Cell-ECM traction force modulates endogenous tension at cell-cell contacts

Abstract

Cells in tissues are mechanically coupled both to the ECM and neighboring cells, but the coordination and interdependency of forces sustained at cell-ECM and cell-cell adhesions are unknown. In this paper, we demonstrate that the endogenous force sustained at the cell-cell contact between a pair of epithelial cells is approximately 100 nN, directed perpendicular to the cell-cell interface and concentrated at the contact edges. This force is stably maintained over time despite significant fluctuations in cell-cell contact length and cell morphology. A direct relationship between the total cellular traction force on the ECM and the endogenous cell-cell force exists, indicating that the cell-cell tension is a constant fraction of the cell-ECM traction. Thus, modulation of ECM properties that impact cell-ECM traction alters cell-cell tension. Finally, we show in a minimal model of a tissue that all cells experience similar forces from the surrounding microenvironment, despite differences in the extent of cell-ECM and cell-cell adhesion. This interdependence of cell-cell and cell-ECM forces has significant implications for the maintenance of the mechanical integrity of tissues, mechanotransduction, and tumor mechanobiology.

Fig. 1.

Traction force imbalance yields the endogenous cell–cell force. (A) MDCK cell expressing GFP-E-cadherin with traction stress vectors (red arrows) superimposed. Green line indicates region used for calculation of total traction force. Stress magnitude and distance scale are indicated by the red arrow and white line, respectively. (B) Heat-scale plot of traction stress magnitudes of the cell shown in A. (C, Top) Schematic of cell on traction gel with traction stress vectors (red arrow). (Bottom) Histogram of the unbalanced traction force across an isolated cell, measured as , and expressed as a percentage (number of cells = 16). (D) A pair of contacting MDCK cells expressing GFP-E-cadherin with traction stress vectors overlaid (red arrows). Outline of the regions used to calculate the force balance within the cell pair or a single cell are indicated by the green and yellow lines, respectively. (E) Heat-scale plot of traction stress magnitudes of the cell pair shown in D. (F, Top) Schematic of side view of the cell pair on traction gel with traction stresses (red arrows) and cell–cell forces (black arrows) depicted. (Bottom) Histogram of the unbalanced traction force, as measured in C for both a cell pair (green) and a single cell within the cell pair (yellow) (number of cell pairs = 24). (G) Net force exerted by cell 2 on cell 1, F_cell1, as a function of the force exerted by cell 1 on cell 2, F_cell2. Dashed line indicates a slope of one. (Inset) Schematic of a cell pair depicting F_cell1 and F_cell2. (H) Histogram of the endogenous cell–cell force; mean ± SD is 100 ± 40 nN. (I) Histogram of the angle between the cell–cell force and the line joining the edges of the cell–cell contact; mean ± SD is 88 ± 18⁰. (Inset) Schematic depicting the angle calculated. Scale bar in A, B, D, and E is 5 μm. Reference traction vector in A and D is 950 Pa.

Fig. 2.

Cell–cell force is stable over time and is concentrated at the contact edges. (A) Time lapse images of randomly migrating MDCK cell pair expressing GFP-E-cadherin at times indicated. (B) Heat-scale map of traction stress magnitudes of the cell pair at times identical to those in A. Cell–cell contact line indicated by white line. (C) Cell–cell force and the cell–cell contact length of a cell pair during time lapse imaging of random migration. Time points corresponding to images in A and B are denoted by an asterisk (*). (D) Localization of GFP-E-cadherin and mApple-actin in an MDCK cell pair. White outline indicates region used for analysis in E. (E) Images of actin (Top) and E-cadherin (Bottom) at times after calcium depletion. E-cadherin images overlaid with traction stress vectors (red arrows) at times after calcium depletion. Arrows indicate location of fiducial marks in F-actin coinciding with the edges of the GFP-E-cadherin plaque at the cell–cell contact. Reference traction vector in E is 250 Pa. (F) Cell–cell force, total traction force, and cell–cell contact length as a function of time after calcium depletion; data calculated from cell shown in E. (G) Percentage drop in cell–cell contact length, total traction force, and cell–cell force within 10 min after calcium depletion for n = 3 cell pairs. Scale bar in A, B, and D is 10 μm and in E is 3 μm.

Fig. 3.

Cell–cell force is directly proportional to the total cell-ECM traction force. (A) Schematic of a cell pair depicting force balance between cell-ECM and cell–cell forces. Cell-ECM traction forces acting at cell periphery (purple arrow, ) can be deconstructed into a component that is parallel (blue arrow,) and perpendicular (red arrow, ) to the cell–cell force. Cell–cell forces (, black arrows) act primarily perpendicular to the cell–cell contact. (B) Cell–cell force as a function of the total traction force per cell directed perpendicular to the cell–cell force. Data are for n = 24 MDCK cell pairs. (C) Cell–cell force as a function of the total traction force exerted per cell. (D) Histogram of the ratio of the cell–cell force to the total traction force exerted per cell, mean ± SD = 0.47 ± 0.07.

Fig. 4.

ECM properties modulate endogenous cell–cell forces. (A) Box plot of the total cell-ECM traction force for cells plated on substrates of different stiffnesses, with a Young’s modulus of 8.4 kPa or 20.7 kPa, or coated with different ligands, collagen I (CnI) or fibronectin (FN). Ligand biochemistry and gel stiffness indicated. Data reflects statistics as follows: FN, 8.4 kPa (n = 18 cell pairs); Cn I, 8.4 kPa (n = 24 cell pairs); Cn I, 20.7 kPa (n = 15 cell pairs). Statistical properties of data shown in box plot are as follows: mean (open square), box (25/75% quantile), whisker (5/95% quantile), and asterisks (maximum/minimum). (B) Box plot of cell–cell force for all three substrate conditions as in A. (C) Cell–cell force as a function of cell-ECM force for all three substrate conditions indicated: FN, 8.4 kPa (open green triangles); CnI, 8.4 kPa (closed red squares); CnI, 20.7 kPa (closed blue circles). Dashed line indicates a slope of 0.5.

Fig. 5.

Total forces exerted on the microenvironment by individual cells in a linear three-cell island are similar. (A) Traction stresses exerted by a linear three-cell island of MDCK cells expressing GFP-E-cadherin. Traction stress vectors are overlaid (red arrows). Reference traction vector is 1,000 Pa; scale bar indicates 10 μm. (B) Heat-scale map of the traction magnitude for the cell island shown in A. Dashed white line indicates cell–cell contacts. The three cells are indicated by text. (C) Relative total cell-ECM traction force (red) and cell–cell force (black) exerted by the three cells shown in A. Values are relative to the average between the total forces exerted by the outer cells. Schematic on the right depicts the three-cell configuration and the cell-ECM (red arrows) and cell–cell (black arrows) forces exerted by each cell. Data reflect the mean and standard deviation for n = 4 cell islands with identical geometry.