Strength dependence of cadherin-mediated adhesions

Abstract

Traction forces between adhesive cells play an important role in a number of collective cell processes. Intercellular contacts, in particular cadherin-based intercellular junctions, are the major means of transmitting force within tissues. We investigated the effect of cellular tension on the formation of cadherin-cadherin contacts by spreading cells on substrates with tunable stiffness coated with N-cadherin homophilic ligands. On the most rigid substrates, cells appear well-spread and present cadherin adhesions and cytoskeletal organization similar to those classically observed on cadherin-coated glass substrates. However, when cells are cultured on softer substrates, a change in morphology is observed: the cells are less spread, with a more disorganized actin network. A quantitative analysis of the cells adhering on the cadherin-coated surfaces shows that forces are correlated with the formation of cadherin adhesions. The stiffer the substrates, the larger are the average traction forces and the more developed are the cadherin adhesions. When cells are treated with blebbistatin to inhibit myosin II, the forces decrease and the cadherin adhesions disappear. Together, these findings are consistent with a mechanosensitive regulation of cadherin-mediated intercellular junctions through the cellular contractile machinery.

Figure 1

Immunofluorescent staining of C2 cells on substrates with different rigidities coated with Ncad-Fc. (A and B) PA gels with E = 95 and 10 kPa, respectively; Arrows in A indicate the location of some cadherin adhesions. Immunostaining for β-catenin (red), F-actin (green), and pillars (blue) was performed on cells spread on Ncad-Fc-coated pillars of two different rigidities (k = 120 for D–F, and 17 nN/μm for G–I) and analyzed by confocal microscopy. Scale bars: 10 μm. (C) Histograms of the projected spreading area of C2 cells on substrates with different stiffnesses; continuous PA gels of 10 and 95 kPa, and micropillars of 17 and 120 nN/μm.

Figure 2

Organization of cadherin adhesions on substrates with various stiffnesses and traction forces. (A and B) Colocalization and intensity fluctuations of β-catenin (red) and F-actin (green) were analyzed along the orange line indicated on the overlay image by line scan (ImageJ software) on stiff (120 nN/μm) and soft (17 nN/μm) substrates, respectively. The images in A and B correspond to the dashed rectangles represented in Fig. 1, F and I, respectively. Scale bar = 10 μm.

Figure 3

Control experiments of cell spreading on substrates coated with either Ncad or fibronectin. C2 cell spread on Ncad (A and C) and fibronectin (B and D) coated substrates were doubly stained for β1-integrin (red) and F-actin (green) or doubly stained for phospho-FAK (red) and F-actin (green). Scale bar = 10 μm. (E) Plot of average force generated per post over time for a cell cultured on Ncad-coated micropillar substrates (73 nN/μm) after the addition of soluble RGD peptides (1 mM) in the medium at T = 0.

Figure 4

(A) Average forces, <F>, exerted by C2 cells though cadherin adhesions on micropillars as a function of substrate rigidity, k (bottom axis), and E_eff (top axis). The • and ▪ symbols correspond to the standard conditions and the cell response after blebbistatin treatment, respectively; ∼10 cells were analyzed for each rigidity. The ▴ and Δ symbols correspond to the forces exerted by C2 cells through integrin-mediated adhesions on fibronectin-coated micropillars (k = 89 nN/μm) without and with blebbistatin treatment, respectively (n = 5). (B) Distribution of the areas of cadherin adhesions analyzed by measuring β-catenin structures on a micropillar substrate (120 nN/μm) coated with Ncad (n = 62).

Figure 5

Inhibition of NMM-II affects cadherin adhesion formation and actin cytoskeleton organization. (A–C) Blebbistatin-treated C2 cells on a micropillar substrate coated with Ncad-Fc did not exhibit organized β-catenin radial structures. Immunostaining for β-catenin (red), F-actin (green), and pillars (blue) was performed on cells spread on a Ncad-Fc-coated pillar (120 nN/μm) and analyzed by confocal microscopy (optical slice focused on the top of the pillars). Scale bar = 10 μm.

Figure 6

Model of cadherin contact formation and strengthening in response to mechanical changes in the cell-cell contacts. The micropillar substrate coated with Ncad represents a neighboring cell. The close-ups of cadherin contacts show the balance of external and internal forces (F_ext and F_cell, respectively). As the cell pulls on the substrate via cadherin adhesions, it induces an increase of its internal tension through the recruitment of adhesion proteins and a buildup of actomyosin contractility. On a stiff substrate (large K), the internal tension (K_int) is supported by the formation of large clusters of cadherin complexes aligned along actin cables. Myosin II may contribute to the contraction of actin bundles, thus promoting cadherin adhesion stabilization and reinforcement. In contrast, if the cellular environment provides less resistance to deformation when the cells pull on it (here, soft substrates with small k), small forces are observed involving a limited number of cadherin links and thus a smaller internal rigidity (k_int).