E-cadherin junction formation involves an active kinetic nucleation process.

Epithelial (E)-cadherin-mediated cell-cell junctions play important roles in the development and maintenance of tissue structure in multicellular organisms. E-cadherin adhesion is thus a key element of the cellular microenvironment that provides both mechanical and biochemical signaling inputs. Here, we report in vitro reconstitution of junction-like structures between native E-cadherin in living cells and the extracellular domain of E-cadherin (E-cad-ECD) in a supported membrane. Junction formation in this hybrid live cell-supported membrane configuration requires both active processes within the living cell and a supported membrane with low E-cad-ECD mobility. The hybrid junctions recruit α-catenin and exhibit remodeled cortical actin. Observations suggest that the initial stages of junction formation in this hybrid system depend on the trans but not the cis interactions between E-cadherin molecules, and proceed via a nucleation process in which protrusion and retraction of filopodia play a key role.