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

Pascale Winckler, Lydia Lartigue, Gregory Giannone, Francesca De Giorgi, François Ichas, Jean-Baptiste Sibarita, Brahim Lounis, Laurent Cognet, Pascale Winckler, Lydia Lartigue, Gregory Giannone, Francesca De Giorgi, François Ichas, Jean-Baptiste Sibarita, Brahim Lounis, Laurent Cognet

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.

Figures

Figure 1. Live cell super-resolution imaging of…
Figure 1. Live cell super-resolution imaging of functional membrane EGFRs newly activated by their ligand.
(a) Principle of the super-resolution method. Oblique illumination (light green) does not excite EGF ligands in solution. (b) uPAINT image of EGFR labeled by EGF-Atto532 acquired on live cells. (c) Same experiment performed using Panitumumab-Atto647N. (d) Competition assay showing specificity of EGF-Atto532 labeling: number of fluorescent EGF detected per frame (50 ms) on the cell membrane during a uPAINT acquisition using EGF-Atto532. After ~8 s and 38 s (red arrows), unlabeled Panitumumab was added in 100-fold excess (40 nM) compared to EGF.
Figure 2. Live cell super-resolution imaging of…
Figure 2. Live cell super-resolution imaging of membrane EGFR dimers based on single-molecule FRET.
Dual color uPAINT imaging of EGFR was performed using a 1:1 mix of EGF-Atto532 and EGF-Cy5 under 532 nm laser excitation. (a) Schematics of single molecule FRET between two fluorescent ligands bound on a EGFR dimer. (b) Donor channel: super-resolved image of EGFR labeled by EGF-Atto532 as in Fig. 1b. (c) Acceptor channel: super-resolved image of EGF activated dimer EGFRs obtained by single molecule FRET. (d) Signature of single molecule FRET: anti-correlated fluorescence signals detected by single molecule fitting in the donor (green line) and acceptor (red line) channels, in corresponding positions. Insets in (b) and (c) represents zooms of highlighted regions showing preferential cell edge localization of the dimers.
Figure 3. Membrane dynamics of EGFR dimers…
Figure 3. Membrane dynamics of EGFR dimers based on single molecule tracking of the FRET acceptor signals.
(a) and (b) color coded trajectories lasting more than 200 ms found in one of the highlighted regions of Fig. 2b and c respectively. (c) Cumulative distribution of D values obtained on a single cell for EGFR dimers alone (red) and for the entire population of EGFR imaged in the donor channel (green).

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