Identification and super-resolution imaging of ligand-activated receptor dimers in live cells

Sci Rep. 2013:3:2387. doi: 10.1038/srep02387.

Abstract

Molecular interactions are key to many chemical and biological processes like protein function. In many signaling processes they occur in sub-cellular areas displaying nanoscale organizations and involving molecular assemblies. The nanometric dimensions and the dynamic nature of the interactions make their investigations complex in live cells. While super-resolution fluorescence microscopies offer live-cell molecular imaging with sub-wavelength resolutions, they lack specificity for distinguishing interacting molecule populations. Here we combine super-resolution microscopy and single-molecule Förster Resonance Energy Transfer (FRET) to identify dimers of receptors induced by ligand binding and provide super-resolved images of their membrane distribution in live cells. By developing a two-color universal-Point-Accumulation-In-the-Nanoscale-Topography (uPAINT) method, dimers of epidermal growth factor receptors (EGFR) activated by EGF are studied at ultra-high densities, revealing preferential cell-edge sub-localization. This methodology which is specifically devoted to the study of molecules in interaction, may find other applications in biological systems where understanding of molecular organization is crucial.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • COS Cells
  • Chlorocebus aethiops
  • Fluorescence Resonance Energy Transfer / methods*
  • Image Enhancement / methods*
  • Microscopy, Fluorescence, Multiphoton / methods*
  • Molecular Imaging / methods*
  • Receptors, Vascular Endothelial Growth Factor / metabolism*
  • Subcellular Fractions / metabolism*
  • Subcellular Fractions / ultrastructure
  • Vascular Endothelial Growth Factor A / metabolism*

Substances

  • Vascular Endothelial Growth Factor A
  • Receptors, Vascular Endothelial Growth Factor