Wienbeuker, E.; Felker, A.; Nevskyi, O.; Harms, S. T.; Marx, D.; Tanzusch, K.; Chizhik, A.; Kurre, R.; You, C.; Enderlein, J.; Piehler, J.

Transmembrane signaling mediated by cytokine receptors orchestrates key cellular processes such as proliferation, differentiation, and immune responses. While numerous high-resolution structures of cytokine receptor ectodomains are available, the structural organization of the largely disordered intracellular domains (ICD) has remained unclear. Here, we interrogate the axial organization of cytokine receptor signaling complexes at the plasma membrane by metal-induced energy transfer (MIET). For this purpose, we leveraged biofunctionalized nanodot arrays (bNDAs) to capture cell surface receptors at a defined distance from the substrate. Readout by fluorescence lifetime imaging microscopy enabled quantifying axial distances of proteins in the plasma membrane of cells at both ensemble and single-molecule levels with a resolution of ~1 nm. Using the prototypic, biomedically highly relevant class I cytokine receptor GP130 as a model system, we uncover by MIET that the ICD extends randomly into the cytosol in the resting state, but surprisingly undergoes an axial compaction upon signal activation. These results highlight the potential of bNDA-supported MIET for resolving the axial architecture of signaling complexes within the cellular context.