Single-Molecule Recreation of the Cadherin/Catenin/Actin Complex

Abstract

Adherens junctions (AJs) comprise protein complexes essential for integrating cell-cell adhesion and actin cytoskeleton functions, and their importance has been shown in many loss-of-function studies. Disruption of acto-myosin tension in living cells impairs formation of AJs, but attempts to reconstitute in bulk assays a direct linkage between a minimal AJ complex (E-cadherin/β-catenin/α-catenin) and actin filaments in vitro have been unsuccessful. Here we present a new optical trap-based single-molecule assay that tests whether this linkage requires tension (Fig. 1A). using a reconstituted E-cadherin/β-catenin/α-catenin complex (∼100 nM), an actin filament held between 2 optical traps, and moving the complex along the actin filament by oscillating the microscope stage, we observe binding events that likely involve multiple complexes and displacements of over 100 nm with loading rates of ∼45 pN/s that can sustain over 15 pN of force. The stepwise release mechanism may reflect sequential detachment of individual E-cadherin/catenin complexes (Fig. 1B). These data indicate that several E-cadherin/catenin complexes can form a robust, load-resistant connection to an actin filament under tension. In ongoing work, we are studying how mechanical load may regulate the affinity between the E-cadherin/catenin complexes and actin filaments.View Large Image | View Hi-Res Image | Download PowerPoint Slide