virus (EV) (Fig. 6C,D). Downregulation of Robo1decreased 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).

DISCUSSIONGradients of molecular cues along the cell migration path is widelybelieved to be the driving mechanism for brain cell migration duringdevelopment and pathogenesis. Our findings suggest that anendogenous or an applied EF can also establish a gradient ofmolecular cues at the cellular level through electrophoresis of HS,and resulted in directional migration. These findings haveimplications in understanding brain homeostasis and may beutilized 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 changesin 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 beenshown to be polarized in an electric field (Poo et al., 1979; Zagyansky

and Jard, 1979; Fang et al., 1999; Finkelstein et al., 2007; Nakajimaet al., 2015). However, the identity of a macromolecule that exhibitsboth electrophoretic polarization and is also necessary forgalvanotaxis has not been reported. For example, the ConAreceptor has been shown to be polarized in an EF and its directionof 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 tobe important for galvanotaxis as pharmacological inhibitionabolished galvanotaxis in keratinocytes, although it is unclearwhether the polarization of EGFR towards the cathode is due to anelectrophoretic force. Here, we present direct evidence in support ofthe electrophoresis in regulating galvanotaxis. As HS and othersulfated GAGs are amongst the most highly negative chargedbiopolymers in nature (Sarrazin et al., 2011) and HS is polarizedtowards the anode-facing side of glial cells regardless of theirdirection of galvanotaxis, localization of HS is probably a physicalprocess involving an electrophoretic force. Furthermore, HS is alsocritical for the cathodic response observed in fNPCs and astrocytes.Taken together, we conclude that HS is a novel EF sensor that relayselectrical signals to directional migration cues through electrophoreticpolarization.

However, since the anodic directional response of astrocytes wasnot affected upon removal of HS, other mechanisms, perhaps incompetition with HS, probably exist and collectively determinethe direction of galvanotaxis. This could explain the surprisingobservation that the directional response of BTICs was reversedfrom cathodic to anodic upon removal of different types of sulfatedGAGs (Fig. 3E); as we weakened the cathodic response mediatedby sulfated GAGs, the mechanisms governing an anodic responsedominated and cells migrated towards the anode. Interestingly, wehave also previously observed that the galvanotaxis of BTICs canalso be reversed by their surrounding microenvironment (Huanget al., 2016); galvanotaxis of BTICs switched from cathodic toanodic upon addition of poly-L-ornithine on top of a laminin-coated substrate, similar to the galvanotropism of neurons reportedpreviously (Movie 5 and Fig. S4) (Rajnicek et al., 1998). Theseresults highlight the existence of potential competing mechanisms.

Fig. 5. Slit2 colocalizes with HS and forms agradient across cellular membranes in thepresence of an EF. fNPCs (A), BTICs (B) andastrocytes (C) were stained for both HS (red) and Slit2(green) to investigate whether localization of HS iscapable of forming a ligand gradient across cellularmembranes. Antibodies raised in different specieswere selected to avoid cross-reaction. Slit2 colocalizedwith HS as indicated in the overlays (yellow) andformed a gradient across cellular membranes in allthree cell types. (D) Chlorate treatments significantlyhindered the affinity of HS to Slit2, as indicated by thedecrease in Slit2 fluorescence signal.

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We hypothesize that one of the contributing factors in mediatingthe competing mechanisms may be the cellular level of cAMP, aspoly- L-lysine was shown to elevate cellular cAMP levels morethan twofold in oligodendrocytes (Vartanian et al., 1988). Cellularlevels of cAMP have been shown to modulate the repulsive versusattractive response towards netrin-1 in Xenopus spinal neurons(Ming et al., 1997) and elevated cAMP levels abolished thegalvanotaxis of keratinocytes and keratocyte fragments (Pullar andIsseroff, 2005; Zhu et al., 2015). Another candidate in regulatingthe anodic galvanotaxis is sialic acid, a negatively chargedsugar molecule that contributes to the overall charge of themembrane. Removal of sialic acid has been shown to reversethe polarization of ConA receptors and impair the cathodicresponse 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 twohypotheses; one involved the function of the core proteins ofHSPGs and the other involved sulfated GAGs. We examined thefirst hypothesis by selectively knocking down the expression levelof five individual syndecans and glypicans, as both types of surface

HSPG have been shown to influence cell migration (Wight et al.,1992). For example, syndecan-1 has been shown to localize at theuropods (trailing edge) of polarized myeloma cells and promote celladhesion (Børset et al., 2000). Similarly, syndecan-4 has also beenobserved to inhibit Rac at the back of neural crest cells in vivo duringdevelopment (Matthews et al., 2008) and mediate the persistentmigration of fibroblasts by locally regulating Rac activity (Basset al., 2007). These results support our observations and hypothesis,where HSPGs were found to be localized at the anodal face (trailingend) while cells migrated towards the cathode. However, knockingdown any individual HSPG alone did not have any significant effecton the cathodic response of BTICs, suggesting that galvanotaxis isunlikely to be dependent on the function of any individual HSPGcore protein. Given that mammalian cells have four syndecanand 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 knownto serve as a co-receptor for many different ligands based on varioussulfation modifications (Sarrazin et al., 2011). Treatment withsodium chlorate completely abolished the cathodic response infNPCs and BTICs (Fig. 4D), suggesting the important role ofsulfated HS in sensing and migrating during galvanotaxis. Inaddition, as Slit2 was found to be colocalized with HS in forming apositive surface gradient towards the anode (Fig. 5A–C) anddownregulation of Robo1 attenuated galvanotaxis (Fig. 6), wepropose a model where galvanotaxis is mediated by theelectrophoresis of HS and its function to serve as a co-receptor forligands such as Slit2 (Fig. 7). However, it is likely that othermechanisms and ligands, such as other family members of Slit andRobo as well as FGF, may have also contributed to the process asdownregulating Robo1 only partially attenuated galvanotaxis.Nevertheless, our results highlight the novel role of HS as an EFsensor during galvanotaxis and provide direct evidence in supportof the electrophoretic galvanotaxis model. These findings couldhave broad implications in many physiological events as cellsubiquitously express HS and small EFs have been associated withneural 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 cellmigration and how an applied EF could potentially be leveraged tomodulate this process provide a rationale for new therapeutics. Forexample, transcranial direct current stimulation (tDCS) has shownclinical benefits for central nervous system diseases (Fregni et al.,2015) and was able to promote cathodal accumulation ofendogenous neural stem cells (Rueger et al., 2012). As more islearned about the role of bioelectricity on cell function it is likelythat new opportunities for interventions will emerge.

MATERIALS AND METHODSCell linesAll cell cultures were established with institutional approval by the JohnsHopkins University Internal Review Board.

fNPCs: F54 cells were derived after 17 weeks of gestation, obtained fromelective abortion (Tzeng et al., 2011) and were maintained in 2:1 high-glucose Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen)/Ham’sF12 (Cellgro) mixture supplemented with 2% B-27, 1% antibiotic–antimyotic, 20 ng ml−1 bFGF, 20 ng ml−1 EGF, 20 ng ml−1 leukemiainhibitory factor (LIF; Millipore, Billerica, MA) and 5 μg ml−1 heparin(Sigma).

Astrocytes: Astrocytes were derived by plating fNPCs in a tissue culture flaskin DMEM/F12 medium (Sigma) supplemented with 10% fetal bovine serum(Sigma) and 1% penicillin-streptomycin (Invitrogen) (Placone et al., 2015).

Fig. 6. Downregulation of Robo1 attenuates the galvanotaxis of BTICs.BTICs were transduced with shRNA against Robo1 (sh-Robo1) to evaluate thecontribution of Slit-Robo signaling in galvanotaxis. (A,B) The efficiency of theknockdown against cells transduced with an empty virus (EV) was evaluatedusing western blot, where the transduction resulted in a 47±9.7%downregulation of Robo1. Trajectories of EV cells (C) and sh-Robo1 cells (D) inthe presence of an EF were analyzed to examine the involvement of Slit-Robosignaling in galvanotaxis. Robo1 knockdown resulted in a decrease in both cellmotility (E) and directedness (F). *P<0.05; Student’s t-test. Statistics wereobtained from two independent experiments with at least 60 cells in eachexperiment. Error bars represent standard deviation.

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BTICs: GBM612 cells were used and previously validated by JohnsHopkins Genetic Resources Core Facility (Li et al., 2014). GBM612 cellsisolated from intra-operative tissue are multipotent and are able to formdiffuse tumors when implanted into animal models (Guerrero-Cázares et al.,2009; Li et al., 2014; Kondapalli et al., 2015). BTICs were grown inculture flasks coated with laminin and cultured in DMEM/F12 mediasupplemented with 2% B-27, 1% antibiotic–antimyotic, 20 ng ml−1 FGFand 20 ng ml−1 EGF.

Two-dimensional galvanotaxis and cell trackingGalvanotaxis experiments were carried out using a customized galvanotaxisdevice reported previously (Huang et al., 2013), with standardmicrofabrication techniques (Fig. 1A). Briefly, a galvanotaxis chip isfabricated from polydimethylsiloxane (PDMS) and oxygen plasma bondedonto a glass coverslip. Ag/AgCl electrodes embedded in agarose are insertedinto the reservoirs, and the exposed glass slide in the channel is coated withlaminin. Before each experiment, different cell media were replaced with astandard medium for 6 h to ensure that the reported phenotypes are notmedium dependent. The standard medium was DMEM/F12 supplementedwith 2% B-27, 1% antibiotic–antimycotic, 20 ng ml−1 bFGF and20 ng ml−1 EGF. The galvanotaxis device was mounted onto an invertedmicroscope (Nikon Ti-E 2000) equipped with a live-cell chamber at 37°Cand 5% CO2. The cells were imaged through the glass slide. Cells werestimulated in a 1 V cm−1 DC EF for 3–9 h before being fixed forimmunofluorescence studies.

The trajectories of cells from time-lapse images were automaticallytracked using MetaMorph software (Molecular Devices, Sunnyvale, CA,USA) to minimize any tracking biases. Only isolated cells that remainedin 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 parametersincluding cell motility and directedness. Here, we define cell motility as thetotal path length traveled by a cell divided by the elapsed time. Thedirectedness is defined as Σcosθi/n and ranges between −1 and +1, where nis the total number of cells and θi is the angle between the vector of celldisplacement and electric field vector (Huang et al., 2013). A directednessclose to zero indicates randommotion, whereas positive and negative valuesindicate cathodic and anodic responses, respectively. To determinestatistical significance, we use a two-tailed Student’s t-test (*P≤0.05;**P≤0.01; ***P≤0.001).

Confocal immunofluorescence imagingFor immunofluorescence imaging, cells were fixed with 3.7% formaldehydeand stained with selected antibodies without a permeabilization step tominimize any fluorescence background arising from inside the cells. Cellswere then blocked with tris-buffered saline with 0.1% Tween 20 (TBST)containing 5% bovine serum albumin (BSA) for 1 h and incubated withmouse anti-HS (1:100, US Biological, 10E4 epitope) or rabbit anti-Slit2

(1:100, Abcam, ab7665) overnight at 4°C. After incubation, cells wereextensively washed with TBST and stained with a secondary antibodyconjugated to a fluorophore (1:200, Invitrogen).

To quantify the localization of membrane proteins, confocal fluorescenceimages were collected to minimize any out-of-focus pixel due to anyvariation throughout the cell surface (TiE, Nikon). Confocal z-stacks at 1 μmper step were collected for each cell at the excitation wavelengthcorresponding to the fluorophore used.

Quantification of protein localizationFor each confocal z-stack, a maximum intensity projection was created usingNIS-Elements software to generate a 2D image and converted to an 8-bitgrayscale to quantify the localization of heparan sulfate. The contour of a 2Dcell image was identified and vertically divided along its geometric centerinto a cathode- and an anode-facing side using ImageJ (Fig. 2G).Subsequently, a Wilcoxon signed rank test was performed among all thepixels in the cathode face to pixels in the anode face using R software, whereP<0.05 is defined as a cell with anodic localization of HS.

Enzymatic removal of surface glycosaminoglycansCells were treated with 12.5 U ml−1 of HPase I and III blend (Sigma) at37°C for 6 h before being subjected to an EF for galvanotaxis experiments.To remove CS and dermatan sulfate, cells were incubated withchondroitinase ABC (chABC) (2.5 U ml−1; Sigma) at 37°C for 6 h.

siRNA transfection using polymeric nanoparticlesPolymers (R646) designed to condense siRNA into bioreduciblenanoparticles were made of poly(β-amino ester) as described previously(Kozielski et al., 2014). The particles have been shown to be capable ofefficient gene knock-down in primary human glioblastoma cells withoutsignificant 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 wereallowed to adhere overnight before transfection. Cells were then transfectedwith siRNAs (OriGene Technologies, Rockville, MD, USA) for SDC1,SDC2, SDC3, SDC4 and GPC1 genes or a scrambled siRNA (SC) at a finalconcentration of 120 nM using polymeric nanoparticles (R646) followingprocedures reported previously (Kozielski et al., 2014). Transfected cellswere collected for western blot and qRT-PCR analysis after 72 h.

Western blot and qRT-PCRCells 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 supernatantswere removed from the pellets and collected. Protein collected from eachlysate was measured with a Pierce BCA protein assay kit (ThermoFisherScientific) to ensure equal loading. Denatured lysates were loaded onto4–15% gradient SDS-polyacrylaminde gels (Bio-Rad) and transferred to anitrocellulose membrane (Bio-Rad). Membranes were blocked with TBST

Fig. 7. Proposed mechanism for cathodicgalvanotaxis mediated by electrophoreticlocalization of HSPG. Electrophoresis of HS in thepresence of an EF redistributes HSPGs towards theanode and consequently establishes a ligand gradient(for example, Slit2) across a cellular membrane, as HSis a co-receptor for many ligands. A ligand gradientacross a cellular membrane asymmetrically activatesdownstream signaling, in this case Slit-Robo repulsiveguidance, and promotes cell migration towards thecathode possibly by locally suppressing the activationof Cdc42 and Rac1 at the anode.

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containing 5% milk at room temperature for 1 h and incubated with primaryantibodies at 4°C overnight. For each experiment, GAPDH-HRP (Santa CruzBiotechnology, FL-335, 1:400) was used as a loading control. The followingantibodies 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) wereused at 1:2000. Immunoactive bands were detected using enhancedchemiluminescence 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 recommendedprotocol and measured using a StepOnePlus system (ThermoFisherScientific). mRNA expression level was calculated using [Δ(ΔCt)]relative to scrambled control, with GAPDH being used as ahousekeeping gene.

Stable transduction of cells with shRNATo induce the knockdown of Robo1 expression, we used pLKO.1 lentiviralparticles 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). Humanfetal neural progenitor cells were treated with one of the three viruses at 1 μlper 2 ml with polybrene (8 μl ml−1) overnight. Virus-containing media werereplaced the next day with complete mediawith 0.25 μgml−1 puromyocin asselection agent. After one week, the concentration of puromycin wasreduced to 0.125 μg ml−1 for maintenance. Knockdown was confirmedusing western blot.

AcknowledgementsWe thank Dr Cheng Ran (Lisa) Huang for scientific discussions.

Competing interestsThe authors declare no competing or financial interests.

Author contributionsConceptualization: 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.

FundingThe work was supported by the National Institutes of Health (grant numbersR01CA170629, R01NS070024). Deposited in PMC for release after 12 months.

Supplementary informationSupplementary information available online athttp://jcs.biologists.org/lookup/doi/10.1242/jcs.203752.supplemental

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