Contact-mediated communication between cells involves the engagement of membrane-associated proteins on the apposed surfaces. The biomolecular architecture of these lipid membranes imparts lateral mobility to these proteins, influencing the interaction and dynamics of these signaling molecules over scales ranging from nanometers to micrometers. However, the full functional implications of this mobility remain incompletely understood. In this report, we use a supported lipid bilayer model to identify such effects in the context of E-cadherin signaling in epithelial cells.
The supported lipid bilayer system consists of a planar assembly of phospholipids in close apposition to a silicon oxide support such as glass or quartz 2; 3; 15; 28
. A balance of molecular forces maintains a thin (~ 1 - 3 nm) layer of water that separates the bilayer and support 17
, allowing lateral mobility of the membrane biomolecules while constraining this structure to the substrate surface. Tethering of a protein to these lipids imparts lateral mobility to that biomolecule, providing a model of the cell membrane that has been used predominantly in the study of immune synapse structure but also other cellular systems to a limited extent 5; 6; 13; 14; 24; 25; 29; 31
. In the context of epithelial cells, the supported lipid bilayer system offers much promise in capturing the dynamics of cell-cell adhesion that normally leads to formation of barrier structures such as adherens junctions. Functional contact between epithelial cells is initiated by homophilic binding of E-cadherin between apposed cells, inducing cell ruffling and lamellipodial extensions that encourage further interactions between the membranes 1; 8
and recruit additional E-cadherin to the developing adhesion site. The role of cadherins in this and other signaling events has been studied extensively by putting cells in contact with either other cells, which capture the dynamic nature of this interface but are compositionally complex, or substrate-attached E-cadherin proteins, which allow precise control over the biomolecular makeup but lack the fluidity of natural membranes 7; 12; 18; 21; 26
. Notably, cells exert forces on substrates through cadherin-based complexes 11; 20
. Following the concept of rigidity sensing that is well established for integrin-based signaling, it is plausible that the response of cadherin complexes to cell-generated forces, such as motion along the apposed cell membrane or substrate support, modulates downstream intracellular signaling. In this direction, Perez et al.
developed a micropatterned protein – lipid bilayer system to present laterally mobile E-cadherin to epithelial cells interacting with a planar substrate 25
. However, this system does not allow control over either the timing or location of E-cadherin engagement, which is important in separating the influences of multiple factors in these experiments. As an alternative configuration, we explore in this report the use of silica beads to present either mobile (membrane-tethered) or immobile (directly adsorbed) E-cadherin to cells pre-seeded on a cell culture surface ().
The influence of ligand mobility on cell response was tested by presenting to cells Ecad/Fc either tethered to a lipid bilayer (mobile) or directly adsorbed (immobile) onto silica beads.
Engagement of E-cadherin initiates a rich set of downstream cell signaling pathways. This report will focus on Rac1, a member of the Rho family small GTPases that holds a particularly central role as both a downstream result of E-cadherin signaling and also upstream effector of cytoskeletal dynamics regulating cadherin function 4; 9; 10; 18; 22
. Live cell imaging 8; 22
showed that Rac1 is present at the edges of developing cell-cell contacts, and we will similarly use the accumulation of this protein around the bead-cell interface as a measure of functional engagement.