In living systems, the contact between cells is the basis of recognition, differentiation, and orchestration of an immune response. Obstacles and barriers to biomolecular motion, especially for receptors at cellular synapses, critically control these functions by creating an anisotropic environment. Whereas conventional fluorescence fluctuation methods, such as fluorescence correlation spectroscopy or fluorescence recovery after photobleaching, can only measure the isotropic diffusion of molecules, the two-dimensional pair correlation function (2D-pCF) approach probes the anisotropic paths at different spatial locations within an image, allowing the creation of high-resolution maps that can visualize and quantify how molecules move in a living cell. In this work, we show how the 2D-pCF method maps the environment in cellular synapses as perceived by natural killer (NK) cell receptors. In cultured human HLA null 721.221 cells, 2D-pCF reveals the motion of inhibitory receptor HLA-Cw4-YFP coexpressed with KIR3DL1 to be highly directional around specific loci, while these restrictions were absent in the case of HLA-B51-YFP coexpressed with KIR2DL1. Further, in freshly isolated educated (H-2Dd) and uneducated (MHC-/-) primary murine NK cells, the 2D-pCF method shows significant differences in the paths taken by activating receptor NKp46 and inhibitory receptor Ly49A in educated compared to uneducated cells. Altogether, we demonstrate that the 2D-pCF method is very powerful in informing about the spatial organization of motion in cells. Our data support the hypothesis that flexibility in the spatial arrangement of membrane receptors, that is, the absence of barriers, is crucial for NK cell function.
Keywords: barrier mapping; fluorescence fluctuation spectroscopy; immune cell receptors; natural killer cells; pair correlation analysis; spatiotemporal correlation.