The costs of close contacts: Visualizing the energy landscape of cell contacts at the nanoscale

Abstract Cell-cell contacts often underpin signalling between cells. For immunology the binding of a T cell receptor (TCR) to an antigen presenting pMHC initiates downstream signalling and an immune response. While this contact is mediated by proteins on both cells creating interfaces with gap sizes typically around 14 nm, many often contradictory observations have been made regarding the influence of the contact on parameters such as the binding kinetics, spatial distribution and diffusion of signalling proteins within the contact. Understanding the basic physical constraints on probes inside this crowded environment will help inform studies on binding kinetics and dynamics of signalling of relevant proteins in the synapse. By tracking quantum dots of different dimensions for extended periods of time, we have shown that it is possible to obtain the probability of a molecule entering the contact, the change in its diffusion upon entry and the impact of spatial heterogeneity of adhesion protein density in the contact. By analysing the contacts formed by a T cell interacting with adhesion proteins anchored to a supported lipid bilayer, we find that probes are excluded from contact entry in a size-dependent manner for gap-to-probe differences of 4.1 nm. We also observed probes being trapped inside the contact and a decrease in diffusion of up to 85% in dense adhesion protein contacts. This approach provides new insights into the nature of cell-cell contacts, revealing that cell contacts are highly heterogeneous, due to topography- and protein density-related processes. These effects are likely to profoundly influence signalling between cells.