Fluorescence-Based Measurements of Two-Dimensional Affinity in Membrane Interfaces.

Binding between ligands and receptors across cell contacts influences a range of biological processes including the formation of the immune synapse. The dissociation constant (K = 1/affinity) of the interaction corresponds to the concentration of ligands where half of the receptors in the contact have bound a ligand. In this chapter, we outline how to measure this two-dimensional affinity using model cell membranes called supported lipid bilayers (SLBs) functionalized with fluorescently labeled ligands that bind to cells containing the corresponding receptor.

Reversible Self-Assembled Monolayers with Tunable Surface Dynamics for Controlling Cell Adhesion Behavior.

Cells adhering onto surfaces sense and respond to chemical and physical surface features. The control over cell adhesion behavior influences cell migration, proliferation, and differentiation, which are important considerations in biomaterial design for cell culture, tissue engineering, and regenerative medicine. Here, we report on a supramolecular-based approach to prepare reversible self-assembled monolayers (rSAMs) with tunable lateral mobility and dynamic control over surface composition to regulate cell adhesion behavior.

Supported Lipid Bilayers and the Study of Two-Dimensional Binding Kinetics.

Binding between protein molecules on contacting cells is essential in initiating and regulating several key biological processes. In contrast to interactions between molecules in solution, these events are restricted to the two-dimensional (2D) plane of the meeting cell surfaces. However, converting between the more commonly available binding kinetics measured in solution and the so-called 2D binding kinetics has proven a complicated task since for the latter several factors other than the protein-protein interaction per se have an impact.

Single-cell measurements of two-dimensional binding affinity across cell contacts.

The two-dimensional (2D) affinity between protein molecules across contacting cells is a key parameter regulating and initiating several cellular processes. However, measuring 2D affinity can be challenging, and experimental data are limited. In addition, the obtained 2D affinities are typically averaged over the cell population.

Effects of a local auxiliary protein on the two-dimensional affinity of a TCR-peptide MHC interaction.

The affinity of T-cell receptors (TCRs) for major histocompatibility complex molecules (MHCs) presenting cognate antigens likely determines whether T cells initiate immune responses, or not. There exist few measurements of two-dimensional (2D) TCR-MHC interactions, and the effect of auxiliary proteins on binding is unexplored. Here, Jurkat T-cells expressing the MHC molecule HLA-DQ8-glia-α1 and the ligand of an adhesion protein (rat CD2) were allowed to bind supported lipid bilayers (SLBs) presenting fluorescently labelled L3-12 TCR and rat CD2, allowing measurements of binding unconfounded by cell signaling effects or co-receptor binding.

Lipid Bilayer-like Mixed Self-Assembled Monolayers with Strong Mobility and Clustering-Dependent Lectin Affinity.

Glycans at the surface of cellular membranes modulate biological activity via multivalent association with extracellular messengers. The lack of tuneable simplified models mimicking this dynamic environment complicates basic studies of these phenomena. We here present a series of mixed reversible self-assembled monolayers (rSAMs) that addresses this deficiency.