Signal transduction across the plasma membrane via cytokine receptors requires assembly of complexes from two or more receptor subunits. Stoichiometry and dynamics of such signaling complexes are defined by subtle, cooperative interactions, which are emerging as key determinants defining signaling specificity. We therefore aim to disentangle and quantify the molecular interactions governing receptor assembly in the cellular context. For this purpose, we have devised multicolor single-molecule imaging techniques for visualizing the assembly of class I cytokine receptor signaling complexes down to nanometer resolution. Highly efficient, cell surface-selective fluorescence labeling of cytokine receptors at physiological expression level was achieved by engineered nanobodies. Exploiting the enhanced capabilities of a newly designed TIRF-SIM microscope, we applied single-molecule co-tracking and single-molecule FRET (smFRET) to quantify interactions in the plasma membrane of live cells. We could not observe significant pre-assembly of receptor subunits in the absence of agonists. However, we found that formation of active signaling complexes critically depends on collective protein-protein and protein lipid interactions encoded in the transmembrane and cytosolic receptor domains and the associated Janus family kinase (JAK). These interaction sites appear to be evolved for low affinity as several oncogenic mutations in these interaction sites enhance agonist-independent receptor dimerization. Time-lapse monitoring of receptor dimers by smFRET enabled quantifying complex lifetimes even for very transiently interacting variants. Furthermore, the smFRET efficiencies report on the nanoscale structural organization and conformational dynamics of receptor dimers diffusing in the plasma membrane. Correlative analysis across spatial and temporal scales uncovered surprising functional heterogeneity of signaling complexes upon oncogenic dysregulation, opening new avenues for therapeutic intervention.

References

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 Bio

Diploma in Chemistry, Tübingen/Germany 1993. PhD in Physical Chemistry, Tübingen/Germany 1997; 1997-2000, Postdoc in Protein Science at the Weizmann Institute of Science/Israel. 2001-2008, Independent Research Group Leader/associate professor for protein biophysics, Frankfurt/Germany. Since 2008, full professor for biophysics, Osnabrueck/Germany.