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

Cell-cell contact often underpins signalling between cells. Contact is mediated by proteins on both cells creating interfaces with gap sizes typically around 14 nm. Protein binding and accumulation leads to the contact becoming crowded, reducing the rate of protein diffusion, even for unbound proteins. Here we show that, by tracking quantum dots of different dimensions for extended periods of time, 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 observe 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. Statement of Significance The spatial distribution and diffusion of proteins has been shown to be important for various signalling machineries. As such size-dependent reorganisation of proteins in the immune cell-contact has been shown to affect activation of immune cells. While these studies relied on bulk measurements to investigate protein exclusion, small scale topographical changes and protein dynamics could not be evaluated. However, recent studies show that T cell activation is mediated by nanoscale structures. In our study the use molecular probes of various sizes to investigate the energy landscape of single molecules in a cell contact. This provides additional information and insights which cannot be determined by performing bulk experiments alone indicates.