Identification and super-resolution imaging of ligand-activated receptor dimers in live cells.
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.
LP2N, University of Bordeaux, UMR 5298, F-33405 Talence, France.
Pascale
P
Winckler
Lydia
L
Lartigue
Gregory
G
Giannone
Francesca
F
De Giorgi
François
F
Ichas
Jean-Baptiste
JB
Sibarita
Brahim
B
Lounis
Laurent
L
Cognet
D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D007089:Image Enhancement / Q000379:methods*; D036641:Microscopy, Fluorescence, Multiphoton / Q000379:methods*; D057054:Molecular Imaging / Q000379:methods*; D040262:Receptors, Vascular Endothelial Growth Factor / Q000378:metabolism*; D013347:Subcellular Fractions / Q000378:metabolism* / Q000648:ultrastructure; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism*
D042461:Vascular Endothelial Growth Factor A; D040262:Receptors, Vascular Endothelial Growth Factor