(15f) Force-Dependent Interactions Between Actin Filaments and a Minimal Adherens Junction Complex

Introduction

Adherens
junctions (AJs) mediate adhesion between neighboring cells and are essential
for coordinating cell-cell adhesion and cytoskeletal dynamics. Disruption of cytoskeletally
generated tension in living cells impairs AJ formation, suggesting that localized
mechanical stress may be required for AJ assembly. Previous attempts to measure
the interaction of the core AJ components E-cadherin, beta-catenin, and alpha-catenin
with actin in vitro have been unsuccessful, perhaps due to the absence
of a mechanical load in bulk biochemical assays. Here we describe a new optical
trap-based single-molecule assay that allows us to directly probe the
interaction of the E-cadherin/catenin complex with filamentous actin as a
function of applied mechanical load (Fig. 1A). Optical traps (also known as
optical tweezers) utilize highly focused laser light to capture and exert force
on micron-sized dielectric objects. The instrument we have constructed consists
of a dual-beam optical trap that can apply up to ~20 piconewtons (pN) of force
while measuring the location of a trapped 1 mm polystyrene bead with nanometer and millisecond resolutions.

Results

Our
assay works as follows: two streptavidin-coated polystyrene beads are trapped
simultaneously using the dual-bead optical trap and a biotinylated actin
filament is stretched between them. This filament is then lowered over a
platform bead immobilized on a coverslip and functionalized with the reconstituted
E-cadherin/beta-catenin/alpha-catenin complex. Oscillation of the
piezo-actuated microscope stage moves the platform bead and attached
E-cadherin/catenin complexes back and forth along the axis of the actin
filament while the optical traps remain stationary. Load is applied when the
immobilized complexes bind to the filament as the stage continues moving and
one of the two beads is pulled away from the trap center. As the bead is pulled
away, the force gradient produced by the focused laser beam attempts to pull
the bead back to the center of the optical trap. The farther the bead is
pulled, the greater the restoring force it and the attached protein complex experience.
The microscope stage velocity and the stiffness of the optical traps thus
determine the force loading rate experienced by the E-cadherin/catenin
complexes. Using high concentrations of the E-cadherin/catenin complex (~10 to 100
nM), we observe binding events that likely involve the attachment of multiple
complexes to a single actin filament. Under these conditions, we observe
displacement events of over 100 nm with loading rates of ~45 pN/s that can
sustain over 15 pN of force. Detachment occurs via a stepwise release mechanism
that may reflect sequential detachment of individual complexes (Fig. 1B).

Conclusions

Our
data indicate that several E-cadherin/catenin complexes can together form a
robust, load-resistant connection to an actin filament under tension. This
observation is consistent with a hypothesized role for alpha-catenin in linking
the adherens junction to the actin cytoskeleton. Ongoing work tests the
possibility that the E-cadherin/catenin complex acts as a mechanosensitive
element whose affinity for actin is directly modulated by mechanical load.