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Downloaded by guest on October 1, 2021 a Shim migration Gawoon cell collective of external programming enhances coordination cell native Overriding PNAS collectively migrate to tissues allows coordi- coupling and This transmission motion. force nated long-range cells for cadherin- allow couple and mechanically instance, together tissues For in leverage. adhesions that cell–cell can mediated control migration and directional cues coordinated, external estab- for have already mechanisms endoge- cells hand, the lished one means the migration attempts cell On for collective behavior. bane nous cell and program boon and a control both to are tissues in present already its with competes that behavior collective behaviors? collective a natural perform to command tissue we when a happens What problem: collectives fundamental cellular a complex raises cell to in them advances directed applying despite even tools, However, sophisticated over 7). has (6, dimensions interfaces control two in bioelectric migration programmable in truly work recent demonstrated collective and contractility direct (5), cell and constrain of growth can control scaffolds dynamic micropatterned (2, allow (4), cells can chemotaxing optogenetics redirect 3), can Tunable generators biotechnologies. gradient modern chemical becom- with is feasible motility wound cell increasingly accelerated over ing control enabling regu- precise as of and such Efficient ways bioengineering healing. processes, new and key fundamentally biology these enable for lating would tool control there- powerful such migration, a as cell be collective cancer would of and fore, Control injury, (1). modes upon invasion self-healing development, embryonic C E-cadherin migration cell collective of key control a migration. external as collective optimizing con- considered when in variable be and design should are systems quantitative collectivity results noncellular endogenous diverse, These that from firm vitro. those in with closure of keeping wound demon- paradigm and accelerated control this improve strate to significantly applied coordination chelators, existing we weakening cell–cell Finally, calcium of controllability. with improved formation disman- junctions rapidly the E-cadherin or tling antibodies disrupting E-cadherin–specific by with junctions either cell– adhesion, E-cadherin–dependent coordination inhibiting preexisting cell specifically by reducing tissue However, the tissue. leading in the the damaging of actively elec- edge conflict resisted this and with coordination control, trotactic natural strong showed cell–cell high adhesion with to Monolayers strength collectivity. adhesion endogenous cell–cell mouse modulate tune can primary we where electrotaxis—to monolayers skin and migratory external stimulation potent a cue—electrical applying by How- We problem system. with this or value. investigate tissue compete the great commands in behaviors external of collective the preexisting strong, are if arise migration can processes cell problems ever, biological program in externally essential methods (received to progression, 2021 cancer is 8, and June healing, Levine migration development, Herbert spanning Member cell Board Editorial collective by accepted As and MA, Boston, Health, 2021) Public 22, of January School review Harvard for Fredberg, J. Jeffrey by Edited 08540 NJ Princeton, University, eateto ehncladArsaeEgneig rneo nvriy rneo,N 84;and 08540; NJ Princeton, University, Princeton Engineering, Aerospace and Mechanical of Department aaoial,toeedgnu olciecl behaviors cell collective endogenous those Paradoxically, essta r seta omliellrlf,spanning life, multicellular to essential are pro- coordinated that intricate, enables cesses migration cell ollective 01Vl 1 o 9e2101352118 29 No. 118 Vol. 2021 | elcl adhesion cell–cell a ael Devenport Danelle , | electrotaxis | oriae motion coordinated b n ailJ Cohen J. Daniel and , | a,1 doi:10.1073/pnas.2101352118/-/DCSupplemental hsatcecnan uprigifrainoln at online information supporting contains article This 1 Editorial under the distributed by BY).y is invited (CC article editor access guest open a This is J.J.F. Submission. Direct PNAS Board. a is article This interest.y competing no declare authors The ulse uy1,2021. 16, July Published and data; analyzed D.J.C. paper. the and wrote D.D. G.S. D.J.C. research; tools; and performed D.D., reagents/analytic G.S., G.S. new research; contributed designed D.J.C. D.J.C. and and G.S. contributions: Author collective controlling of capable perturbation both circumvent needed programmable we we questions, can a these How endoge- investigate to 2) To an ability limitations? migration? such our of cell limit strength collective tissue its a the control in does two behavior migration much address collective We How nous tissue. 1) col- the questions. underlying in the key present of already strength behaviors the lective and direct migration to cell attempting command collective external applied, an between play applied the of potency the of and strength stimulus. and processes resilience collective the natural between the tug-of-war likely a control external on to how depends be may Hence, system (12–14). collective a locations cells “susceptible” necessary ensure to to gastrulation migrate embryos to collectively other prior and junctions zebrafish cell–cell for weaken for need to signals the by to to shown responding via as from established fluidity migration, cells coordination hinder the Strong can 11). lack coupling strongly (10, physical physically so group a they are as that cells migrate where confined tissue and a solid-like attached create or can collective coupling state” cell existing “jammed Tight an conflicts. over the generate behavior On may new behavior 9). a (8, imposing might hand, cells other individual than and better cohesion far maintain and organization distances large over directionally and owo orsodnemyb drse.Eal [email protected] Email: addressed. be may correspondence whom To rlapoc oetral otoln ihycoordinated highly controlling gen- externally a tissues. to offer results approach These migration. eral ability cell collective our control improves to adhesion cell–cell disrupting by that E-cadherin–mediated coordination show even cell we natural and However, weakening specifically commands damage. cellular the significant to causing commands, mouse responsiveness migration primary tissue external reducing of with cell layers competes endogenous coordination strong cultured using how demonstrating large, question monolayers, skin in program this migration externally investigate cell stimulus—electrotaxis—to We hap- bioelectric tissue. what a a of about collective in are natural known with behaviors it clash is “commands” reprogram external little when to pens However, approaches interest. healing, pro-great as multicellular for such critical is cesses migration cellular collective As Significance ee eseiclyivsiaeterltosi n inter- and relationship the investigate specifically we Here, y b eateto oeua ilg,Princeton Biology, Molecular of Department https://doi.org/10.1073/pnas.2101352118 y raieCmosAtiuinLcne4.0 License Attribution Commons Creative . y https://www.pnas.org/lookup/suppl/ | f11 of 1

APPLIED BIOLOGICAL SCIENCES migration and a physiologically relevant model system allowing nous collective behavior is well characterized (24, 30), they have for tunable “collectivity.” Here, we use collectivity to describe a strong electrotactic response (6), and their cell–cell adhesion how strongly cells are coordinated with their neighbors dur- levels can be easily tuned via calcium levels in the culture media ing migration—highly collective cells exhibit strong, coordinated (28, 31). Previous work has indicated that cell–cell adhesions via motion and vice versa. As a perturbation, we harnessed a bioelec- calcium-dependent proteins, E-cadherin adhesion being one of tric phenomenon called “electrotaxis”—directed cell migration the best studied, are essential in interconnecting individual cells in direct current (DC) electric fields—using our SCHEEP- and maintaining coordination within the monolayers by coupling DOG bioreactor (6). Briefly, electrotaxis arises when endoge- mechanical information via the cadherin–catenin–actin complex nous, ionic fields form during healing or development (∼1 (32–35). Hence, we hypothesized that modulation of cell–cell V/cm) and apply gentle electrophoretic or electrokinetic forces adhesion levels via calcium control would allow us to tune the rel- to receptors and structures in cell membranes, causing them ative strength of collective couplings and collective migration in to aggregate or change conformation to produce a front–rear primary keratinocyte layers, giving us a precise and reproducible polarity cue (15, 16). Components spanning phosphatidylinositol system to explore questions of collective control. phosphates (PIPs), extracellular signal-regulated kinase (ERK), To establish quantitative standards for collective strength phosphatidylinositol 3-kinase (PI3K), phosphatase and tensin in our keratinocyte model, we engineered arrays of identical homolog (PTEN), and small guanosine triphosphate (GTP)ases 2- × 2-mm2. keratinocyte tissues using tissue stenciling methods have been implicated in the transduction process, while gap junc- (6, 36). Tissue arrays were then cultured for 14 h in high-calcium tions appear to have an inconclusive role (8, 17–19). Crucially, (1.0 mM), medium-calcium (0.3 mM), or low-calcium (0.05 mM) electrotaxis may be one of the broadest and most conserved conditions to allow junction formation (Fig. 1A). These cal- migratory cues, having been observed in vitro in over 20 cell types cium levels are standard conditions that span the physiological across multiple branches of the tree of life (20–22). As electro- range based on phenotypes and marker expressions (28, 31, 37, taxis in vitro appears to globally stimulate all cells equally and 38). Using nuclei counting, we confirmed that density across still induce directional motion, it is distinct from more locally conditions did not vary significantly (SI Appendix, Fig. S1). As dependent cues such as chemotaxis and haptotaxis. However, as E-cadherin is a major calcium-dependent adhesion protein, we no other reported cue has as much versatility and programmabil- used immunostaining to quantify and confirm the direct rela- ity, electrotaxis is an ideal choice for a broadly applicable cellular tionship between calcium level and E-cadherin recruitment to control cue in this study. cell–cell junctions (Fig. 1 A and B and SI Appendix, Fig. S2). To complement electrotaxis, we chose primary mouse skin for We generated collective motion data for each tissue by process- our model system as skin injuries were where the endogenous ing phase-contrast time-lapse movies captured using automated electrochemical fields that cause electrotaxis were first discov- imaging with particle image velocimetry (PIV) to generate veloc- ered (in vivo, the wound boundary is negative relative to the ity vector fields for each time point (Materials and Methods). To surrounding epidermis), and we and others have shown layers ensure PIV fairly captured the range of conditions, we performed of keratinocytes to exhibit strong electrotaxis (6, 23–25). Criti- preliminary validations against analyses based on nuclear track- cally, primary mouse keratinocytes have tunable collectivity in ing and found no appreciable difference (SI Appendix, Fig. S3). culture as the cadherin-mediated cell–cell adhesion strength in The vector fields were then analyzed to visualize and quantify this system can be easily tuned by varying calcium levels in the the strength of coordinated motion within a given tissue over media—with low-calcium media thought to mimic conditions in time (SI Appendix, Fig. S4) (6, 23, 36). First, we calculated the the basal layers of the epidermis with weak adhesions and high- directionality of cellular movements to visualize domains of coor- calcium media akin to conditions in the uppermost layers of skin dinated migration within tissues. Directionality (Eq. 1) is defined with strong adhesions (26–28). as the average of the cosine of θ, the angle between each PIV Together, these experimental approaches allowed us to pre- velocity vector and the horizontal x axis, while N is equal to the cisely explore how the ability to externally “steer” collective total number of velocity vectors in the frame. As the electric migration in a living tissue using a powerful bioelectric cue field command is in the horizontal direction (1 F), the direction- depends on the native collectivity of the underlying tissue. First, ality can also indicate how well aligned the cellular migration we quantify collective strength in cultured skin layers by measur- is with the field direction under stimulation. Directionality can ing neighbor coordination of cellular motion [a standard metric vary between −1 (cell motion to the left; perfectly antiparallel for collective motion adapted from collective theory (29)] and with field) and 1 (cell motion to the right; perfectly parallel with then, validate that the collectivity can be tuned in our model sys- field). Additionally, we quantified the collectivity by calculating tem of mouse keratinocyte monolayers by calibrating junctional the overall coordination within a tissue using the polarization E-cadherin levels. Next, we demonstrate how applying the same order parameter (Eq. 2) from collective theory, where vi indi- electrical stimulation conditions to tissues with differing native cates the ith velocity vector (29). A coordination value of one collectivity results in radically different outputs, with weakly col- indicates perfect coordination and anistropy across the whole lective tissues precisely responding to our attempts to control tissue, while zero indicates wholly isotropic motion: their motion, while strongly collective tissues exhibited detrimen- N tal supracellular responses resulting in tissue collapse. We then 1 X Directionality = cos (θ) [1] prove that E-cadherin is responsible for these differences, ruling N out any effects of calcium signaling per se. Finally, we leverage i=1 these findings to develop an approach that allows us to effec- N −→ tively control mature, strongly collective tissues, which we utilize 1 X vi Coordination = . [2] to demonstrate that we can accelerate wound repair in vitro. N kvi k i=1 Results Our data (Fig. 1 C and D) clearly demonstrate that increasing Establishing Baseline Collective Migration of Primary Keratinocyte calcium levels increases collectivity within the tissue. Both the Layers. To determine how natural collective cell behaviors com- general size of coordinated domains, represented by large zones pete with externally imposed control of collective behavior, we of either red or blue in Fig. 1C, and the coordination parameter first need to establish baseline data of endogenous collective varied directly with calcium levels (Fig. 1D). Velocity correlation behavior in the absence of guidance cues. We used monolayers of with nearest neighbors also increases in value with increased cal- mouse primary keratinocytes as a model system as their endoge- cium levels (SI Appendix, Fig. S11). However, we also noted that

2 of 11 | PNAS Shim et al. https://doi.org/10.1073/pnas.2101352118 Overriding native cell coordination enhances external programming of collective cell migration Downloaded by guest on October 1, 2021 Downloaded by guest on October 1, 2021 vriigntv elcodnto nacsetra rgamn fcletv elmigration cell collective of programming external enhances coordination cell native SCHEEPDOG Overriding our al. et of Shim version modified a using elec- using cue unidirectional tissues a trotactic these delivered in we Here, attempted migration stimulation. collective next bioelectric drive we and E-cadherin, strong program to junctional to levels high calcium and high and collectivity E-cadherin junctional low and Cell Collective Program to Migration. Difficult More It Makes Collectivity Strong collective how susceptibility. investigated electrotactic next regulates strength we quantify tissues. established, to unstimulated baselines of and motion With endoge- collective layers natural tune keratinocyte the profile to in and ability strength our collective validated nous and data levels studies these cadherin prior between coordination, and correlation calcium strong with a E- directly demonstrating that vary indicating also of data levels collection our cadherin dense with a Together like agents. more individualistic far and behaving fluctuations tissues supracellular low-calcium high- exhibiting with visually levels, and tissues calcium cell calcium different in shift the clear across a morphology is tissue there Notably, trade-off (39). common motion collective a in motion, cellular impeded cell adhesion average cell–cell 1E reduced (Fig. of edge cost speed the the migration indicate at outlines came Pastel tissues. coordination line. across increased dotted SD white represent the bars at Error starts h. stimulation 9 kymographs Electrical throughout displacement stimulation. levels edge of test. calcium trailing Mann–Whitney h same and nonparametric 8 of unpaired Leading and monolayers (G) stimulation) unstimulated displacement). low-calcium (no of edge and control displacement trailing of and mM), h (leading (0.3 1 cathode throughout the medium-calcium toward mM), migration monolayer (1.0 (C keratinocyte signal. high-calcium image; immunofluorescence phase in E-cadherin the junctional cultured 45 indicates normalized Gray monolayers for h. keratinocyte plot 14 tribution mouse for media primary mM) for (0.05 imaging E-cadherin and 1. 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Fig. junctions reassemble or then and reshape tissue, to susceptible tran- electrotaxis more to the use us move to junctions, allow cell–cell to sought strategy disrupt we stimulation siently formation, general barrier is more E-cadherin and a that develop function yet, skin and for skin as in essential control Tissue electrotactic a limit can of Strategy. Reassembly Control and a Transport, Collective Disassembly, cause even and retraction. as controllability such effects reduce mechanical can adverse sug- here, specifically data mediated E-cadherin these coordination, of by DECMA-1 native all strong only Together, overly S8). that when Fig. gest than Appendix, calcium (SI effective medium used less was in down-regulate also adhesion to was P-cadherin effort monolayers an and in E-cadherin together both PCD-1 S7 and phe- Fig. as DECMA-1 undesirable Appendix, conclusive exacerbated as (SI cases, nor notypes certain significant con- in as electrotactic and, neither in E-cadherin is role role minor its while some trollability, play However, to P-cadherin/E-cadherin. seems both P-cadherin of blockade dual vriigntv elcodnto nacsetra rgamn fcletv elmigration cell collective of programming external enhances coordination cell native Overriding al. et Shim ****P < P F E-cadherin GFP (A) junctions. E-cadherin altering acutely by rescued quickly and easily be can monolayers coordinated highly of Controllability 0.0001. ausaecluae sn h nardnnaaercMn–hte test; Mann–Whitney nonparametric unpaired the using calculated are values nwn ohta togcl–eladhesion cell–cell strong that both Knowing E D C A X velocity (um/ hour) Time (8h) 100 120 140 20 40 60 80 -eoiyhampkymographs heatmap X-velocity edn deretraction edge Leading -ahrnwt BAPTA with E-cadherin 0 + -eoiydnmc ihstimulation with dynamics X-velocity 0101020303040480 420 360 300 240 180 120 60 0 * efso 1.0mM = Perfusion t and (Left 0 = ih1h with 0 = t .Uaeo both of Usage S7). Movie ie(minutes) Time n ih1ho AT ramn (Right treatment BAPTA of h 1 with and ) hs mg fhg-acu eaioyemnlyr at monolayers keratinocyte high-calcium of image Phase (G) E. - BAPTA + F * ) n etrdi ihclimmdafr1 (t h 14 for media high-calcium in restored and h), 9 = efso 0.05mM = Perfusion ietoaiya 4h at Directionality AT.(C BAPTA. µM B ihBPAadfield and BAPTA with 1.0mM Coordination -eoiyha a yorp o AT-rae ihclimmonolayers high-calcium BAPTA-treated for kymograph map heat X-velocity ) re ht1ho AT ramn ple otsuswith tissues to 5 (Fig. applied junctional coordination reduce treatment reduce and transiently BAPTA E-cadherin could of junctions E-cadherin h of strong 1 con- imaging keratinocytes that Fluorescence E-cadherin firmed (GFP) (54). protein tissues junc- fluorescent E-cadherin green preestablished disrupted in it how tions examined (Materials and chelator Methods), calcium-specific and to extracellular exposure an brief (BAPTA), tested control to we trying alternative, are 1,2-bis(o an we tissues As very effectively. the more in have value overall cell–cell to how their strong appear ing control established antibodies 4 to already (Fig. 3) difficult penetrating junctions and time is junctions; difficult it block a will 2) it expensive; long are it approach: general E-cadherin, Antibodies a of as used role 1) be the to limitations revealing significant three at DECMA- had effective While location. was target treatment its 1 reached had tissue the when AT o ,wse u h hltr n eundthe returned and chelator, the with out monolayers washed h, treated 1 we for monolayers, BAPTA collective strongly - -120um/h 120um/h ots o ai hlto fetdtecnrlaiiyof controllability the affected chelation rapid how test To 8 n G + N -aminophenoxy)ethane- 2t 5frec odto.(cl as=500 = bars (Scale condition. each for 15 to 12 = t=0 t=1h .(cl as 20 bars: (Scale ). t=23h t=9h irto ihBAPTA with Migration D–F and oriainvle o high-calcium for values Coordination (B) µm.) ,teeylimit- thereby ), S8 Fig. Appendix, SI 3h.Errbr ersn Dacross SD represent bars Error h). 23 = t https://doi.org/10.1073/pnas.2101352118

rae o ihBPA(t BAPTA with h 1 for treated 0 =

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APPLIED BIOLOGICAL SCIENCES monolayers to BAPTA-free, high-calcium media for electrical Accelerating Bioelectric Healing in Vitro by Manipulating the stimulation; 1 h of BAPTA treatment boosted controllability in Strength of Cell–Cell Adhesion. Combining pharmacological per- strongly collective monolayers, with treated monolayers exhibit- turbations with bioelectric cues to improve tissue response sug- ing both significantly increased migration speed and reduced gests practical avenues to engineering the behavior of otherwise leading edge retraction (Movie S8). However, these benefits recalcitrant tissues for practical purposes. To test this, we cre- were short lived, as speed and displacement drastically decreased ated a wound gap across a strongly collective, high-calcium skin over time (Fig. 5 C and D, orange) likely as cell–cell junctions layer and reconfigured SCHEEPDOG to have a central negative eventually reengaged due to the high calcium concentration (SI electrode and peripheral positive anodes, generating an elec- Appendix, Fig. S10). To prevent the gradual restoration of junc- tric field that converged on the middle of the wound to drive tions, we maintained tissues in low-calcium media after washing each side of the tissue (Materials and Methods) (55). In this out BAPTA. These tuned tissues exhibited a nearly 5× increase case, na¨ıve stimulation would trigger a collapse or at best, no in maximum speed, strong leading edge displacement, and high edge outgrowth (Figs. 2 and 3), but the disassembly/reassembly alignment with the field command (Fig. 5 C–F, purple). process described above should enable complete, expedited heal- Having confirmed that transient chelation could dramatically ing. Identical to the scheme described above, strongly collective increase controllability, we then examined if we could restore monolayers were treated with BAPTA for 1 h, stimulated in low- the monolayer to its initial, highly coordinated state by removing calcium media, and restored in high-calcium media. The increase the electrical field and returning disrupted monolayers to high- of wound closure rate for BAPTA + electrically stimulated calcium media, allowing the calcium to reestablish junctions. E- tissues compared nonstimulated strongly collective monolayers cadherin fluorescence imaging shows that disrupted monolayers is clearly visible in the time-lapse panels (Fig. 6 and Movie returned to high-calcium media overnight regained their contact S10). Monolayers moved toward each other rapidly during the with neighbors and reestablished strong E-cadherin junctions (SI 12-h stimulation and successfully merged soon after they were Appendix, Fig. S10). Time-lapse imaging of the entire process— returned to high-calcium media to restore their initial state. BAPTA treatment of strongly collective monolayers, migration These data demonstrate both how controllability of tissues can in low-calcium media, and restoration in high-calcium media— be dynamically tuned and how such tuning can be used for prac- demonstrates how a difficult to control tissue can be transformed tical effects—in this case, increasing the baseline wound closure to a more susceptible tissue, maneuvered to a desired location rate by 2.5×. an arbitrary distance away, and then reassembled (Fig. 5G and Movie S9). In this case, while we do still note a thin zone of mem- Discussion brane damage at the initial leading edge (Movie S9, red band “If you cannot join it, then beat it.” Our work demonstrates at the rightward edge), this no longer causes retraction, and the that the more strongly collective a given tissue is—determined

tissue instead surges forward as a cohesive unit. here by cell–cell adhesion and native coordination levels—the

+ A No stimulation vs. with BAPTA and stimulation - No Stimulation With BAPTA + Stimuation + + t=0+ t=0

6h t=7h stim

12h 13h = t stim

Restored 27h = t

B Kymograph for convergent stimulation No Stimulation Time (27h)

With BAPTA + Stimulation ++- Time (27h)

Fig. 6. Accelerated in vitro wound healing. (A) Fluorescence images of unstimulated high-calcium monolayers (Left) and a high-calcium monolayer treated with BAPTA, convergently stimulated in low-calcium media for 12 h, and incubated in high-calcium media for 14 h (Right). (Scale bars: 500 µm.) (B) Kymographs of an unstimulated high-calcium monolayer (Upper) and a high-calcium monolayer treated with BAPTA, convergently stimulated in low- calcium media for 12 h, and incubated in high-calcium media for 14 h (Lower). The green dashed line indicates when the stimulation was switched on and media were changed to low-calcium media, and the yellow dashed line indicates when the stimulation was switched off and monolayers were returned to high-calcium media. (Scale bars: 500 µm.)

8 of 11 | PNAS Shim et al. https://doi.org/10.1073/pnas.2101352118 Overriding native cell coordination enhances external programming of collective cell migration Downloaded by guest on October 1, 2021 Downloaded by guest on October 1, 2021 vriigntv elcodnto nacsetra rgamn fcletv elmigration cell collective of programming external enhances coordination cell native Overriding al. et Shim lcrclydietemr otolbetsu oatre location target a to tissue bio- controllable 2) 1) more tissue, the tissues: a drive solu- collective in electrically coupling strongly a collective controlling internal suggested weaken of transiently externally problem the tissue to a tion messenger stimulate electrically second and and ion cell–cell facilitate of which effects signaling. junctions, the gap respect with explore especially to electrotaxis, further con- on broadly can electrotactic more interactions effective work window and con- Future “Goldilocks” strength affect trol. a adhesion detrimentally be cell–cell to can for appears adhesion little there too Hence, cell–cell either trollability. much electrotaxis, too collective a the play regulating or be behaviors, in to collective appears role E-cadherin baseline major while dif- and that to shows type story due cell emerging comparison the direct in in ferences complications (8, the knock- cells are despite epithelial E-cadherin results 61), immortalized in where the electrotaxis findings When diminished down tissue. prior the alongside inter- of electrotaxis, weaker considered now prerogatives of the collective favor outcompete to nal E- in cue reducing balance electrical the that the allowing concept tipped the adhesion support cadherin (), junctions linking E-cadherin data Our (Figs. 60). coordination 1 baseline (59, weaker to motion levels E-cadherin and collective mechani- reduced coordination of and long-range emergence forces enabling the physical cells, junc- across cell–cell of signaling as coupling migration cal intimate cell collective allow in tions adhe- role tissue E-cadherin, critical cell–cell by a whole regulated weaken plays often a adhesion, to Cell–cell across E-cadherin strength. sion motion targeting specifically directed by sustained restore and collapse the in cytoskeleton the observe. the of nec- we role seems at the tissues clarify behavior better migrating to collectively essary and driven, morphology of edge cytoskeletal leading on (56–58). fields work such in Future topical be electri- could how retraction suggest modulated may cally results as budding, our stratification, formation, such epidermal tumor and developmen- morphogenesis, dynamics, as in such present cell-substrate processes also tal in are changes transitions, elec- wetting–dewetting As collective inhibiting that (42). abolish indicating not electro- trotaxis data does suboptimal prior contractility for with myosin-mediated consistent blame is to and not that taxis suggests is problem the contractility inhibit- worsened collective that 3) Further, (Fig. reported. contractility previously cell ing been we collapse not the elec- has but 36), here prior less 23, see several (6, seem models in different tissue noted in studies been a trotaxis bulk—has of the edges than the responsive across tissue—where behavior 2 in electrotaxing differences (Figs. con- an supracellular edge also, of leading but level the Some tissue at 3). the and cells of in edge damage in leading electrical membrane resulted the siderable same of layers collapse skin the only collective clean, not strongly migration, for to tissue allowed applied cells stimulation layers over skin of control mouse tissue electrotaxis primary large-scale a programmed coupled of While weakly imperative drastic. of natural quite and be command can the between between flict conflict of chance the mitigate imperatives exter- to collective imperatives. appropriate system internal, target an the the applying modifying of both “controllability” and by the stimulus results collective nal optimize our cellular better particular, than a In can rather of it.” we that, “beat benefi- that is or be demonstrate override, this can com- weaken, it to behavior behaviors to behavior collective corollary cial native existing the A the an with other. program and synergizing command each externally the with to as pete be tissue may that of it difficult more B–D hsaiiyt neednl ueitra olciestrength collective internal tune independently to ability This collapse edge mitigate successfully to able were we However, con- potential the ignoring of consequences the Surprisingly, ,aogwt h eut fseicihbto of inhibition specific of results the with along 4C), and a oee fe h CEPO ltomaddrcl sebe onto onto assembled directly seeded and platform SCHEEPDOG were the after electro-bioreactor plastic keratinocytes The modeled media. culture in was calcium-supplemented mouse in tissue cultured available fibronectin-coated and Primary dishes on are stencils Methods. methods polydimethylsiloxane and and als materials Full Methods and Materials program to ability our improve to there from tissues. that that inspiration processes take likely collective can natural is we from it guidelines more systems, Given many collectives. collective are of across controllability similarities reduc- exam- and coordination key these and responsiveness properties collectivity of the the stronger each ing define with In group, behaviors allow the (72). collective of and underlying in of state the outside die jammed ples, the to this from group fluidize circulation the transiently their push of to and symmetry outside neighbors break the clusters penguin that on unless a exposure transition penguins exhibit jamming cause a huddles would creating penguin tendency, Finally, clustering (69–71). natural that group from showed the bees scout swarms guiding redi- prevents individuals bee to among more of attraction and difficult strong denser models too is more mathematical swarm be the and of aligned, to structure of natural shown the swarms when been Similarly, rect and have (68). cues nymphs exit conditions locust strength of escape inhibiting advantage the group, during take efficiently increase the doorways to within can ability motion group’s groups correlated the sys- human of multiple a distance in across and perturbations, Panic data external (67). experimental to tems with system consistent the finding reduce of can coupling responsiveness collective swarm the strong example, diverse overly For that with control. predicts line and theory in motion collective are of and examples systems collective across principles improved for rules” “design formal (25). of underly- performance lack the observing and and mechanisms characterizing results ing of promising difficulty despite the stalled to been due appli- has that clinical process for here a biointerfaces presented cations, next-generation paradigms control enable and help results can (12, our efficiency hope chemotactic We modulate 66). to shown col- been endogenous has as cue, especially lectivity be “controller,” programmable also of can role and the gradients in chemotactic potent explored as such a stimuli alternative pos- albeit metrics and one cue, establishing simply stimulatory is as electrotaxis well sible Similarly, as collectivity. systems, quantify model to other cell–cell coordination various endogenous beyond tune findings, in to factors controlled the be identifying can of of that for generality adhesion importance effects the the their to stress clarifying respect we as maintain With geo- well to research. density, as crucial future cell constant is as it parameters such proliferation, such factors and by confinement, affected metric cellular be in collectivity can As migration electrotaxis. collec- with of as controllability such migration, optimizing tive when to variable needed independent as an as modified treated be be should it how and nation (62–65). vivo in been wound healing has improving bioelectric there of developing capable as dressings toward exciting effort is control but recent behaviors this tremendous cell vs. optimize collective 2 to control begin (Fig. only also, collectives cannot we weak That as 5). began Fig. that directionality, and tissues speed with both per- compared of terms collective electrotactic in adjusting improved, dynamically that was of strength noted process this we quickly during as Unexpectedly, twice formance least control. collec- at healed the strongly it a that as such of layer process skin injured healing tive, collective us the allowed accelerate ultimately approach to This tissue location. and new coupling the cell–cell at integrity restore fully 3) and configuration, or oebody u nig ihih neligfundamental underlying highlight findings our broadly, More coordi- cell native of importance the demonstrate results Our https://doi.org/10.1073/pnas.2101352118 IAppendix, SI PNAS | Materi- f11 of 9

APPLIED BIOLOGICAL SCIENCES the tissue culture dish, delivering current from a Kiethly sourcemeter to High resolutions versions of the figures in this work are available the silver chloride electrode pairs. Field strength was maintained consis- on Zenodo (https://zenodo.org/record/4730646#.YObrZehKiHs) and GitHub tently at 2 V/cm using a custom MATLAB script. Cells were imaged using (https://github.com/CohenLabPrinceton/PNAS-ShimEtAl2021). an automated Zeiss inverted fluorescence microscope. Image analysis and quantification were performed with FIJI (ImageJ) and MATLAB. ACKNOWLEDGMENTS. Support for this work was provided in part by the Princeton Catalysis Initiative, NIH Award R35 GM133574-03, and National Science Foundation CAREER Award 2046977. We thank Lena Basta and Katie Data Availability. All study data are included in the article and/or Little at Princeton University for providing primary keratinocytes and cul- SI Appendix. Raw data/images and MATLAB scripts data have been ture support. We also thank members of the laboratory of D.J.C., especially deposited in Zenodo (https://zenodo.org/record/4730646#.YObrZehKiHs). Dr. Thomas Zajdel, for their assistance and advice.

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