Repetitive change in the monomer to dimer ratio of EGFR revealed by RICS analysis.
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(A) (left) Plasma membrane localization of eGFP-EGFRwt in CHO-K1 cells. Confocal images of the basal plasma membrane before EGF addition. The bottom and right panels show cross sections of the cell. The scale bar indicates 10 μm. (right) Example of 50 frames of eGFP-EGFR in CHO-K1 basal plasma membrane used to calculate the RICS auto-correlation. The scale bar indicates 5 μm. (B) RICS auto-correlation (left) of eGFP-EGFRwt in CHO-K1, calculated for a time bin of 50 frames (for ξ and ψ from -16 to 16 pixels, respectively), fitted to the 2D diffusion model with residual plot (right). The residuals of the fitting being less than 10% of the magnitude of the function indicate the appropriate fit. (C) Reproducible time trace of eGFP-EGFRwt diffusion coefficient. At the t = 0 EGF was added. Each point represents one time bin in RICS analysis, consisted of 50 frames which corresponds to 83 s, with 5 frame (8.3 s) interval. The EGFR monomer to dimer ratio at the regions indicated by arrows was calculated by N&B analysis. The experiment was repeated 20 times with essentially the same result. (D) Schematic structure of eGFP-EGFR monomer and dimer based on following PDB files: 3EVP, 1EGF, 2JWA, 1M17, 2M20, 2GS2 and 3GOP. EGF and eGFP are shown in red and green, respectively. 2JWA structure based on ErbB2 was used to display transmembrane helix dimer. (E) N&B analysis of eGFP-EGFRwt before (left) and after (right) EGF addition (indicated with red boxes in C). Histograms of apparent molecular brightness are shown. Green and blue lines represent the distribution of the EGFR monomer (ε ≈ 0.1, B ≈ 1.1) and dimer (ε ≈ 0.2, B ≈ 1.2), respectively; red line shows the cumulative distribution of both monomer and dimer.