Fig. 1: Live-cell labeling of receptors with multiple ligands.
From: Insights into receptor structure and dynamics at the surface of living cells
a Structure of IL-4Rα ectodomains (D1 and D2; PDB: 1IAR, backbone in ‘cartoon’ representation) and natural ligand IL-4 aligned for binding (black arrows) placed to scale on a schematic plasma membrane (PM; EC, extracellular; CP, cytoplasmic compartments). BCNK incorporation (indicated by B) sites (Cβ atoms in ‘spheres’ representation) of the receptor mutants are indicated by mature residue numbers (one-letter-code; Uniprot: P24394) and color-coded by domain: D1 (purple), the linker between D1 and D2 (pink), the activation loop in D2 (blue), D2 sites distant from the activation loop (teal), and neutralizing sites at the IL-4 binding interface (wheat). His-tag at the N-terminus ((His)6, gray ‘sphere’), and maleimide-coupled ATTO647N within IL-4 (N38C, gray ‘sphere’) serve as functional labels. The PM is traversed by a single transmembrane domain (TMD, dashed blue arrow). b Schematic representation of reference channels to validate functional subpopulations of labeled receptors: expression (eGFP), surface accessibility (trisNTA), and structural integrity (IL-4). c Representative equatorial confocal cross-sections showing multi-labeled receptors at the plasma membrane of a single HEK293T cell. Scale bar: 5 µm. d Example correlation functions with a finite cross-correlation amplitude (black arrow) indicating co-diffusion of IL-4Rα* and IL-4-ATTO647N. e Ratio of cross- and autocorrelation amplitudes reflecting the fraction of receptors occupied with IL-4-ATTO647N or clicked with Cy5-tet2 relative to IL-4Rα* (dashed line). Receptor mutants K97B and Y13B contain BCNK, K97B-Cy5 represents receptor mutant K97B clicked with Cy5-tet2, and Lyn-eGFP serves as plasma membrane marker (illustrated in Supplementary Fig. 4a). Experiments were performed once (Lyn-eGFP, Y13B) or twice (else). Source data are provided as a Source Data file.
