Regulation of cell motile behavior by crosstalk between cadherin- and integrin-mediated adhesions

Abstract

During normal development and in disease, cohesive tissues undergo rearrangements that require integration of signals from cell adhesions to neighboring cells and to the extracellular matrix (ECM). How a range of cell behaviors is coordinated by these different adhesion complexes is unknown. To analyze epithelial cell motile behavior in response to combinations of cell-ECM and cell-cell adhesion cues, we took a reductionist approach at the single-cell scale by using unique, functionalized micropatterned surfaces comprising alternating stripes of ECM (collagenIV) and adjustable amounts of E-cadherin-Fc (EcadFc). On these surfaces, individual cells spatially segregated integrin- and cadherin-based complexes between collagenIV and EcadFc surfaces, respectively. Cell migration required collagenIV and did not occur on surfaces functionalized with only EcadFc. However, E-cadherin adhesion dampened lamellipodia activity on both collagenIV and EcadFc surfaces and biased the direction of cell migration without affecting the migration rate, all in an EcadFc concentration-dependent manner. Traction force microscopy showed that spatial confinement of integrin-based adhesions to collagenIV stripes induced anisotropic cell traction on collagenIV and migration directional bias. Selective depletion of different pools of alphaE-catenin, an E-cadherin and actin binding protein, identified a membrane-associated pool required for E-cadherin-mediated adhesion and down-regulation of lamellipodia activity and a cytosolic pool that down-regulated the migration rate in an E-cadherin adhesion-independent manner. These results demonstrate that there is crosstalk between E-cadherin- and integrin-based adhesion complexes and that E-cadherin regulates lamellipodia activity and cell migration directionality, but not cell migration rate.

Fig. 1.

(A) EcadGFP-expressing MDCK cells on collagenIV:EcadFc, collagenIV:Fc, or collagenIV:PEG (the collagenIV stripe is represented in red, and the opposing stripe in black) seen in widefield epifluorescence. (B) Left axis: EcadGFP recruitment to the stripe opposing collagenIV as a function of the EcadFc/Fc ratio (or PEG). I_E, I_C are the fluorescence intensities of EcadGFP on EcadFc/Fc (or PEG stripes) and collagenIV stripes, respectively. Right axis: cell spread area on EcadFc/Fc (EcadFc %) or PEG, and collagenIV patterns. S_E, S_C are cell surface areas on EcadFc/Fc (or PEG) stripes and collagenIV stripes, respectively. A positive value denotes a larger area spread on noncollagenIV stripes than on collagenIV stripes. Data show mean ± SEM; line is a smooth fit to guide the eye. n = 12 (PEG), 32 (Fc), 14 (EcadFc 25%), 13 (EcadFc 33%), 21 (EcadFc 50%), and 44 (EcadFc 100%) for Ecad-GFP recruitment. n = 10 (PEG), 25 (Fc), 11 (EcadFc 25%), 9 (EcadFc 33%), 15 (EcadFc 50%), and 42 (EcadFc 100%) for contact area. *P < 0.05, **P < 0.01, ***P < 0.001, with respect to Fc data; Mann–Whitney test (two-tailed). (C) β-Catenin-GFP and α-E-catenin-GFP–expressing cells on collagenIV:EcadFc, seen in total internal reflection fluorescence (TIRF). (D) F-actin organization on collagenIV:EcadFc, seen in widefield epifluorescence and TIRF. (E) E-cadherin (n = 19), β-catenin (n = 14), α-E-catenin (n = 10), and UtrCH (n = 13, 13, 10; see D) mobility on collagenIV:EcadFc assessed by fluorescence recovery after photobleaching. Bar: 10 μm.

Fig. 2.

(A–D) Vinculin–GFP (A) or paxillin–GFP (C) recruitment on collagenIV versus EcadFc (A) or EcadBiot (E-cadherin extracellular fragment fused to biotin) (C). Note that the preferential localization of FAs on collagenIV did not depend on the FA marker used or the E-cadherin chimeric protein on the functionalized surface. Vinculin recruitment to EcadFc (in foci) versus collagenIV in FAs per cell (fluorescence intensities weighted by surface area covered by all foci and all FAs, respectively) (B: n = 3) and per focus and FA (fluorescence intensities unweighted) (D: EcadFc n = 6; collagenIV n = 7). The difference between B and D reflects the larger surface covered by all FAs on collagenIV than covered by all vinculin foci on EcadFc in a given cell. (E) Vinculin mobility on collagenIV with (circle, n = 7) and without (square, n = 8) juxtaposed EcadFc stripes, as assessed by fluorescence recovery after photobleaching. Vinculin mobility on EcadFc stripes (cross, n = 6). (F) VinGFP-expressing MDCK cells on collagenIV:EcadFc, collagenIV:Fc, or collagenIV:PEG. (Maximum intensity projection over time. Left, original fluorescence intensity signal; Right, FA localization by edge detection.) Bar: 10 μm. Data show mean ± SEM. *P < 0.05; ***P < 0.001, with respect to indicated controls; Student's t test.

Fig. 3.

(A) Measurement of lamellipodia activity (average area of cell surface difference between 10 s intervals; Movie S1 and Movie S2). (B) Lamellipodia activities L_C and L_E on collagenIV (red) and EcadFc/Fc or PEG stripes (black), respectively, as a function of non-collagenIV stripe functionalization. n = 10 (PEG), 26 (Fc), 11 (EcadFc 25%), 9 (EcadFc 33%), 15 (EcadFc 50%), and 42 (EcadFc 100%). (C) Measurement of cell migration (average cell velocity sampled at 10 min intervals; Movie S3, Movie S4, and Movie S5). (D) Migration rate on patterned surfaces as a function of non-collagenIV stripe functionalization and on plain EcadFc surfaces. (E) Migration direction on patterned surfaces as a function of non-collagenIV stripe functionalization. A positive migration direction index denotes that the migration rate component parallel to pattern direction V_// is higher than perpendicular component V_┴. n = 43 (PEG), 24 (Fc), 23 (EcadFc 25%), 15 (EcadFc 50%), 12 (EcadFc 100%), and 16 (plain EcadFc) for D and E. (F–H) Traction force microscopy. (F) Traction stress map: local traction stress magnitude and orientation (black vectors, a.u.) exerted by a single cell (delineated by the green line) on collagenIV-printed (inside dashed lines) polyacrylamide gel sheet. (G) Traction stress orientation distribution: count of local traction stress vectors as a function of their orientation on collagen stripes (left, n = 1,431) and on nonpatterned collagen (right, n = 1,518). (H) Quantification of traction stress direction bias. A positive value denotes a stronger traction in the direction of the stripes than in the perpendicular direction (collagen stripes: n = 1,431; non-patterned collagen: n = 1,518). Bar: 10 μm. Scatter plots show mean ± SEM; line is a smooth fit to guide the eye. *P < 0.05, *P < 0.01, ***P < 0.001, with respect to indicated control; Mann–Whitney test (two-tailed).

Fig. 4.

(A) Effects of vinculin and αE-catenin depletions on lamellipodia activity and (B) on EcadGFP recruitment and contact area. n = 20 (siRNA ctrl), 11 (siRNA Vin), 10 (shRNA ctrl), 9 (shRNA α-E-cat), and 11 (β-cat ActA) for A; n = 23 (siRNA ctrl), 13 (siRNA Vin), 11 (shRNA ctrl), 11 (shRNA a-cat), and 11 (β-cat ActA) for EcadGFP recruitment; n = 18 (siRNA ctrl), 10 (siRNA Vin), 10 (shRNA ctrl), 8 (shRNA α-E-cat), and 11 (β-cat ActA) for contact area. (C and D) Effects of vinculin and αE-catenin depletions on cell migration (C) rate and (D) direction. Note that control clone (expressing VinGFP) for αE-catenin depletion migrates faster than control clone (EcadGFP) for vinculin depletion. n = 16 (siRNA Vin), 22 (siRNA ctrl), 39 (shRNA α-E-cat), and 9 (β-cat ActA). *P < 0.05, **P < 0.01, ***P < 0.001, with respect to controls; Mann–Whitney test (two-tailed).

Fig. 5.

(A–C) Coordination and rate of cell migration in an epithelial sheet (Materials and Methods). (A) α is the angle between velocity vectors of two cells and r the distance between these cells. (B) Coordination index as a function of the distance between cells. <cos α> is the coordination index between all cells at a distance r from each other at a given time, averaged over time. Lines are fits of experimental data (dots) with an exponential decay function <cos α> = exp(−r/ξ), where ξ is the coordination length beyond which cells have lost about two-thirds of their coordination. (C) Average migration rate in the epithelial sheet. Data are displayed as a box-whisker plot (50% of the data within the box, 100% within the whiskers). ***P < 0.001, Mann–Whitney test (two-tailed). (D). Model of functional regulation of cell motile behavior by crosstalk between E-cadherin– and integrin-mediated adhesions (see text for details).