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

Abstract

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. We here present a method to measure 2D affinity on single cells binding to polyhistidine-tagged fluorescent ligands anchored to a supported lipid bilayer (SLB). By decreasing the density of ligands in the SLB using imidazole, a new steady-state accumulation in the contact is obtained, and from this change, both the 2D affinity and the number of receptors on the cell can be determined. The method was validated on an SLB containing rat CD2 binding to the rat CD48 mutant T92A expressed on Jurkat T cells. The addition of imidazole did not influence the average 2D affinity (1/K d ), and the spread in affinities within the cell population was low, K d ¼ 4.9 5 0.9 molecules/ mm 2 (mean 5 SD), despite an order of magnitude spread in ligand accumulation because of differences in receptor density. It was also found that cell contact size increased both with ligand density and with the number of receptors per cell but that the contact size stayed approximately constant when lowering the ligand density, above a density of around 10 rat CD2 molecules/mm 2 , after the contact first had formed, indicative of a heterogeneous process. In summary, this method not only allows for single-cell affinities to be measured, but it can also reduce measurement and analysis time and improve measurement accuracy. Because of the low spread in 2D K d within the cell population, the analysis can further be restricted to the cells showing the strongest binding, paving the way for using this method to study weak binding events.

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

“…Junghans et al (2020) used this approach to determine 2D K d values of different ligandreceptor pairs with values similar to the ones obtained by the classical Zhu-Golan approach. However, in this approach the K d obtained from single cell-SLB contacts was found to vary quite significantly (Junghans et al, 2020;Chouliara et al, 2021), making this approach less suitable to determine single-cell K d values. For this purpose, it instead appears advantageous to start at a high density of ligands in the SLB and reduce the density of polyhistidine-tagged ligands in steps using, for example, imidazole as done by Chouliara et al (2021).…”

“…However, in this approach the K d obtained from single cell-SLB contacts was found to vary quite significantly (Junghans et al, 2020;Chouliara et al, 2021), making this approach less suitable to determine single-cell K d values. For this purpose, it instead appears advantageous to start at a high density of ligands in the SLB and reduce the density of polyhistidine-tagged ligands in steps using, for example, imidazole as done by Chouliara et al (2021). This was used to obtain accurate single-cell affinities of rat CD2 binding rat CD48 T92A expressed on Jurkat T cells, showing only a minor spread in 2D K d values over the whole cell population.…”

“…A period of ~30 min is typically required for ligandfunctionalized bilayers to reach steady state upon interaction with receptor-expressing Jurkat T cells (Dustin et al, 1997;Zhu et al, 2007;Chouliara et al, 2021). The Zhu-Golan analysis requires the measurement of the concentration of bound and free ligands, as well as the cell-area fraction that is in contact with the SLB.…”

“…Obtaining accurate values for binding affinities using the Zhu-Golan method requires measuring over several cell-SLB contacts for each ligand concentration. This is due to an inherent spread in accumulation for each cell contact caused by the spread in receptor density in the cell population (Jönsson et al, 2016;Chouliara et al, 2021). It has further been shown that cells expressing a higher number of surface receptors have a higher probability to bind to SLBs with low ligand densities, which can influence the outcome of the Zhu-Golan analysis (Dustin et al, 1997).…”

“…Interestingly, these effects are not generally observed on cell-SLB systems. For example, Chouliara et al (2021) showed an almost constant affinity vs the density of bound rat CD2 ligands in the range of 200-600 bound molecules/µm 2 when binding to rat CD48 T92A on Jurkat T cells (Chouliara et al, 2021). It cannot be ruled out that cooperative effects occur at lower ligand densities, but it could be that the interaction with the cytoskeleton makes the cell membrane less sensitive to membrane fluctuations.…”