E-Cadherin Tethered to Micropatterned Supported Lipid Bilayers as a Model for Cell Adhesion

Abstract

Cell-cell adhesion is a dynamic process requiring recruitment, binding, and reorganization of signaling proteins in the plane of the plasma membrane. Here, we describe a new system for investigating how this lateral mobility influences cadherin-based cell signaling. This model is based on tethering of a GPI-modified E-cadherin protein (hEFG) to a supported lipid bilayer. In this report, membrane microfluidics and micropatterning techniques are used to adopt this tethered protein system for studies with the anchorage-dependent cells. As directly formed from proteoliposomes, hEFG exhibits a diffusion coefficient of 0.6 ± 0.3 μm 2 /s and mobile fraction of 30-60%. Lateral structuring of the supported lipid bilayer is used to isolate mobile proteins from this mixed mobile/immobile population, and should be widely applicable to other proteins. MCF-7 cells seeded onto hEFG-containing bilayers recognize and cluster this protein, but do not exhibit cell spreading required for survival. By micropatterning small anchors into the supported lipid bilayer, we have achieved cell spreading across the bilayer surface and concurrent interaction with mobile hEFG protein. Together, these techniques will allow more detailed analysis of the cellular dynamics involved in cadherin-dependent adhesion events.

E-cadherin junction formation involves an active kinetic nucleation process

“…This observation suggests that some sort of nucleation event is essential, but is not well reconstituted with the highly fluid supported membrane. We note that adhesion observed in early studies on live cells (26) or giant unilamellar vesicles (47,48) and supported membranes did not resemble the extensive junction formation we report here, and we suggest that those systems failed to achieve the previously unknown nucleation step described here.…”

“…The all-or-nothing nature of junction formation combined with the observed sensitivity to supported membrane mobility and active cellular processes indicates an active nucleation step, which can be defeated if the supported membrane E-cadherin exhibits too much freedom of motion. Previous efforts to reconstitute cadherin in supported bilayers have used an Fc-conjugated extracellular domain of E-cadherin linked to the bilayer via either GPI or biotin−streptavidin interaction (24)(25)(26). As will be directly evidenced in the results presented here, these early efforts most likely did not achieve the nucleation threshold required to establish extended E-cadherin-mediated junctions.…”

How cells respond to environmental cues – insights from bio-functionalized substrates

“…E-cad cluster formation was found to be highly dependent on the viscous drag felt by aggregated ligands on the bilayer. On fluid bilayers, the clusters were unstable and transient (Biswas et al, 2015;Perez et al, 2005). In contrast, reduced diffusion of E-cad (by varying the bilayer fluidity or binding to the cytoskeleton) stabilized the cluster, strengthened the adhesion and redistributed the adhesive zones along the edge of the surrogate contact into a morphology that is reminiscent of the apical actin belt (Biswas et al, 2015).…”

Force Measurement Tools to Explore Cadherin Mechanotransduction

“…To model lateral mobility of cadherins using a substrate, Perez et al (2005) tethered E-cadherin-Fc molecules through glycosylphosphatidyl inositol to support lipid bilayers. Islands of anchored fi bronectin were patterned into the bilayer.…”