Year: 2025
Type: article
Abstract: Ligand-receptor interactions are critical for cell communication, with membrane receptors such as the Epidermal Growth Factor Receptor (EGFR) mediating responses to external signals. Super-resolution microscopy techniques in principle allow the visualization of these interactions at single-molecule resolution. While DNA-Points Accumulation for Imaging in Nanoscale Topography (DNA-PAINT) super-resolution microscopy has been successfully used to image receptors, specific labeling of cognate ligands, such as EGF, with DNA has remained challenging. Here, an approach to label and image the small extracellular ligand EGF using site-specific tagging and DNA modification is presented. Functional, site-specifically tagged EGF constructs, including DNA-conjugated and ALFA-tagged EGF, are generated. When compared to the native ligand, only the ALFA-tagged EGF maintains full functionality such as efficient EGFR clustering and internalization, while the DNA-conjugated EGF exhibits reduced EGFR oligomerization. 3D DNA-PAINT imaging of the ALFA-tagged EGF, when bound to EGFR, reveals spatial arrangements of EGF-EGFR complexes and captures different stages of receptor internalization. The labeling approach enables precise visualization of ligand-receptor interactions at high resolution and, in principle, can be extended to other ligand-receptor systems.
Source: Small Methods
Language: English
Subfield: Biophysics
Domain: Life Sciences
Open Access status: hybrid
Estimated APC paid: $4,640
Funders: International Max Planck Research School for Environmental, Cellular and Molecular Microbiology, Max-Planck-Förderstiftung, European Commission, National Research Foundation, Volkswagen Foundation, Danmarks Grundforskningsfond, Bundesministerium für Bildung und Forschung, Max-Planck-Gesellschaft, International Max Planck Research School for Advanced Methods in Process and Systems Engineering, European Research Council, HORIZON EUROPE Marie Sklodowska-Curie Actions
OpenAlex ID: W4409181249