Receptor dimerization probed by single-molecule colocomotion analysis. (a and b) Functional properties of U5A cells, which were stably complemented with tagged IFNAR1 and IFNAR2 (U5A^{IFNAR1/IFNAR2}) for posttranslational labeling and single-molecule imaging. (a) Western blot analysis of STAT phosphorylation, USP18 expression, and differential desensitization to IFNα2 and IFNβ. (b) IFN-induced translocation of STAT1-EGFP into the nucleus. (c) IFN-induced receptor dimerization revealed by single-molecule colocomotion experiments. Trajectories (80 frames, ∼2.5 s) of individual TMR-labeled IFNAR1 (red), DY647-labeled IFNAR2 (blue), and co-trajectories (magenta) in the absence and presence of 50 nM IFNα2 are shown. The diagram above indicates the possible different species detected in each channel before (left) and after (right) addition of the ligand, taking unlabeled IFNAR1 and IFNAR2 into account. (d) Formation and dissociation of an individual IFNAR1-IFNAR2 dimer in the presence of IFNα2 as observed by an overlay of the individual trajectories (left) and by a distance analysis (right). Shown is a representative curve from >25 curves analyzed. (e) Relative number of colocomotion trajectories for dual-labeled IFNAR2 (positive control) and noninteracting proteins (negative control), as well as IFNAR1 and IFNAR2, in the absence and presence of IFNα2. The box plot indicates the data distribution of the second and third quartile (box), median (line), mean (filled square), and whiskers (1.5× interquartile range). (f) Diffusion properties represented as step-length distribution of IFNAR1 (left; from >800 trajectories) and IFNAR2 (right; from >500 trajectories) in the absence and presence of IFNα2. For comparison, the step-length distribution of colocomotion trajectories (+IFNα2) is shown (from ∼100 trajectories).