Integrins and syndecan-4 make distinct, but critical, contributions to adhesion contact formation

Abstract

During cell adhesion to fibronectin there is a major reorganisation of the actin cytoskeleton and concomitant formation of adhesion complexes. Conflicting studies of adhesion receptors report that either integrin alone, or both integrin and syndecan-4 mediate the formation of vinculin-containing adhesions, and differences in these studies have been attributed to the density and conformational integrity of ligands used. We have endeavoured to resolve these issues by ELISA analysis of immobilised polypeptides, and found that ligands of both integrin alpha(5)beta(1) and syndecan-4 are necessary for focal adhesion formation under conditions of equivalent density of folded ligand. We also demonstrate that integrin and syndecan-4 play quite distinct roles in adhesion contact maturation and are not interchangeable. These results help us to understand how cells respond efficiently to changes in matrix environment, which should prove useful for developing approaches to aid wound healing.

Fig. 1

Receptor cooperation rather than ligand density determines focal adhesion formation. (A–C) Fibronectin polypeptides (10 μg ml⁻¹ unless stated) were coated onto surfaces, and the density of folded ligand was measured by ELISA using a conformation-specific antibody. (A) Polypeptides coated onto glass either directly or following treatment with sulfo-MBS. (B) Polypeptides coated onto plastic either directly or following treatment with sulfo-MBS. (C) Polypeptides coated onto sulfo-MBS-derivatised glass at a range of concentrations. (D + E) Human fibroblasts were spread for two hours on polypeptide-coated, derivatised glass before staining for vinculin and scoring 100 spread cells for the ability to form adhesions that exceeded 2% of cell area (* p = 0.009, ** p = 0.003 in comparison with 5 μg ml⁻¹ fibronectin). Error bars represent the standard error of four experiments, results were obtained on three separate occasions.

Fig. 2

Focal adhesion formation depends upon both an immobilised integrin ligand and a syndecan-4 ligand. Human fibroblasts were spread for 2 hours on immobilised ligands (bold) in the presence of soluble ligand (brackets) before fixing and staining for vinculin and actin. Images are representative of 400 cells photographed on four separate occasions. Bar = 20 μm.

Fig. 3

Integrin and syndecan-4 make divergent contributions to adhesion. (A) MEFs were spread for 10–120 minutes on Fn6–10 or plasma fibronectin either with or without 200 nM BIM-I, before measuring the average area of 50 cells. (B) MEFs were spread for 2 hours on fibronectin either with or without 200 nM BIM-I, before fixing and staining for vinculin and actin to determine focal adhesion formation. Error bars represent the standard error of four experiments, images are representative of 100 cells and results were obtained on three separate occasions, bar = 20 μm.