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Nanomechanical Force Mapping of Restricted Cell-To-Cell Collisions Oscillating between Contraction and Relaxation Benhui Hu, Wan Ru Leow, Pingqiang Cai, Yong-Qiang Li, Yun-Long Wu, and Xiaodong Chen ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.7b06063 • Publication Date (Web): 13 Nov 2017 Downloaded from http://pubs.acs.org on November 14, 2017

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Nanomechanical Force Mapping of Restricted Cell-To-Cell Collisions Oscillating between Contraction and Relaxation Benhui Hu, Wan Ru Leow, Pingqiang Cai, Yongqiang Li, Yun-Long Wu and Xiaodong Chen *

Innovative Center for Flexible Devices (iFLEX), School of Materials Science and Engineering, Nanyang Technological University, 50 Nanyang Avenue, Singapore 639798, Singapore Correspondence should be addressed to X. C. ([email protected]). KEYWORDS: cell traction force, cellular bridge, nanomechanics, cell migration, biophysics, oscillation ABSTRACT Contact-mediated cell migration strongly determines the invasiveness of the corresponding cells, collective migration and morphogenesis. The quantitative study of cellular response upon contact relies on cell-to-cell collision, which rarely occurs in conventional cell culture. Herein, we developed a strategy to activate a robust cell-tocell collision within smooth muscle cell pairs.

Nanomechanical traction force

mapping reveals that the collision process is promoted by the oscillatory modulations between contraction and relaxation and orientated by the filopodial bridge composed of nanosized contractile machinery. This strategy can enhance the occurrence of cellto-cell collision, which renders it advantageous over traditional methods that utilize micropatterned coating to confine cell pairs. Furthermore, modulation of the balance between cell tugging force and traction force can determine the repolarization of cells and thus the direction of cell migration. Overall, our approach could help to reveal the mechanistic contribution in cell motility and provide insights in tissue engineering.

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Oscillatory modulations of multicellular dynamics are rich in living systems, in order to maintain the tissue integrity and functionalities of corresponding organisms.14

Through proper spatial and temporal organization of periodic cell-cell

communications, single-cell level oscillations can give rise to collective oscillations, which promote the long-distance coordination of cell movements within tissues and even drive morphogenesis for development.5 Understanding the key steps of these complex processes would aid in developing strategies for tissue engineering and regenerative biology.6-9 To date, one of the most salient progressive findings in biomedical fields is that the mechanisms driving development are not only biochemical or genetic, but also mechanical.10-16 The contribution of mechanical principles, which includes the activities of cytoskeletons such as the actin-myosin network and the migratory behaviors of cells,17-21 has become a promising area for investigation through various nanotechnologies.22-32 It must be noted that in vivo, live cells are mechanically coupled to both the extracellular matrix (ECM) and the adjacent cells.33-35

Therefore, the mechanical coupling requires maintenance of

connection between neighboring cells, and is essential for vital activities such as vasoconstriction and the beating of the heart.36 Intercellular adhesion, which constitutes the link between solitary and collective cellular behaviors, is cell-contact mediated.37-40 In particular, the cellular bridge, which is usually spanning two distant cells, would produce robust intercellular adhesion.

For example, smooth muscle cell (SMC) pair would each develop a

significantly extended protrusion composed of actomyosin stress fibers to form the cellular bridge and connection via a junctional complex. In most instances, the connection within a cell pair could maintain temporarily due to contact inhibition of locomotion (CIL), a process in which a cell would cease migration and repolarize

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upon contact with another cell.41,42 The diminution of CIL behavior would enable a cell to invade healthy tissues, resulting in enhanced metastatic property.43 Cell-to-cell collision is thus required for the quantitative analysis of the contact-mediated cell migration. However, cell-to-cell collision rarely occurs in conventional cell culture, which increases the difficulty of in vitro observation.44 Although micropatterned substrates have been used to confine a couple of cells and to enhance the chances of collision,45-47 these methods rely on geometrical restriction and may limit cell spreading and adhesion.48 Herein, we developed a strategy to promote restricted cell-to-cell collision by inducing oscillation between contraction and elongation. Briefly, a minimal model system was established via studying the oscillation within a SMC pair, in order to address the critical questions raised from oscillatory regulation, such as how contractile stress is generated, and how intercellular communication is impacted by the stress. This is a strategy that not only enhances the occurrence of cell-to-cell collision, but also manipulates cell movements based on the intercellular adhesion rather than cell-ECM adhesion as in other methodologies involving chemotaxis,49 haptotaxis,50 necrotaxis,51 electrotaxis,52 and durotaxis.47 The nanomechanical force mapping of the collision process with piconewton resolution reveals that the force transmission through the cellular bridge plays a key role in the repolarization of cells and directs the trajectory for each cell. Our approach may further indicate alternative cues for elucidating collective cell migration since the contraction-elongation type of motion has been observed in migrating cell sheets constrained within narrow strips.53

RESULTS AND DISCUSSION

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Cellular Bridge in SMC Pair Guides the Cell-to-cell Collision. The junction between two adjacent SMCs provided the cell tugging force to mediate the intercellular mechanical communication. CIL and cell-to-cell collision would occur via either extending or shortening of the cellular bridge. To form the SMC pair, a suitable density (~2k/cm2) of human aortic SMCs were grown on a collagen-coated polydimethylsiloxane (PDMS) substrate and allowed to fully attach. The extended connection between cell pair could be generated either through cytokinesis or from cellular exploratory machinery like the filopodia (Figure 1a).54,55 Using scanning electron microscopy (SEM) to characterize the overall topographic structure of the SMC pair, it was observed that the extended connection was spanning two distant cells, forming the cellular bridge (Figure 1a). Super-resolution microscope reveals the existence of both smooth muscle myosin-II and actin at the cellular bridge (Figure 1b). Similarly, abundant myosin II was also found in the cleavage furrow,56 which is a precursor that initiates the formation of the cell pair. When combined with the actin filaments, these actomyosin bundles demonstrated great capability for generating contractile forces along the junction (Figure 1b). The cell tugging force is established for intercellular communication via the linkage of cadherin-catenin complex,57 and counterbalanced by the cell-ECM tractions.58 Inducing calcium ionophore (A23187, 5 nM, an overdose would lead to severe cell blebbing and even detachment from ECM) would result in an influx of calcium, which would increase the intracellular calcium concentration and hence cell contraction.59

During cell contraction, a

synchronous retraction would be initiated at both cell rears, which would promote the subsequent cell-to-cell collision guided by the cellular bridge.

However, the

contraction would only last for a time window of roughly 10 mins; a spontaneous relaxation would subsequently occur, which would elongate both cells and stabilize

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their adhesion on the ECM. Repeating the contraction would induce the oscillation between contraction and relaxation, which would further promote cell-to-cell collision. To investigate the dynamics of the cellular bridge during collision, the transfection technique was applied to obtain red fluorescent protein (RFP)-tagged actin for both SMCs. Figure 1c showed that the straight cellular bridge was maintained to guide the collision. Figure 1d showed the outlines of the cell pair at different times, showcasing the stepwise completion of cell-to-cell collision based on a chronological sequence, in which the cell contraction acts as a propellant and relaxation acts as a stabilizer. It can be seen that after three cycles of oscillation, the two cells collided with enhanced contact.

Figure 1. Cell-to-cell collision within a SMC pair and the cytoskeleton distribution. (a) SEM image of a fixed SMC pair. Scale bar indicates 10 μm. (b) Immunostaining of smooth muscle myosin II (green) and actin (red) for individual SMC pair, the white square indicates the cellular bridge and the following three pictures indicates the magnified view (by super-resolution microscope). Scale bar indicates 50 μm in the first image and 10 μm in the following images. (c) 3D dynamics for the cell-to-cell

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collision with SMCs expressing RFP-Actin. Every cycle is induced by A23187. Scale bar indicates 50 μm. (d) Graph represents the cell-to-cell collision is completed by three cycles of oscillation between contraction and relaxation. The outlines of the SMC pair are in dark blue, light blue, green, yellow, red, pink and white based on the chronological order.

Mechanosensing

Platform for

Measuring

Cell-ECM

Traction

and

Intercellular Tugging Force. To elucidate the force regulation during this oriented cell migration, the cellular traction forces (CTF) were directly measured on our mechanosensing platform with a monolayer of fluorescence beads uniformly distributed beneath cell-ECM interface (Figure 2a). Multi-protein complexes (yellow) and integrin (green) facilitated the force transduction from actomyosin fibers (red) and led to the elastic deformation of PDMS (Figure 2b).

The deformation is

monitored by fluorescent beads and further turned into traction force mapping by tracking displacement of every bead (Figure 2c). When combined with confocal microscope imaging, the platform can achieve high-accuracy traction force mapping with piconewton resolution. For a cell pair grown on this platform, the net force encompasses cell traction force and intercellular tugging force and remains zero. Hence, the tugging force is equal in magnitude but opposite in direction to the net traction force.60

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Figure 2. Mechanosensing Platform based on Cell-Matrix Traction with accuracy of piconewton. (a) Schematic of the platform to measure cell traction forces. A thin layer of PDMS is placed on top of glass coverslip, with collagen coated on PDMS surface. Fluorescent nanobeads are embedded in the top of PDMS layer with a uniform distribution. For a cell pair grown on this platform, the net force encompasses cell traction force and intercellular tugging force. (b) Scheme of force transduction. The contractile force generated by acto-myosin fibers (red) transmits to ECM (white) through multi-protein complexes (yellow) and integrin (green). (c) Principle of mechanosensing. Cell traction forces are measured by detecting precise displacement of fluorescence beads induced by elastic deformation of PDMS.

Contact Inhibition of Locomotion Resulted in the Separating of the Cell Pair and the Breaking of the Filopodial Bridge.

It was observed that CIL

spontaneously occurred when the occasional contact was made between the two SMCs, which cause an extension and eventual dissociation of the filopodial bridge between them (Figure 3a and Figure S1).

Both cells were polarized along the

direction of the bridge during the CIL, as shown in the outlines in which the protruding region is represented by the red solid line, and the retracting region is

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represented by the black dash line (Figure 3a). It was worth noting that the length for each cell during CIL changed only by small fluctuations, in contrast to that of the cells involved in restricted cell-to-cell collision (Figure 3b). As shown in the lower line of Figure 3b, the cell in restricted collision shortened during contraction and elongated during relaxation. Furthermore, the cell front remained expanded with a wide protrusion during CIL, which indicated significant traction on the underlying surface. CTF mapping confirmed that the SMCs retained significant traction force on the substrate and promoted the separation movement (Figure 3c). To prove a better understanding on the role of the bridge in resisting CIL, we performed a simulation in which both cells are assumed to be elastic. The results showed that the separation movement exhibited a ‘stretching’ effect at the junction, such that the largest strain concentration is at the narrowest part, which is the middle of nanosized filopodial bridge (Figure 3d).

Figure 3. Contact inhibition of locomotion relies on the protruding of cells and dissociates the bridge. (a) Successive edge configurations during CIL with the

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protruding regions shown in red and retracting regions in black. Blue arrow represents the single cell length. (b) Migrating cells remain elongated during CIL, whereas the lengths of the cells during restricted collision decrease in contraction and increase in relaxation. (c) The overall CTF remains in little fluctuation during CIL. (d) The bright field image of a cell pair, which is about to break the bridge during CIL (left), and a model showing the largest strain is concentrated in the middle part of nanosized filopodial bridge during CIL (right). Scale bar indicates 10 μm.

Bidirectional Migration of Two Cells Is Mediated by Tug-of-War Between Cell-ECM Force and Cell Tugging Force. The balance between the cell tugging force and the cell-ECM traction was manifested as a tug-of-war, and determined if the cells would perform the approaching movement (cell-to-cell collision) or separation movement (CIL). Cramer et. al. concluded that directed cell migration required the breaking of cell symmetry triggered by the formation of either cell front or cell rear.18 Here, the CIL was mainly promoted by cell front protrusion.61 Conversely, the cellto-cell collision was formed through contraction, which reversed the sequence of symmetry breaking by first forming the cell rear (Figure 4a). The CTF for the overall cell pair exhibited an oscillating curve over the whole observation period for the cellto-cell collision (Figure 4b, repeatable curves shown in Figure S2). A closer look at the oscillating curve revealed that the period for every single cycle, which comprised contraction and elongation, was around 20 mins, and that the CTF would decreased significantly during contraction and return to the initial level during elongation. The changes in CTF could be ascribed to cytoskeletal remodeling.62 During contraction, the enhanced actomyosin-based contractility would shorten the cellular length and cause the actin stress fibers to gradually reduce their attachment to focal adhesion

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sites,63 thus resulting in the reduction in CTF. Conversely, in the following relaxation process, both cells would become elongated with increased CTF. The oscillation of the individual SMC also showed a similar trend with respect to CTF changes (Figure S3). CTF polarity (defined by the main axis orientation of the CTF vector sum) remained parallel to the extended cellular connection with only small fluctuations (±16°), which indicated that the cell-to-cell collision was highly directed towards their junction (Figure 4c and Figure S4). It is worth noting that the polarization of the cells along their bridge was essential for successful oscillation driven cell-to-cell collision. In contrast, when the cells were polarized perpendicular to the junctional direction (the vector from the center of mass of the corresponding cell to the center of the junction), the bridge could be easily broken during oscillation (Figure S5). Since cells in a pair would be mechanically coupled to both the ECM and each other, the coordination between cell-ECM adhesion (determined as CTF) and cell-cell adhesion (determined as cell tugging force) must mediate the migration orientation (Figure 4d, upper). To address this correlation, we further calculated the cell tugging forces during the whole process based on the equilibrium formula raised by Margaret L. Gardel.64 Since the cell tugging force was perpendicular to cell-cell contact and only counterbalanced the cell-ECM traction in the antiparallel direction (Figure 4d), the CTF vectors exerted on each individual cell could be characterized as to cell tugging force) and

(antiparallel

(perpendicular to cell tugging force). Accordingly, the

magnitude of cell tugging force would be a fraction of the cell-ECM traction and equal to

(Figure 4d, lower), while

would be equal to zero due to internal

balance. The consistent changing of CTF over the cell-to-cell collision would result in fluctuations for the ratio of

and

, and in turn for the ratio between cell tugging

force and CTF for each cell (Figure 4e). In addition, the migration velocity over this

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period were carefully recorded through manual tracking analysis by ImageJ. There appeared to be good correlation between the force ratio and migration velocity (Figure 4e), which suggested that the cell-to-cell collision could be promoted by cell tugging force but impeded by cell-ECM traction. Force analysis confirmed that CIL was dominantly driven by cell-ECM traction, rather than the cell tugging force. To sum up, we have established a mechanical strategy to regulate the bidirectional migration of cells by either maintaining cell-ECM tractions or reducing them through contraction.

Figure 4. Quantitative analysis of cell-to-cell collision within a SMC pair. (a) The cell-to-cell collision was completed by four cycles of oscillation between contraction (induced by A23187) and elongation. The five images show the force mapping for

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cells in elongated states; stress strength indicated by color bar; scale bar indicates 10 μm. (b) The overall CTF dynamics over the whole cell-to-cell collision process. CTF decreases during cell contraction and increases during its elongation. A23187 was induced at the elongated states indicated by red arrows. (c) CTF polarity (orientation of main axis) dynamics over the whole process. Radius length shows the magnitude of CTF; color bar indicates the time scale. (d) Schematic depicts the SMC pair configuration, the cell tugging force (red arrow) and cell-ECM forces (blue arrow) exerted by each cell. Calculated cell tugging force (red column) and measured cellECM forces (black column) over the whole observation period. (e) The correlation between the migration velocity and the force ratio (cell tugging force divided by CTF) for the lower cell shown in (a).

Microtubule Depolymerization Enhances Cell-ECM Traction.

As the

reversal from cell separating migration to approaching migration is initiated by reduction of cell-ECM force, we sought to determine whether enhancement of cellECM force prior to contraction could inhibit the reversal.

Traction forces are

produced from actin cytoskeleton in conjunction with myosin II,65 and could be regulated by the microtubule network.66 In addition, both contraction and elongation are consequences of actomyosin interaction, but the involvement of microtubules remained unclear, especially for our cell pairs. Hence, we focused on the use of microtubules to regulate cell-ECM traction and its mediation in cellular migration. To achieve this, we treated cells with nocodazole to induce microtubule depolymerization. Immunostaining of microtubule (green) and actin (red) showed the microtubule was depolymerized completely in the central connection part and severely at focal adhesion sites for SMC pair after approximately 5 mins treatment of

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nocodazole (100 nM), whereas the actin bundles remained intact. Meanwhile, the control cell pair exhibited clear microtubule and actin bundles both in the middle and focal adhesion sites (Figure 5a). The magnified view at the cellular bridge further confirmed the nocodazole induced microtubule depolymerization (Figure 5b). Quantitative statistical analysis of CTF showed that the cell pairs that have undergone nocodazole (100 nM) treatment for 5mins exhibited ~39% larger cell-ECM tractions (Figure 5c). The overall CTF for SMC pairs exhibited a reproducible tendency; initial stability at a plateau, then a gradual increase upon nocodazole (100 nM) treatment and finally a dramatic decrease after inducing contraction by A23187 (5 nM) (Figure 5d). The signal mechanisms involved in traction increase by microtubule depolymerization has been report by Wang.67

Figure 5. CTF increase induced by nocodazole treatment for SMC pair. (a) Top: Immunostaining of microtubule (green), actin (red) and nucleus (blue) for untreated SMC pair; below: Immunostaining of microtubule (green), actin (red) and nucleus (blue) for SMC pair treated by nocodazole (100 nM) for 5 mins. Scale bars indicate

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20 μm. (b) Sectional view and magnified view for cellular bridge, indicating the microtubule is disassembled by nocodazole treatment. Scale bars indicate 20 μm. (c) CTF of individual cell pair before and after nocodazole (100 nM) treatment for 5 mins. Data indicates the mean value and standard deviation for n=7 cell pairs. (d) CTF dynamics for SMC pairs treated with nocodazole (100 nM, red arrow), followed by A23187 (5 nM, blue arrow) treatment.

Role of Compression Forces from Microtubules in Maintaining the Cell Junction.

Strikingly, the cell junction was dissociated after microtubule

depolymerization, indicating a key role of microtubules in maintaining intercellular adhesion and facilitating cell-to-cell collision.

In fact, our study provided more

evidence to support the tensegrity model in mechanobiology.68 This model illustrates that the forces balance for a cell is based on tension and local compression, in which actomyosin apparatus generates tension and microtubule provides compression, respectively. This explains the weakening or even loss of compression force, as a result of nocodazole induction, from the microtubule enhanced anchorage between cell and ECM at focal adhesion sites. This would lead to the subsequent pulling of the two cells apart due to the enlarged tension upon cell-cell junction. Despite the regulation of forces, the cell-cell junction is tightly associated with the microtubule network, which transports and facilitates recruitment of the intercellular adhesion protein in SMCs.69,70 Hence, the disassembly of microtubule network could result in junction turnover. Based on this, we proposed a schematic diagram to illustrate the function of compression forces, which serve to prevent excessive traction exerted on ECM by cells and thus maintain cell junction survival to promote cell-to-cell collision. The elimination of compression forces within the cell

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pair would enlarge the risk of dissociating cell-cell junction, which would eventually lead to cells separation (Figure 6).

Figure 6. Schematic of proposed mechanism for regulating maintenance of adherent junction during cell contraction. Initially, the relatively low cell tractions exerted on the ECM by resistance from microtubule provided compression force; after nocodazole was induce to depolymerize microtubule, the cell-ECM traction was enhanced and the junction was eventually dissociated.

CONCLUSION Our experimental approach, which utilizes the oscillation between contraction and relaxation, complemented by force analysis, has enabled us to investigate the role of cell tugging force in generating alternative cues for directional cell migration. We demonstrated that such oscillation induced a restricted cell-to-cell collision in SMC pair, which enabled it to overcome CIL and thus promote the maintenance of tissue integration. The switch from CIL to restricted cell-to-cell collision was initiated by contraction, which reduced the cell-ECM tractions and thus caused both cells to

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retract, and further could be stabilized by elongation.

Hence, based on the

accumulating effect of repeated contractions and subsequent elongations, the restricted cell-to-cell collision occurred and finally brought two distant cells into collision, with a significantly enlarged contact.

We also observed that the cell

connection based on pseudopodia protrusion was straight during the whole progress, indicating that the tensile force along it promoted and guided the cell-to-cell collision. Depolymerization of the microtubules further revealed the importance of compression force in maintaining cell junction during contraction, as it counteracted the traction force and prevented the increased CTF from pulling the two cells apart. To sum up, our results developed a method to obtain directed cell migration based on cell communications. This mode of migration provided a practical way to guide cell movement targeted at other cells and arrange cellular organization.

EXPERIMENTAL METHODS Preparation of PDMS Substrate for Traction Force Microscopy. The elastic substrate for recording cell traction force is composed of chemically modified glass coverslips, PDMS and fluorescent beads (FluoSpheres microspheres, 0.1 m, carboxylate-modified, 0.1 vol %; Invitrogen, Carlsbad, CA). Briefly, coverslips were rinsed by acetone and isopropanol and then activated by oxygen plasma before being placed

in

a

vacuum

desiccator

containing

5

L

of

1H,1H,2H,2H-

perfluorooctyltrichlorosilane for half an hour. The coverslips were removed from the desiccator and 100 L of diluted yellow-green FluoSpheres (0.5 vol %) aqueous solution was applied to cast a single layer of fluorescent microspheres on the top of glass coverslips. A thin film of PDMS was formed by spreading 18 L of PDMS precursor (uncured, 1:60 w/w of crosslinker to monomer ratio) on the fluorescent beads coated glass coverslips. Subsequently, a single layer of Scotch tapes was used 16


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as a spacer before putting the other clean glass coverslip on the top of the spread PDMS film. The PDMS precursor was further flattened by the load of the upper coverslip. The entire assembly was then placed in an incubator at 37 C overnight before peeling off the upper bonded PDMS thin film with fluorescent beads embedded within its top.71 To promote cell adherence on the PDMS substrate, UV sterilization was applied for the substrate.

After sterilization, the substrate was

incubated with collagen solution (50 g/mL in 0.02 M acetic acid) overnight before further rinsing by phosphate buffered saline (PBS, pH 7.4; GIBCO) solution, and then stored in 4 C fridge before use. Cell Culture and Drug Stimulus. Human aortic smooth muscle cells (Lonza, Basel, Switzerland) with passages below 10 were cultured in SmGM-2 smooth muscle growth medium supplemented with 10% fetal bovine serum (Sigma, St. Louis) at 37 C in cell incubator with 5% CO2.

Cells were then seeded on

polydimethylsiloxane (PDMS; Sylgard 184, Dow Corning, Midland, MI) substrate coated with collagen (type I, Rat tail; GIBCO, Gaithersburg, MD). For chemical stimuli loading, calcium ionophore A23187 (Sigma) was solved in DMSO (Sigma) with concentration at 10-5 M.

To trigger cell contraction, 0.5 L of 10-5 M

A23187/DMSO solution was added into cell incubator containing 1mL cell culture medium. After repeated stimulus, the cells were detached from substrate by Trypsin (0.05%; GIBCO) to obtain the bead reference images.

To depolymerize the

microtubule, nocodazole (0.1 M) was used to induce the junction dissociation of the SMC pair. Sample Preparation for the SEM Imaging. Cells with a cellular bridge in between were imaged on a Zeiss SUPRA 55 field emission scanning electron microscope. Before imaging, the sample has been fixed, washed, dehydrated and

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dried. Briefly, the sample was fixed by 2.5% glutaraldehyde and washed by PBS. After washing, the sample was dehydrated by a series of ethanol in distilled water with gradually increased concentration (50%, 70%, 80%, 90%, 96% and 100%, 15mins duration for every concentration). The following chemical drying is implemented by hexamethyldisilazane (HMDS). The sample was transferred from 100% ethanol into a 1:2 solution of HMDS: 100% ethanol and leave for half an hour, then to a 2:1 solution of HMDS: 100% ethanol for another half an hour and finally to 100% HMDS for overnight. After the evaporation of HMDS, the sample is ready for sputter coating and SEM imaging. Confocal Microscopy and Time-Lapse Imaging. Cells fully attached on PDMS substrate were imaged on a Leica TCS SP8 laser scanning confocal microscope (Leica Microsystems, Mannheim, Germany) equipped with stage top incubator (37 C and continuous pumping in of 5 % CO2; Tokai Hit, INUB-WELS-F1 series). The images were captured utilizing a 40/1.40 NA oil-immersion objective lens with scanning resolution at 20482048 pixels. The fluorescent beads embedded in substrate were excited by an Argon laser with wavelength at 488 nm and the corresponding emission was collected in the range of 500 nm to 600 nm. Furthermore, the differential interference contrast (DIC) images were also captured for the location and morphology of cells. Image Analysis and Force Calculation. Calculation of cell traction force and polarity (ratio of force along principle axe to force along vice axe which is vertical to principle one) were based on the elasticity of PDMS substrate and displacements of fluorescent beads. Briefly, the traction force at discrete point , located at the position (xi,

yi)

was

calculated

based

on

, where

18

the

following

formulation:

denoted the Greens’ tensor and


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denoted the experimental displacements of fluorescent beads at position (xi, yi). The overall force of the cell (F) is an integral of the traction field magnitude over the area, , where

is the

continuous field of traction vectors defined at any spatial position (x,y) within the cell.72 The traction fields were calculated by Matlab (Mathworks, Natick, MA) and cell tugging force was deducted based on equilibrium formula by summing traction force vectors experienced by each cell in SMC pair. Cell migration velocity was calculated by ImageJ with manual tracking plugin. Staining and Transfection. Cells were grown on bare PDMS flat substrate. Before staining, the cells were fixed in 4% paraformaldehyde for 15 mins and permeabilized by 0.2% Triton X-100 in PBS for 10 mins. Cells were then incubated with Alexa Fluor 594 phallodin to stain actin filaments. For myosin visualization, cells were incubated monoclonal anti-myosin (smooth) antibody at 4 °C overnight followed by incubation with Alexa Fluor 488 goat anti-mouse for 1 h at room temperature. Zeiss ELYRA PS.1 Super-resolution Microscope with structured illumination (SIM) have been utilized to investigate the distribution of myosin-II and actin at the cellular bridge. To stain the microtubule, control cells and cells treated with Nocodazole (Sigma) for 5 mins before fixation were incubated with anti-αTubulin-Alexa 488 for 20 mins, and 4'-6-Diamidino-2-phenylindole (DAPI) was used to stain cells nuclei for 5 mins. Images were captured on Leica confocal microscope (SP8). For visualizing the actin and talin cytoskeleton, red/green fluorescent protein (RFP/GFP) transfection was applied by incubating cells with CellLight Actin-RFP or CellLight Talin-GFP (Thermo Fisher Scientific) for 16 h, respectively. Simulation. To estimate the strain concentration in the cell pair with a cellular bridge, a simulation (ABAQUS, Pawtucket, RI) was conducted through finite element

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modeling (FEM) analysis. As cells are assumed to be elastic incompressible material in our modeling, the Poisson's ratio is chosen as 0.499 based on the stability criterion in simulation. The elastic modulus of the cell was chosen as 1 kPa.

ASSOCIATED CONTENT The authors declare no competing financial interest.

Supporting Information The Supporting Information is available free of charge. Real-time bright field images of CIL, schematic of the platform for measuring CTF, Statistical study of CTF during cell-to-cell collision and single cell oscillation, and an orientation-dependent maintenance of cellular bridge (PDF).

AUTHOR INFORMATION Corresponding Author Email: [email protected]. Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

ACKNOWLEDGMENT This project was supported by the NTU-Northwestern Institute for Nanomedicine and the National Research Foundation, Prime Minister’s Office, Singapore, under the NRF Investigatorship (NRF-NRFI2017-07).

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Table of Content

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Locally Orchestrates a Cell Migration Pattern for Re-Epithelialization. Adv. Mater. 2017, 29, 1700145.

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