Cadherin-based intercellular adhesions organize epithelial cell–matrix traction forces
Edited by David A. Weitz, Harvard University, Cambridge, MA, and approved November 28, 2012 (received for review October 19, 2012)
Abstract
Cell–cell and cell–matrix adhesions play essential roles in the function of tissues. There is growing evidence for the importance of cross talk between these two adhesion types, yet little is known about the impact of these interactions on the mechanical coupling of cells to the extracellular matrix (ECM). Here, we combine experiment and theory to reveal how intercellular adhesions modulate forces transmitted to the ECM. In the absence of cadherin-based adhesions, primary mouse keratinocytes within a colony appear to act independently, with significant traction forces extending throughout the colony. In contrast, with strong cadherin-based adhesions, keratinocytes in a cohesive colony localize traction forces to the colony periphery. Through genetic or antibody-mediated loss of cadherin expression or function, we show that cadherin-based adhesions are essential for this mechanical cooperativity. A minimal physical model in which cell–cell adhesions modulate the physical cohesion between contractile cells is sufficient to recreate the spatial rearrangement of traction forces observed experimentally with varying strength of cadherin-based adhesions. This work defines the importance of cadherin-based cell–cell adhesions in coordinating mechanical activity of epithelial cells and has implications for the mechanical regulation of epithelial tissues during development, homeostasis, and disease.
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Mechanical interactions of individual cells have a crucial role in the spatial organization of tissues (1, 2) and in embryonic development (3–5). The mechanical cooperation of cells is evident in dynamic processes such as flow-induced alignment of vascular endothelial cells (6) and muscle contraction (7). However, mechanical interactions of cells within a tissue also affect the tissue’s static mechanical properties including elastic modulus (8), surface tension (9), and fracture toughness (10). Little is known about how these tissue-scale mechanical phenomena emerge from interactions at the molecular and cellular levels (11).
Tissue-scale mechanical phenomena are particularly important in developmental morphogenesis (12), homeostasis (13), and wound healing (14) in epithelial tissues. Cells exert mechanical force on each other at sites of intercellular adhesion, typically through cadherins (15, 16), as well as on the underlying extracellular matrix (ECM) through integrins (17–19). Cadherin-based adhesions can alter physical aspects of cells such as the surface tension of cellular aggregates (20) and the spreading (21) and migration (22) of single cells adherent to cadherin-patterned substrates. Integrity of intercellular adhesions may also contribute to metastatic potential (23). We and others have shown that epithelial cell clusters with strong cell–cell adhesions exhibit coordinated mechanical behavior over length scales much larger than a single cell (24–27). Several studies have implicated cross talk between cell–ECM and cell–cell adhesions (28, 29) that can be modulated by actomyosin contractility (30). Recent data suggest that integrin-mediated adhesions can modulate the composition (31, 32) and tension (25, 33, 34) of cell–cell junctions. Although cadherins have been shown to modify local traction forces (35) and monolayer contractility (36), the effects of intercellular adhesions on the spatial organization of cell–ECM forces remain unexplored.
In this paper, we address the impact of intercellular adhesions on cell–ECM traction forces in colonies of primary mouse keratinocytes. We measure tractions of colonies of keratinocytes before, during, and after formation of cadherin-mediated intercellular adhesions. As cadherin-dependent junctions form, there is dramatic rearrangement of cell–ECM traction forces from a disorganized, punctate distribution underneath the colony to an organized concentration of force at the colony periphery. Through perturbations of cadherin-based adhesions, we demonstrate an essential role for cadherin in organizing cell–matrix mechanics. Finally, the spatial reorganization of cell–matrix forces is reproduced by a minimal physical model of a cell colony as 2D objects connected by springs and adherent to a soft substrate. Although downstream signaling pathways regulate responses to cadherin-based–junction formation, our experimental data and physical model suggest that the simple physical cohesion created by intercellular adhesions is sufficient to organize traction forces. These results have implications for intercellular adhesions’ role in the mechanical relationship of tissues to their surroundings and the emergence of tissues’ bulk material properties.
Results
Traction Stresses Dynamically Reorganize in High-Calcium Medium.
To investigate the relationship between cadherin-based intercellular adhesions and cell–matrix traction stresses, we induced the formation of cadherin-based adhesions in primary mouse keratinocytes by elevating extracellular-calcium concentrations. In low-calcium medium, keratinocytes plated at low density proliferated into colonies of cells with weak cell–cell interactions. Exposing keratinocytes to high-calcium medium resulted in formation of cadherin-based cell–cell adhesions after 6–12 h (Fig. S1).
We quantified the effect of cell–cell adhesions on cell–matrix forces using traction force microscopy (TFM) (37). We plated keratinocytes onto a fibronectin-coated, elastic silicone gel coupled to glass. To quantify gel deformation due to cell–ECM traction force, we imaged fluorescent beads embedded in the silicone gel and measured the beads’ displacements relative to their positions after removing the cells with proteinase K. We calculated in-plane traction stresses, σiz, from bead displacements and the substrate’s elastic properties (38, 39) (SI Text).
Over 12 h in high-calcium medium, keratinocytes developed cell–cell junctions (40) and contracted (41) (Fig. 1 A–C and Movie S1). Before adhesion formation, in-plane traction stresses emanated from both the colony periphery and the interior junction of the three cells in a colony. Forces at the colony periphery pointed radially inward, while interior forces pointed in various directions (Fig. 1D). During the time course, traction stress in the middle of the colony gradually weakened (Fig. 1E), and by 12 h after calcium elevation, interior traction stress all but disappeared (Fig. 1F).
Fig. 1.
From substrate displacement and traction stresses, we calculated the strain energy density, w, the mechanical work per unit area performed by the colony to deform the substrate (42) (SI Text). Shortly after calcium elevation, high strain energy was localized both underneath and at the periphery of the colony (Fig. 1G). Twelve hours after calcium elevation, strain energy was limited to the colony edge (Fig. 1I and Movie S1).
To quantify these spatial changes, we calculated azimuthal-like averages of strain energy during the time course. We eroded the colony outline inward by distance, Δ, in discrete steps, δ, until the entire colony area was covered (Fig. 1J). We calculated the average strain energy, 
, in each of these concentric, annular-like regions and plotted it as a function of distance from the colony edge, Δ (Fig. 1K). During the first 3 h after calcium elevation, three peaks exist in the strain energy profiles, corresponding to localization of strong strain energy at the colony periphery (Δ = 0) and center. Between 5 and 9 h, the center strain energy peak diminishes and disappears, and high strain energy is only at the colony periphery. We measured some strain energy outside the colony (Δ < 0) due to the finite spatial resolution of our implementation of TFM.
, in each of these concentric, annular-like regions and plotted it as a function of distance from the colony edge, Δ (Fig. 1K). During the first 3 h after calcium elevation, three peaks exist in the strain energy profiles, corresponding to localization of strong strain energy at the colony periphery (Δ = 0) and center. Between 5 and 9 h, the center strain energy peak diminishes and disappears, and high strain energy is only at the colony periphery. We measured some strain energy outside the colony (Δ < 0) due to the finite spatial resolution of our implementation of TFM.Although strain energy localization changed after calcium elevation, the colony’s overall average strain energy density was relatively consistent during the time course (Fig. 1L). Hotspots of strong strain energy (Fig. 1G, yellow regions) were no longer present by the end of the experiment (Fig. 1I), but overall average strain energy density was compensated by a decrease in colony area.
Traction Stresses Systematically Reorganize in High-Calcium Medium.
To probe how intercellular adhesions alter traction forces across a large range of colony geometrical size and cell number, we analyzed the magnitude and localization of traction force in 32 keratinocyte colonies in low-calcium medium and 29 keratinocyte colonies after 24 h in high-calcium medium. A total of 117 low-calcium cells and 150 high-calcium cells comprised these colonies, each containing 2–27 cells, and spanned a geometrical dynamic range of nearly a factor of 100 in spread area.
In general, low-calcium colonies exhibited traction stresses throughout the colony, usually pointing radially inward from the colony edge and in various directions in the interior (Fig. 2A). Regions of high strain energy were found throughout the interior (Fig. 2B). In contrast, high-calcium colonies displayed traction stresses generically pointing radially inward from the colony edge (Fig. 2C) with hardly any strain energy beyond the colony edge (Fig. 2D). This observation is reminiscent of measurements on cohesive Madin–Darby canine kidney cells showing enhancement of traction force at the edges of cell pairs (25) and large cell sheets (24).
Fig. 2.
, giving each colony the same height on the graphs, indicated by the vertical scale bar. For clarity, profiles are spaced vertically according to colony size, with profiles for larger colonies (terminating at larger values of Δ) appearing higher up the y axis. Profile colors correspond to colony cell number given in the legend. (G) Quantification of relative distance from colony periphery (Δ/R) corresponding to 75% of total strain energy, 3W/4, in colonies in low- or high-calcium medium. Small colonies (R < 50 μm, below hash marks in E and F), in low- (n = 8) or high-calcium (n = 8) medium showed no significant difference, whereas large (R > 50 μm) low-calcium colonies (n = 24) had significantly more strain energy closer to colony center than large high-calcium colonies (n = 21). Statistical significance between low- and high-calcium populations is indicated by asterisks (P < 0.001). Error bars indicate 1 SD. (H) Relationship between total strain energy, W, and area, A, of colonies in low- and high-calcium medium. Open symbols correspond to low-calcium colonies, closed symbols to high-calcium colonies. Symbol colors indicate colony cell number, given in the legend. (I and J) Keratinocytes in low-calcium medium (I) or after 24 h in high-calcium medium (J) labeled with anti–E-cadherin and anti-paxillin antibodies and stained with phalloidin to mark F-actin. (Scale bars: A–D, I, and J, 50 μm.) Data for high-calcium colonies in F–H are adapted from ref. 27.To quantify these spatial distributions, we plotted average strain energy density as a function of distance, Δ, from the colony edge (as depicted in Fig. 1J). Average strain energy densities, 
, were normalized by the average strain energy density at the colony periphery, 
. These profiles (Fig. 2 E and F) terminate where inward erosion covered the entire area of the colony, at Δ ∼ R, where R is the effective radius of the colony, given by the radius of the disk with the same area as the colony.
, were normalized by the average strain energy density at the colony periphery, . These profiles (Fig. 2 E and F) terminate where inward erosion covered the entire area of the colony, at Δ ∼ R, where R is the effective radius of the colony, given by the radius of the disk with the same area as the colony.In most low-calcium colonies, we observed some localization of strain energy at the colony periphery (Δ = 0) and high amounts of strain energy throughout the colony (Δ > 0), sometimes at the colony center (Δ ∼ R) (Fig. 2E). In contrast, the strain energy of nearly all of the high-calcium colonies was strongly localized at the colony periphery, generally decaying to zero toward the colony center (Fig. 2F). Although this trend seems to hold regardless of number of cells in the colony, the difference is much less pronounced for the smallest colonies (R ≲ 50 μm). The radii of small colonies are comparable to the traction stress penetration length, ℓp, which measures how far from the periphery traction stresses penetrate the colony. Thus, in small colonies, the stress measurements do not readily distinguish the colony center and periphery. In our previous study on high-calcium keratinocytes, we measured ℓp ≈ 11 μm (27).
Next, we quantitatively compared the spatial distributions of strain energy across these two colony populations with and without cadherin-based intercellular adhesions. We calculated the total strain energy, W, exerted by each colony and the relative distance into the colony from its periphery, Δ/R, required to capture 75% of the total strain energy, 3W/4. We separated larger colonies (R ≫ ℓp, or R > 50 μm) of the low- and high-calcium populations. Large, low-calcium colonies required on average 10% more inward erosion (statistically significant, P = 0.0002) to achieve 75% of the total colony strain energy than large, high-calcium colonies, whereas there was no significant difference in strain energy distribution for the populations of small (R < 50 μm) colonies (Fig. 2G) (P = 0.43). These data suggest that formation of cadherin-based adhesions in high-calcium medium results in a shift in localization of traction stress from internal regions of the colony to the periphery.
The low- and high-calcium colonies did not seem to exhibit different amounts of average strain energy density. A plot of total strain energy versus colony area, A, although scattered, shows no apparent difference between these populations (Fig. 2H). In both cases, larger colonies tended to perform more work on the substrate.
Because low- and high-calcium keratinocyte colonies have different arrangements of cytoskeletal and adhesion proteins, we characterized spatial localizations of actin, E-cadherin–mediated cell–cell adhesions, and focal adhesions in keratinocyte colonies using phalloidin staining and immunohistochemistry (SI Text). E-cadherin is highly expressed in keratinocytes, mediates adhesive activity, and is essential for adherens-junction formation. In high-calcium colonies, E-cadherin was localized at keratinocyte junctions (Fig. 2I). Positions of actin stress fibers were correlated with areas of strong E-cadherin localization, and there was coordination of the orientation of actin fibers across multiple cells, consistent with earlier reports on cytoskeletal rearrangement after calcium elevation (13). Although traction stresses of low- and high-calcium colonies had different spatial distributions, focal adhesions, marked by paxillin, were concentrated at the colony periphery in both cases.
Cadherin-Based Adhesions Are Required for Organization of Traction Stresses in High-Calcium Medium.
Because elevation of extracellular calcium modulates cellular properties in addition to cadherin-based–adhesion induction (43, 44), we sought to isolate the role of cadherin in spatially organizing traction forces. We used two different methods to inhibit formation of cadherin-based adhesions in the presence of high-calcium medium. First, we used the function-blocking antibody DECMA-1, which prevents homophilic binding between extracellular domains of E-cadherin (45). DECMA-1 was added to keratinocyte colonies with high-calcium medium for 24 h. Immunostaining of these colonies showed strong reduction of E-cadherin at intercellular contact (Fig. 3A). Despite this change, we observed minimal coordination of actin across multiple cells in a colony, and focal adhesions were present at the colony periphery and throughout the colony interior. In keratinocytes in high-calcium medium with DECMA-1, we measured traction stress and strain energy throughout the colony, in particular at cell–cell contacts (Fig. 3 B and C). Strain energy profiles of 15 DECMA-1–treated colonies (all with R > 50 μm) show many cases of high strain energy transmitted in the colony interior (Fig. 3D).
Fig. 3.
We further investigated the role of classical cadherins using primary keratinocytes from an epidermal-E-cadherin–knockout (KO) mouse (46). We used shRNA to knock down (KD) the other classical cadherin expressed in these cells, P-cadherin, which is up-regulated in E-cadherin–null cells (47) (SI Text). We analyzed cell–cell and cell–matrix adhesions by immunostaining KO/KD cells cultured in high-calcium medium for 24 h. Colonies of KO/KD cells showed no E-cadherin expression, did not coordinate their actin cytoskeletons across multiple cells, and displayed a slight reduction of focal adhesions underneath the colony (Fig. 3E). As with DECMA-1–treated colonies, KO/KD colonies in high-calcium medium for 24 h showed traction stresses and strain energy underneath cell–cell contacts (Fig. 3 F and G). Strain energy profiles of 14 KO/KD colonies in high-calcium medium (all with R > 50 μm) show strong strain energy transmitted throughout the colony (Fig. 3H).
DECMA-1–treated colonies needed on average 6% more inward erosion than large high-calcium wild-type colonies to achieve 75% of the total colony strain energy (statistically significant, P = 0.048). KO/KD colonies required on average 10% more inward erosion than large high-calcium wild-type colonies to achieve 75% of the total colony strain energy (statistically significant, P = 0.002) (Fig. 3I). Compared with large low-calcium colonies using this same measure, neither DECMA-1–treated colonies (P = 0.14) nor KO/KD colonies (P = 0.94) showed significant differences in spatial distributions of strain energy. Thus, keratinocytes in high-calcium medium are unable to organize traction forces to the colony periphery in the absence of cadherin-based cell–cell junctions.
Minimal Physical Model Captures Cadherin-Dependent Organization of Traction Stresses.
Because of the simple spatial trends of traction stresses observed in colonies with and without intercellular adhesions, we examined whether a minimal physical model could reproduce the experimental results. We model each cell in a colony as a homogeneous and isotropic elastic material (48, 49). In our model, each cell exerts a contractile “pressure” opposed by strong adhesion to a compliant substrate (50). At each point within a cell, we require that these opposing forces balance. This model ignores all active processes modulated by cell–cell adhesions, including downstream signaling, and represents each intercellular adhesion as a purely physical connection characterized by a spring constant, k (51).
To make predictions with this model, we use a numerical solution of the 2D governing equations (SI Text). To mimic the cell geometry in the time course experiment (Fig. 1), we consider the case of three hexagonal cells (Fig. 4A). We find that, for increasing cell–cell–coupling strength, k, traction stress and strain energy disappear under cell–cell junctions (Fig. 4 B–D), recapitulating the transition seen in real cells stimulated by calcium elevation (Fig. 1 D–F). The similarity between model and experiment is also evident in plots of strain energy density as a function of distance from the colony edge (Figs. 4 E–G and 1K).
Fig. 4.
The model demonstrates the importance of intercellular-adhesion strength in spatially organizing cell–ECM forces. For weak cell–cell coupling (small k), individual cells deform the substrate independently of each other, with significant substrate deformation at all edges of each cell. However, strongly coupled colonies (large k) behave as a cohesive, contractile unit, with substrate deformation only at the colony periphery.
Discussion
Our results show that cadherin-based cell–cell adhesions modulate force transmission to the ECM. In particular, our traction force data on cohesive cell colonies suggest that intercellular-adhesion formation through classical cadherins reorganize the spatial distributions of traction stress. In colonies of cells with strong E-cadherin–based adhesions, cell–ECM traction stresses are localized in a ring around the colony periphery. In weakly cohesive colonies, regions of high traction stress appear throughout the colony. Furthermore, traction stresses cannot reorganize in high-calcium medium when cadherin-based adhesion is inhibited. Comparison of our experimental data with our minimal physical model suggests that strong physical cohesion between cells is sufficient to drive the relocalization of cell–ECM forces to the periphery of cell colonies. Although our data show that E-cadherin is necessary to reorganize traction forces, E-cadherin alone may not be sufficient. Further study is required to determine whether additional adhesive processes downstream of adherens junctions, such as the formation of desmosomes by nonclassical cadherins (47), are necessary to achieve sufficient cohesion.
Our findings resonate with recent studies on cellular adhesion pointing toward cross talk of cadherin- and integrin-based adhesions. Focal adhesions have been observed to disappear underneath cell–cell contacts (31, 52), but this effect may depend on substrate stiffness (29) and the extent of cell spreading (53). Recent work has also suggested that forces transmitted through focal adhesions can modulate intercellular forces (25, 29), which in turn can modulate intercellular-junction assembly and disassembly (15). Our study highlights intercellular adhesions’ ability to impact cell–ECM force generation, which allows for bidirectional feedback between cell–cell and cell–matrix forces. Indeed, tension at cadherin junctions (13, 54) is known to elicit cell-signaling events and actin dynamics (52, 55–58) and contribute to collective cell migration (26, 59, 60). In light of these prior results on integrin–cadherin feedback, it is somewhat surprising that a minimal physical model can capture the observed dependence of cell–matrix forces on the strength of cadherin-mediated cell–cell adhesions.
Reorganization of cell–ECM forces is likely one important mechanism by which cadherin-based adhesions drive tissue morphogenesis and homeostasis. In development, differential adhesion has been shown to play an important role in cell sorting (61–63), and the reorganization of intercellular forces in this context is entirely unexplored. Furthermore, in wound healing, we expect strong cell–ECM forces to be generated at a wound edge due to the local loss of intercellular adhesion. These forces could act as a signal, inducing migratory behavior in epithelial cells (24, 64), activating responses of stromal cells, and organizing the ECM (65–67). A key avenue for future investigations will be to explore how organization of force stimulates cellular responses within tissues.
Materials and Methods
Primary wild-type and E-cadherin–KO mouse keratinocytes were isolated as described (68, 69) and plated on fibronectin-coated silicone gel with Young’s modulus of 3 kPa. Fluorescent beads within the gel were imaged using confocal microscopy, and traction stresses and strain energies were calculated from measured bead displacements and the gel’s elastic properties. Images of immunohistochemical staining were acquired using confocal microscopy. Further details of substrate preparation, confocal microscopy, live-cell imaging, TFM calculations, primary keratinocyte culture, immunohistochemistry, and statistical analyses are included in SI Text.
Acknowledgments
We are grateful to Margaret L. Gardel (University of Chicago) and Alpha S. Yap (University of Queensland) for helpful discussions. We thank Barbara Boggetti (University of Cologne) for preparation of KO/KD cells. This work was supported by a National Science Foundation Graduate Research Fellowship (to A.F.M.), German Cancer Aid and Sonderforschungsbereich Grants 829 A1 and Z2 (to C.M.N.), and National Science Foundation Grants DMR-0806511 and DMR-1004789 (to M.C.M.) and DBI-0619674 (to E.R.D.). V.H. is a Pew Scholar in Biomedical Research and is funded by National Institutes of Health Grant AR060295 and Connecticut Department of Public Health Grant 12-SCB-YALE-01. We also acknowledge support from Yale University's Raymond and Beverly Sackler Institute for Biological, Physical, and Engineering Sciences.
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Published online: December 31, 2012
Published in issue: January 15, 2013
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Acknowledgments
We are grateful to Margaret L. Gardel (University of Chicago) and Alpha S. Yap (University of Queensland) for helpful discussions. We thank Barbara Boggetti (University of Cologne) for preparation of KO/KD cells. This work was supported by a National Science Foundation Graduate Research Fellowship (to A.F.M.), German Cancer Aid and Sonderforschungsbereich Grants 829 A1 and Z2 (to C.M.N.), and National Science Foundation Grants DMR-0806511 and DMR-1004789 (to M.C.M.) and DBI-0619674 (to E.R.D.). V.H. is a Pew Scholar in Biomedical Research and is funded by National Institutes of Health Grant AR060295 and Connecticut Department of Public Health Grant 12-SCB-YALE-01. We also acknowledge support from Yale University's Raymond and Beverly Sackler Institute for Biological, Physical, and Engineering Sciences.
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This article is a PNAS Direct Submission.
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The authors declare no conflict of interest.
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Cadherin-based intercellular adhesions organize epithelial cell–matrix traction forces, Proc. Natl. Acad. Sci. U.S.A.
110 (3) 842-847,
https://doi.org/10.1073/pnas.1217279110
(2013).
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