Adherens Junctions spatially guide ECM deposition by stimulating integrin activation and clustering

Abstract

The extracellular matrix (ECM) plays an essential role in all multicellular organisms as a substrate for cell anchorage and tissue scaffolding. It is involved in diverse processes such as cell proliferation and survival, tissue morphogenesis, cell fate determination, and wound healing. Given the critical role of the ECM, the mechanisms regulating its formation have been studied extensively both in vitro and in vivo. Tissue tension and cadherin-based interactions were early on identified as critical factors through which ECM deposition was regulated. However, the mechanism through which adherens junctions influence ECM deposition was unclear and believed to be indirect, through the establishment of tissue-level tension. Unpublished data from our group however revealed that AJs can stimulate and spatially guide integrin activation creating spatially restricted pools of high ligand affinity receptors at cell-cell junctions. In this study we addressed the possibility that these AJ-driven pools of high ligand affinity integrins can a) spatially influence ligand binding on the cell surface and b) impact ECM deposition. To address these questions, we initially developed a protocol to allow monitoring fibronectin assembly both during live imaging as well as in fixed samples. Specifically, we generated fluorescently labelled fibronectin – YPET FN conditioned media using HEK293 cells. We showed that these conditioned media allow labelling of FN fibrils in different cell lines and used this assay to select appropriate cells for our experiments. Subsequently, HeLa and NIH 3T3 fibroblasts were used to show selective accumulation of FN on cell-cell contacts in various contexts. These data conclusively confirm our hypothesis that the AJ-driven active integrin pools in fact spatially influence ligand binding on the cell surface. Interestingly, use of a number of conformational specific integrin antibodies showed that at early time points only integrins with an extended closed headpiece conformation are recruited at cell-cell junctions, while accumulation of FN, leads to headpiece opening. We propose that the selective recruitment of ligands at cell-cell junctions is due to the recruitment and stabilization of extended conformation integrins at these sites. Given the fact that AJ-driven integrin activation is believed to be driven by PM tension and subsequent topological adaptation of the transmembrane domains we then asked if Talin is necessary for this process. Using Talin null cells we show that ligand accumulation on cell-cell contacts is strictly dependent on Talin raising the possibility that additional mechanisms may contribute to the process of integrin activation at AJs. Finally, to directly examine the role of AJs in ECM formation and FN fibrillogenesis we modulated AJs using various approaches including pharmacological agents and function-blocking cadherin mutants. These data show that AJs have a profound influence not only on ligand binding on the cell surface but also on the 3D architecture of the ECM generated by cells in culture. Collectively our data demonstrate that the impact of AJs on ECM formation and remodeling goes well beyond the establishment of tissue-level tension and provides mechanistic insight into the critical integrin cadherin cross talk.