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

Area of Science:

• Biophysics
• Cell Biology
• Surface Science

Background:

• Two-dimensional (2D) affinity between cell-surface proteins is crucial for cellular processes.
• Measuring 2D affinity at the single-cell level is experimentally challenging and often yields population-averaged data.

Purpose of the Study:

• To develop and validate a novel method for quantifying single-cell 2D affinity on supported lipid bilayers (SLBs).
• To determine both the 2D affinity and the number of cell receptors simultaneously.
• To investigate the relationship between ligand density, receptor number, and cell contact dynamics.

Main Methods:

• Utilizing polyhistidine-tagged fluorescent ligands anchored to an SLB and their interaction with cell-expressed receptors (rat CD2 on Jurkat T cells).
• Employing imidazole to modulate ligand density on the SLB, inducing steady-state ligand accumulation in cell contacts.
• Analyzing ligand accumulation changes to calculate 2D affinity (1/K_d) and receptor density per cell.

Main Results:

• The method accurately determined 2D affinity (K_d = 4.9 ± 0.9 molecules/µm²) with low variation within the cell population.
• Ligand accumulation varied significantly due to receptor density differences, but average 2D affinity remained consistent.
• Cell contact size correlated with ligand density and receptor number, showing complex dynamics upon ligand density changes.

Conclusions:

• The developed method enables precise single-cell 2D affinity measurements, reducing time and improving accuracy.
• The technique's ability to resolve low spread in affinities facilitates the study of weak binding events.
• This approach offers a powerful tool for dissecting molecular interactions governing cell-cell adhesion and signaling.