Spatial organization of the extracellular matrixregulates cell–cell junction positioningQingzong Tsenga, Eve Duchemin-Pelletierb, Alexandre Deshiereb, Martial Ballandc, Hervé Guilloud,e, Odile Filholb,and Manuel Thérya,1

aLaboratoire de Physiologie Cellulaire et Végétale, Unité Mixte de Recherche (UMR) 5168, Institut de Recherches en Technologies et Sciences pour le Vivant(iRTSV), Commissariat à l’Energie Atomiques et aux Energies Alternatives (CEA), Centre National de la Recherche Scientifique (CNRS), Institut National de laRecherche Agronomique, Université Joseph Fourier (UJF), 38054 Grenoble, France; bLaboratoire de Biologie du Cancer et de l’Infection, Unité 1036, iRTSV,Institut National de la Santé et de la Recherche Médicale, CEA, UJF, 38054 Grenoble, France; cLaboratoire Interdisciplinaire de Physique, UMR 5588, UJF, CNRS,38402 Saint Martin d’Heres, France; dUnité Mixte International 2820, Laboratory for Integrated Micro Mechatronic Systems, CNRS, Institute of IndustrialScience, University of Tokyo, Meguro-ku, Tokyo 153-8505, Japan; and eLaboratoire de Thermodynamique des Petits Systémes, Institut Néel, CNRS, UJF, BP166,38042 Grenoble, France

Edited by Alexander D. Bershadsky, Weizmann Institute of Science, Rehovot, Israel, and accepted by the Editorial Board November 22, 2011 (received forreview April 21, 2011)

The organization of cells into epithelium depends on cell in-teraction with both the extracellular matrix (ECM) and adjacentcells. The role of cell–cell adhesion in the regulation of epithelialtopology is well-described. ECM is better known to promote cellmigration and provide a structural scaffold for cell anchoring, butits contribution to multicellular morphogenesis is less well-under-stood. We developed a minimal model system to investigate howECM affects the spatial organization of intercellular junctions. Fi-bronectin micropatterns were used to constrain the location ofcell–ECM adhesion. We found that ECM affects the degree of sta-bility of intercellular junction positioning and the magnitude ofintra- and intercellular forces. Intercellular junctions were perma-nently displaced, and experienced large perpendicular tensionalforces as long as they were positioned close to ECM. They remainedstable solely in regions deprived of ECM, where they were submit-ted to lower tensional forces. The heterogeneity of the spatial or-ganization of ECM induced anisotropic distribution of mechanicalconstraints in cells, which seemed to adapt their position to mini-mize both intra- and intercellular forces. These results uncovera morphogenetic role for ECM in the mechanical regulation of cellsand intercellular junction positioning.

tissue architecture | cytoskeleton | actin | traction force | integrin cadherincrosstalk

Epithelial sheets lie on a layer of extracellular matrix (ECM),the so-called basement membrane. In such epithelia, cells

establish integrin-based adhesions on the basal part of the cell incontact with ECM, and cadherin-based intercellular adhesionson the apical part of contacting lateral domains, away fromcontact with ECM. The two adhesion systems display non-overlapping spatial distributions. Both cell–cell and cell–ECMadhesions are required to establish proper epithelium morphol-ogy (1). They both participate in mechano-transduction of ex-ternal physical cues into intracellular signaling (2). The biochemicalnature of adhesion molecules engaged in intercellular adhesion,the energy of the interaction, as well as the mechanical tensiondeveloped along intercellular junctions have been shown togovern epithelial cell shape and orient intercellular junctionsin various systems (3–6). However, whereas the contribution ofcell–cell adhesion to epithelial topology has been the focus of manystudies, much less attention has been paid to the role of ECM.ECM is a dynamic scaffold that is actively remodeled duringmorphogenesis, where it plays major roles in stimulating andguiding cell migration as well as orienting stem cell fate (7, 8).ECM is also known to impart morphoregulatory signals to epi-thelia, and thereby regulates tissue morphogenesis (8, 9). How-ever, the mechanism by which ECM guides cell positioning at thesingle-cell scale is still not known. ECM geometry has beenshown to regulate intracellular architecture (10) and provide

spatial information for cell polarization (1, 11, 12), but how itregulates cell positioning and thereby spatially organizes multi-cellular architectures remained to be investigated.

ResultsIntercellular Junctions Are Stabilized in Regions Deprived of ECM.The effect of the spatial distribution of ECM on the localizationof MCF10A intercellular junctions was investigated by control-ling the location of ECM with fibronectin micropatterns (13, 14)(SI Methods). To provide a maximum degree of freedom to theintercellular junction and to minimize the number of parametersparticipating in its positioning, we focused our analysis on celldoublets, formed by daughter cells after mitosis. The positioningof cells, as revealed by the spatial coordinates of their nucleus,was recorded by time-lapse microscopy during a complete cellcycle and automatically quantified (Fig. S1, Movie S1, and SIMethods). We measured the angular distribution of the nucleus–nucleus axis as well as the proportion of time during which cellswere moving. As expected from previous work (15), cells werealmost randomly positioned and turned steadily around eachother on [square]-shaped micropatterns (Fig. 1A and Movie S2).Under such conditions, ECM is present all along the contour ofthe cell doublet and provides a continuous peripheral track forcell movement. Because in epithelia, cadherins tend to flow fromthe ECM-rich basal pole to the ECM-free apical pole (16), wetested whether the absence of ECM could stabilize the in-tercellular junction and interfere with cell movement. [H]-shapedmicropatterns were designed to provide two large regions devoidof ECM. In striking contrast to their behavior on [square], a largeproportion of cell doublets on [H] did not move at all, resultingin highly stable configurations in which cells were positioned oneach side of the gap (Fig. 1B and Movie S3). We further testedwhether the nuclear axis was perpendicular to the intercellularjunction by staining E-cadherin on fixed cells. We confirmed thatthe intercellular junctions were positioned over the gap whereECM was absent (Fig. 1 D and E).On [H], the absence of ECM was probably not the only factor

contributing to cell-doublet stabilization. The presence of thelarge gap and the absence of a continuous peripheral track along

Author contributions: Q.T. and M.T. designed research; Q.T., E.D.-P., H.G., O.F., and M.T.performed research; A.D. and M.B. contributed new reagents/analytic tools; Q.T., O.F.,and M.T. analyzed data; and M.T. wrote the paper.

The authors declare some conflict of interest because M.T. is involved in the companyCYTOO, which commercializes micropatterns.

This article is a PNAS Direct Submission. A.D.B. is a guest editor invited by the EditorialBoard.1To whom correspondence should be addressed. E-mail: manuel.thery@cea.fr.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1106377109/-/DCSupplemental.

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the cell-doublet contour may also have physically separated cellsand prevented cell movement. [Hourglass]-shaped micropatternswere designed to provide both a continuous peripheral track forcell movement and two regions along the cell-doublet contour inwhich ECM was absent (Fig. 1C). Thereby we could test thespecific effect of ECM on intercellular junction positioning un-der conditions where migration was not impaired. Analysis ofboth nucleus position and junction position confirmed that celldoublets tend to stabilize their positions in configurations wheretheir intercellular junction lies above regions deprived of ECM(Fig. 1 C and F). Under these conditions, the central part of thejunction lies above ECM. However, cadherins tend to concen-trate at the extremity of the junction (17), where most of theintercellular force is applied (18), and we could show that it isindeed the absence of ECM at the extremity and not in thecentral part of the junction that stabilizes junction displacements(Fig. S2). Notably, these orientations of cell doublets did notmerely result from the orientation of the mother cell division butinstead implied an active repositioning mechanism of the in-tercellular junction in response to ECM geometry (Fig. S3).

If epithelial cells actively tend to form their intercellularjunction away from ECM, the geometry of ECM and the size ofECM-deprived regions should directly regulate the degree ofstability of the intercellular junction positioning. We tested thishypothesis by varying the size of the gap in [C]-shaped micro-patterns (Fig. 2). As the gap size increased from 7 to 20 μm, theproportion of cells with the intercellular junction positioned overthe gap increased progressively, whereas the proportion of cellmovement gradually decreased (Fig. 2).

Intercellular Junctions Are Destabilized by the Presence of ECM. Tofurther confirm whether ECM promotes intercellular junctiondisplacement, we tested a complementary approach in whichECM was added along a stable intercellular junction position. Tothat end, we first designed a micropattern geometry in which celldoublets could adopt two stable configurations. On [cross], theintercellular junction was positioned along the midlines, eitherhorizontally or vertically, within the two large regions devoid ofECM (Fig. 3A). Cell-doublet angular speed was high when theintercellular junction passed over ECM-rich diagonals. It sloweddown when the junction passed over ECM-free midlines (Fig.3C). When ECM was introduced along the horizontal midline,the cell–cell junction was no longer slowed down along this axis,and cell doublets could not adopt the corresponding orientationanymore (Fig. 3 B and C).From these experiments, we concluded that the proximity of

cell–cell adhesion sites with ECM promotes the displacement ofthe intercellular junction, whereas the distance between celladhesion sites and ECM stabilizes it.

Regulation of Tensional Forces Is Implicated in Intercellular JunctionGuidance by ECM.To further investigate the molecular mechanismgoverning intercellular junction positioning in response to ECMgeometry, we inactivated some of the signaling pathways spe-cifically associated with cell adhesion and modified the formation

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Fig. 1. The cell–cell junction is stabilized in regions deprived of ECM. (A–C) Time-lapse acquisition in phase contrast of an MCF10A cell doublet ona [square]- (A) or [H]-shaped micropattern (B). Time frame is 15 min. Au-tomated movie analysis of Hoechst-stained cells (Fig. S1) provided theangular distribution of the nucleus–nucleus axis orientation and quanti-fication of cell-doublet movements on [square]- (A), [H]- (B), and [hour-glass]-shaped micropatterns (C). (D–F) Examples of immunofluorescentstainings of E-cadherin on cell doublets (Upper) and averaged staining overseveral images (Lower) on [square] (A), [H] (B), and [hourglass] (C). Micro-pattern width is 35 μm.

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Fig. 2. The distance from ECM regulates the stability of intercellular junc-tion position. Analyses of cell-doublet positioning from time-lapse acquisi-tion (Fig. S1) provided angular distributions of nucleus–nucleus axisorientations as well as the distributions of the proportion of moving framesin [gapped-square] shapes with gap sizes of 7, 14, and 20 μm (from left toright). Micropattern width is 35 μm.

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or maturation of cell–cell and cell–ECM adhesion constituents.We tested the effects of these treatments on cell doublets platedon the [hourglass], because this geometry guides intercellularjunction positioning without impairing cell migration. We quan-tified the orientation of the intercellular junction and the precisepositions of its extremities on fixed cells stained for α-catenin(Fig. 4A). Under control conditions, most intercellular junctionextremities were positioned in the region deprived ofECM, on bothsides of the narrow part of the [hourglass] (Fig. 4B and Fig. S4).This phenotype was not significantly perturbed by the inhibition ofFAK, ERK, JNK, Rac, or Src, which are the main kinases con-veying biochemical signaling from cell adhesions (19) (Fig. 4B andFig. S4). However, intercellular junction position and orienta-tion were strongly perturbed by the inactivation of cell con-traction using either Rho kinase inhibition or myosin IIinhibition (Fig. 4 B and E and Fig. S4). Down-regulation of focaladhesion proteins, on which traction forces are applied, im-paired cell spreading and precluded the analysis of their specificcontribution to intercellular junction positioning. However, cellspreading on laminin, which is known to engage a distinct subsetof integrins than fibronectin (20), significantly perturbed junc-tion positioning (Fig. 4 C and E and Fig. S4) and thereby showedthat this positioning was directly regulated by the nature of thecell–ECM interactions. We then tried to perturb the assembly ofjunctional complexes that are able to transfer the forces appliedon ECM to the adjacent cells. Down-regulation of p120-cateninby siRNA treatment is known to affect intercellular junctionturnover and actin dynamics (21). It significantly perturbedjunction positioning (Fig. 4 D and E and Fig. S4). These results

suggested that the production of mechanical forces on in-tercellular junctions was responsible for junction positioningaway from ECM.

Intercellular Tension Is Reduced in Regions Deprived of ECM. Tomeasure intercellular tensional forces, we grafted ECM micro-

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Fig. 3. Contact with ECM destabilizes the cell–cell junction. (A and B)Analyses of cell-doublet positioning from time-lapse acquisition (Center)(Fig. S1) and averaged staining of E-cadherin over several cells (Right) on[cross]- (A) and [cross+bar]-shaped micropatterns (B). Micropattern width is35 μm. (C) Quantification of averaged angular rotation speed of cell dou-blets with respect to axis orientation. Arrows indicate cell acceleration whenthe junction passes over ECM along diagonals on [cross] (red arrows), andalong diagonals and horizontal bars on [cross+bar] (blue arrow).

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Fig. 4. Cell contractility regulates intercellular junction positioning. (A)Immunostaining of α-catenin on cell doublets plated on [hourglass] (Left)(α-catenin in green, DNA in blue) allowed the detection and measurement ofintercellular junction positions (Right). (B–D) The positions of junction ex-tremities (black dots) were measured on cells treated for 6 h with PF-573228(1 μM) to inactivate FAK or Y27632 (5 μM) to inactivate ROCK (B), cells platedon laminin-coated micropatterns (C), or cells pretransfected with siRNAagainst GFP or p120-catenin (D). Images are examples of treated cells(α-catenin in green, DNA in blue). More representative stainings are shownin Fig. S4. Micropattern width is 35 μm. (E) Curves indicate the proportion ofjunctions for each angular sector with the same set of data as in B and C.Differences between the two curves were compared using the Kolmogorov–Smirnov test; **, 0.5% and ***, 0.1% error probability in the rejection of thehypothesis that the two distributions are identical; ns indicates that theprobability would be higher than 5%.

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patterns on soft polyacrylamide gels (22) (SI Methods). Defor-mations of polyacrylamide gels were used to measure the forcesexerted by cell doublets on the substrate (23–25) (Fig. 5A) andindirectly derive the forces they exert on each other (26) (SIMethods). The analysis of force balance in cell doublets on[square] and [H] revealed that intercellular forces were signifi-cantly reduced when intercellular junction extremities lay aboveECM gaps (Fig. 5B). Because intercellular tension reduction on

[H] could result from a decrease in global traction force ratherthan a specific decrease of the intercellular force, we calculatedthe ratio between the intercellular force and the total tractionforce. Like the intercellular force, this ratio was lower when thejunction was stabilized over ECM gaps (Fig. 5B). To furtherexplain how large traction forces on sites flanking ECM gapscould be associated with reduced levels of intercellular forcesover such gaps, we analyzed force orientations depending on the

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Fig. 5. Junction positioning away from ECM is associatedwith relaxation of intercellular forces. (A) Fibronectinmicropatterns with [square] and [H] shapes on poly-acrylamide gels and traction maps, averaged over severalcells, on the corresponding geometries. The color codeindicates the local traction in Pascal. (B) Cell–cell forcemeasurement. The mechanical balance in each cell imposesthat the force exerted between cells counterbalances thesum of traction forces exerted on the substrate. Cell–cellforce was measured and compared between cells plated on[square] and [H]. Each dot corresponds to a measure ona single cell. The ratio between the intercellular force andthe total traction force (sum of all force magnitude over themicropattern) was calculated and plotted to confirm thespecific reduction of cell–cell forces on [H]. (C) Traction forcefields averaged over several cells plated on [square] (Left) and[H] (Right). Magnifications correspond to the white squareregions on global maps. Arrows indicate force orientation;color and length both represent local force magnitude in pN.(D) Decomposition of traction forces into “intra” forces ori-ented toward intracellular space and “inter” forces orientedtoward the intercellular junction. These forces were noted“adhesive” or “nonadhesive” whether they were orientedalong a cell edge in contact or not with ECM. The absence ofECM is associated with relaxed intercellular forces. (E) Forcemeasurements along cell edges on various micropatternshapes ([square], [X], and [H]; Fig. S6) were combineddepending on their orientation (intra or inter) and the pres-ence of ECM along their length (with or without ECM). Thesegraphs represent three separate experiments and100 cells percondition. The presence of ECM showed opposite effects onintra- and intercellular tension. All statistical comparisonswere Student’s t tests, **P < 0.01, ***P < 0.001, ns, P > 0.05(F) Physical modeling. The various orientations of the in-tercellular junction can be described by a simple energyfunction H taking into account the length L and the tension Jalong all edges of the cell doublets. The two curves corre-spond to numerical simulations of the energy function forvarious junction orientations when the tension depends(red) or not (blue) on the local presence of ECM. A favoredorientation (junction orientation at 0°), corresponding to ex-perimental observations, only occurs when tension dependson ECM.

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presence or absence of ECM at the extremities of the junction(Fig. 5 C and D). Traction forces at cell apices were decomposedinto a component perpendicular to the junction, considered ascontributing mainly to intercellular forces, and a componentparallel to the junction, considered as contributing mainly tointracellular forces (Fig. 5D). This component of the tractionperpendicular to the junction was a good approximation of theglobal cell–cell force (Fig. S5). On [square], intercellular forcesalong ECM-rich edges were equivalent to intracellular forces.However, on [H], intercellular forces along ECM-free edgeswere almost half the strength of intracellular forces (Fig. 5D).We further compared the intra- and intercellular forces on var-ious micropattern shapes (Fig. S6) and found that the presenceof ECM had an opposite effect on each: It reduced intracellularforces but increased intercellular forces (Fig. 5E).

Overall Tension Minimization Accounts for Cell Positioning. To testwhether the regulation of intra- and intercellular forces in re-sponse to the presence of ECM could account for the favoredcell positioning we observed, we performed numerical simu-lations to take into account all cortical forces and compare theenergetic costs of all cell positions. We used a well-establishedphysical model of the energy associated with the development ofcortical forces (4, 27) and applied it to the simplified case of cellshaving a fixed area (28) (Fig. 5F and SI Methods). Numericalsimulations showed that when tension was considered to be in-dependent of the presence of ECM, all cell orientations hadapproximately the same energetic cost. However, when we in-corporated into the model the relative differences of tension wemeasured in response to the presence of ECM, the tension alongcell edges became anisotropic, and the configurations associatedwith the lower total energy actually corresponded to those weobserved experimentally (Fig. 5F and Fig. S7). This suggestedthat cell positioning could actually result from minimization ofglobal tension in response to ECM geometry.

DiscussionHere we described the development of an experimental systemto study multicellular morphogenesis. Although this system partly

lacks some of the physiological characteristics found in situ in an-imal systems (3) and in vitro in 3D cyst formation (29, 30), it offersthe possibility of quantifying cell movement and accurately mea-suring the spatial distribution of forces in a large number of re-producible assays. In addition, the fine manipulation of ECMgeometry allows precise control of the degree of freedom for cellmovements. Thus, although spatially confined, multiple cells canreveal their natural self-assembly process.ECM has been shown to guide tissue morphogenesis by modu-

lating cell–cell interactions (31, 32). The biochemical, structural,and mechanical interactions between cell–ECM and cell–celladhesions have been well-characterized (2, 33, 34). However, howthese interactions translate into spatial organization of multicellu-lar arrangements remained to be clarified. Our results suggest thatintercellular junction positioning away from ECM did not resultfrom the action of a single ECM signal. Indeed, most of the mainsignaling pathways associated with cell–ECM adhesion could beimpaired individually without affecting junction position, at least inthe range of inhibitor concentration and the timewindowwe tested.Neither did it result from oriented cell divisions. Instead, it ap-peared to stem from ECM-regulated production of intra- and in-tercellular forces and overall minimization of global tension.We found that the contact of intercellular junctions with ECM

caused the displacement of the cell–cell junction and the pro-duction of large perpendicular tensional forces and constantmotion of the cells. In support of these findings, work fromothers has shown that cell adhesion to ECM alters cell–cell ad-hesion and promotes the development of large tensional forcesresponsible for junction disruption and subsequent cell migration(35, 36). In addition, we found that when cells place their in-tercellular junction on ECM-deprived regions, intra- and in-tercellular tension gets reduced and cell position is stabilized(Fig. 6). How cell interaction with ECM regulates inter- andintracellular forces in an opposed way remains to be elucidated.We suspect this involves complex interplays between the growthand dynamics of several types of actin-based structures formed inproximity to cell–ECM and cell–cell contacts.Anisotropic distribution of forces along the cell periphery has

been shown to regulate cell shape, junction remodeling, and cellspatial repositioning within tissues (3). Our results suggest thatECM could participate in similar morphogenetic processes. Inresponse to heterogeneous distribution of ECM, cell doubletsdeveloped anisotropic force fields and adopted stable positionsalong the axis of low tension. According to this mechanism, cellstend to stabilize the position of their intercellular junctions awayfrom ECM. This could account for many important processesduring epithelium morphogenesis (Fig. 6). For example, duringtubulogenesis, cells first form a single-file chain of cells. Thisstage, during which cell–cell junctions are close to ECM, is onlytransient. Cells reorganize and move their junctions away fromECM to form a multilayered cord with a central lumen (30, 37).During this transformation, intracellular forces that were de-veloped across the ECM gap get relocalized along ECM fibers,whereas intercellular forces undergo the opposite reorientation.According to the mechanism we describe, this transformationwould be favored by the relaxation of both intra- and inter-cellular tensions (Fig. 6).Although much attention has been paid to the role of cell–

cell interaction in the shaping of epithelia, our results suggestthat we should also consider the organizing role of ECM, which,far from being a mere supporting scaffold, plays an instructiverole in regulating mechanical forces and orienting multicellu-lar assembly.

MethodsCell Culture. The culture of mammary epithelial cells MCF10A was describedpreviously (38). Cells were seeded on patterned substrates at a density of 8 ×104/cm2. Cells not attaching to the adhesive region on the substrate were

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Fig. 6. Summary and illustration. On micropatterns, cells develop elevatedintra- and intercellular tension when the intercellular junction is in contactwith ECM (Left) and reduced tension when it is far from ECM (Right). In situ,as shown here in the case of tubulogenesis, this mechanism would promotethe relocalization of junctions away from ECM and the formation of multi-layered structures of opposed polarities. ECM is shown in red, intercellularjunction complexes are in green, and nuclei are in blue.

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washed away 1 h after seeding. After cell spreading on micropatterns,Hoechst 33342 was added at 5 ng/mL to label the nucleus during time-lapse acquisition.

Chemical Inhibitors. Chemical inhibitors were added 24 h after cell plating onmicropatterns at the following concentrations: PF573228, 1 μM (FAK in-hibition); PD98059, 2 μM (ERK1 inhibition); SP600125, 1.8 μM (JNK in-hibition); SU6656, 5 μM (Src inhibition); NCS23766, 5 μM (Rac inhibition);Y27632, 5 μM (ROCK inhibition); blebbistatin, 15 μM (myosin II inactivation);ML7, 5 μM (MLCK inhibition). Cells were fixed 6 h later.

Micropatterning. Glass coverslip micropatterning has been described else-where (14). Micropatterned polyacrylamide gels were made as previouslydescribed (22) (SI Methods).

Immunofluorescent Staining. Thirty hours after plating cells on a micro-patterned coverslip, cells were either extracted in cytoskeleton buffercontaining 0.5% Triton X-100 and fixed in 4% paraformaldehyde or fixed inmethanol at −20 °C. Fixed cells were incubated with a 1:200 dilution of anti-α-catenin (B52975; Calbiochem) or a 1:50 dilution of anti-E-cadherin(sc8426; Santa Cruz Biotechnology) for 1 h, and then incubated with cor-responding secondary antibodies and FITC-phalloidin (Invitrogen) at 1 μg/mLfor 30 min.

Traction Force Microscopy. Images of fluorescent beads with and without celldoublets were first aligned to correct experimental drift using the ImageJplugin “align slices in stack.” The displacement field was subsequentlycalculated by a custom-written particle image velocimetry (PIV) programimplemented as an ImageJ (http://rsb.info.nih.gov/ij) plugin. The PIV wasperformed through an iterative scheme. In each iteration, the displacement

was calculated by the normalized correlation coefficient algorithm, so thatan individual interrogation window was compared with a larger searchingwindow. The next iteration takes into account the displacement field mea-sured previously, so that a false correlation peak due to insufficient imagefeatures is avoided. The normalized cross-correlation also allowed us todefine an arbitrary threshold to filter out low correlation values due to in-sufficient beads presented in the window. The resulting final grid size forthe displacement field was 1.63 × 1.63 μm, with six beads per interrogationwindow on average. The erroneous displacement vectors due to insufficientbeads present in the window were filtered out by their low correlation valueand replaced by the median value from the neighboring vectors.

With the displacement field obtained from the PIV analysis, the tractionforce field was reconstructed by the Fourier transform traction cytometry(FTTC) method with regularized scheme (25) on the same grid (1.63 ×1.63 μm) without further interpolation or remapping. The regularizationparameter was set at 9 × 10−10 for all traction force reconstructions. TheFTTC code was also written in Java as an ImageJ plugin, so that the wholetraction force microscopy procedure from PIV to force calculation could beperformed with ImageJ. The entire package of traction force microscopysoftware is available at https://sites.google.com/site/qingzongtseng/tfm.

ACKNOWLEDGMENTS. We thank Laurent Blanchoin and Thomas Lecuit forinteresting discussions about this work, Benedikt Sabass for help on the JavaFTTC code, as well as Matthieu Piel, Alexandra Fuchs, and Susana Godinho forcritical reading of the manuscript. Kevin Berton, Philippe Huber, and JulienVerove kindly provided reagents. This work was supported by Agence Nationalpour la Recherche Grants ANR-PCV08-322457 (to H.G., O.F., and M.T.) andANR-08-JC-0103 (to M.T.), the Ligue Nationale contre le Cancer (O.F.), anda PhD fellowship from the Irtelis Program of the Commissariat à l’EnergieAtomique et aux Energies Alternatives (to Q.T.).

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