Single cell 3-D platform to study ligand mobility in cell–cell contact

Abstract

Lateral mobility and dimensionality have both been shown to influence cellular behavior, but have yet to be combined and applied in a single in vitro platform to address, e.g., cell adhesion in a setting mimicking the three-dimensional environment of neighboring cells in a reductionist way. To study the effect of the lateral mobility of cell adhesive ligands in three dimensions we present and characterize a platform, which enables patterning of single cells into microwells presenting a cell membrane mimetic interface pre-patterned to its walls. Soluble E-cadherin extracellular domains coupled through an optimized streptavidin-antibody linkage to lipids in a supported lipid bilayer (SPB) were presented on the microwell walls as either laterally mobile or immobile ligands. The fluidity was controlled through a small change in temperature by choosing phospholipids for the SPB with a lipid phase transition temperature around 30 °C. The platform thus enabled the investigation of cell adhesion to either laterally immobile or mobile E-cadherin ligands presented on the same cell membrane mimetic surface. Chinese hamster ovary (CHO) cells engineered to express E-cadherin that were cultured on the platform demonstrated that enhanced cadherin lateral mobility significantly decreased the formation of actin bundles and resulted in more diffuse actin organization, while constraining the cell shape to that of the microwell. This example highlights the potential to use in vitro cell culture platforms to mimic direct cell-cell interaction in a controlled environment that nevertheless captures the dynamic nature of the native cell environment.

E-cadherin junction formation involves an active kinetic nucleation process

“…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.…”

Epithelial self-healing is recapitulated by a 3D biomimetic E-cadherin junction

“…Prior reductionist assays have elucidated the role of cadherins in cell-cell junction dynamics using 2D substrates conjugated with purified N-cadherin or E-cadherin (17-21), 2D substrates copatterned with E-cadherin and extracellular matrix (ECM) (15,22), or 3D microwells containing single cells uniformly coated with a single protein type (23,24). However, none of these interfaces are designed to be homotypic to epithelial cells, because 2D copatterned surfaces lack the 3D geometry of a native junction and microwells coated with a single protein lack the bifunctionality of native junctions (basal ECM vs. lateral cadherin adhesions).…”

Photoresponsive Cucurbit[8]uril‐Mediated Adhesion of Bacteria on Supported Lipid Bilayers

“…Previous QCM-D analysis of gel-state SLBs in the presence of serum showed negligible protein fouling on such surfaces. [ 23 ] In addition, a cell adhesion study performed on polymerized zwitterionic SLBs consisting of Bis-SorbPC showed a high degree of cell repellence towards murine-derived macrophages despite the absence of lipid lateral mobility. [ 46 ] Clearly, signifi cant fouling was observed on gel-state SLBs and SAM PEG layers, whereas on the liquid-state SLB no fouling was observed in the case of nonspecifi c adhesion of ConA.…”