Binding of ZO-1 to α5β1 integrins regulates the mechanical properties of α5β1-fibronectin links

Abstract

Fundamental processes in cell adhesion, motility, and rigidity adaptation are regulated by integrin-mediated adhesion to the extracellular matrix (ECM). The link between the ECM component fibronectin (fn) and integrin α5β1 forms a complex with ZO-1 in cells at the edge of migrating monolayers, regulating cell migration. However, how this complex affects the α5β1-fn link is unknown. Here we show that the α5β1/ZO-1 complex decreases the resistance to force of α5β1-fn adhesions located at the edge of migrating cell monolayers while also increasing α5β1 recruitment. Consistently with a molecular clutch model of adhesion, this effect of ZO-1 leads to a decrease in the density and intensity of adhesions in cells at the edge of migrating monolayers. Taken together, our results unveil a new mode of integrin regulation through modification of the mechanical properties of integrin-ECM links, which may be harnessed by cells to control adhesion and migration.

FIGURE 1:

ZO-1 forms a complex with α5β1 at the edge of monolayers that affects cell motility. (A) ZO-1 and α5β1 staining of cells at the edge and center of monolayers of cells transfected with nontargeting siRNA (siCT), ZO-1 siRNA (siZO-1), and the ZO-1 plasmid (S168A) seeded on 12-kPa gels coated with fn. FLAG antibody was used to stain for ZO-1 S168A. Insets show the area marked with a red square. Scale bar, 50 µm. (B) Normalized profile plots of the ZO-1 and α5β1 intensity profiles shown in the red lines in A. Scale bar, 5 µm. (C) Pearson’s r of ZO-1 and α5β1 stainings at the edge and center of monolayers. (D) ZO-1 Western blot of cells transfected with nontargeting siCT, ZO-1 siZO-1, and S168A. (E) Quantification of the Western blot of cells transfected with siCT, siZO-1, and S168A. (F) Directional persistence of migrating single cells (effective distance/total distance) on 12-kPa gels coated with fn. Significant differences were found between siCT and other conditions (p = 0.0123 and 0.0001, respectively). (G) Effective distance of migrating single cells on 12-kPa gels coated with fn. Significant differences were found between S168A and other conditions (p < 0.0001). (H) Migrating single-cell tracks (480 min) for each condition (n = 30). Error bars represent the SEM of n number of data points. Images are representative from three experiments.

FIGURE 2:

Disruption of the α5β1/ZO-1 complex increases α5β1–fn link resistance to forces only at the edge of monolayers. (A) Cartoon depicting the experimental setup with magnetic tip pulling on beads attached to cells both at the edge and center of cell monolayers. (B) Magnetic tip applying a magnetic field producing 0.5 nN of force to beads both at the edge (left) and at the center (right) of monolayers. (C) Time taken to detach beads for each condition either at the center or at the edge of monolayers. Beads were coated with FN7-10 (with or without incubating with Aα5β1) or bBSA. No significant differences were found between centers (p = 0.7887) among conditions. Scale bar, 50 µm. Images are representative from four experiments.

FIGURE 3:

Disruption of the α5β1/ZO-1 complex decreases α5β1–fn recruitment at the edge of monolayers. (A) β1 and ZO-1 staining of silica beads coated with FN7-10 attached to monolayers. (B) β1 recruitment to silica beads coated with FN7-10 attached at the edge and at the center of monolayers. (C) ZO-1 recruitment to silica beads coated with FN7-10 attached at the edge and at the center of monolayers. FLAG antibody was used to stain for ZO-1 S168A. Scale bar, 50 µm. Insets show the area marked with red/blue squares. Images are representative from three experiments.

FIGURE 4:

Disruption of the α5β1/ZO-1 complex leads to a higher number of smaller nascent adhesions. (A) Paxillin staining at the subconfluent edge and at the center of monolayers. (B) Quantification of adhesion intensity. (C) Quantification of adhesion density. (D) Quantification of adhesion length. Scale bar, 50 µm. Insets show the area marked with a red square. Images are representative from three experiments.