Fig. 4
From: The architecture of EGFR’s basal complexes reveals autoinhibition mechanisms in dimers and oligomers
The architecture of ligand-free head-to-head polymers. a Polymer chain formed by repeating the head-to-head interface based on separations in Supplementary Fig. 5a, b and Supplementary Tables 1 and 2 (DII and DIV excluded for simplicity). The intensity is graded according to pbICS results in b, which show the data in Fig. 2c re-normalised to reveal the fractions of oligomer species on wtEGFR-expressing CHO cells treated with 100 nM Alexa 488-Affibody. Results are the mean of seven replicates. Error bars show SD. c FLImP distribution (grey) of DIII–DIII separations in ΔC-EGFR-expressing CHO cells treated with 4 nM CF640R-Affibody, from 41 FLImP measurements (CI ≤ 6 nm). The inset shows positions and error estimates (additional statistics in Supplementary Fig. 7). d As c but from ΔC-EGFR-expressing cells treated with 8 nM CF640R-EgB4-NB (DI–DI separations), from 32 FLImP measurements (CI ≤ 8 nm). Differences with the DIII–DIII distribution are significant (Supplementary Fig. 8). e Left and centre: Cartoons showing a side view of DI and DIII separations from the membrane in head-to-head complexes in the presence and absence of bound 9G8-NB based on Supplementary Fig. 5c–f and Supplementary Table 3. Right: FRET-derived separations from the membrane-DiI to DI (Alexa 488-EgB4-NB, blue) or DIII (Alexa 488-Affibody, red). The bar chart was derived from the measurements in Supplementary Fig. 10. (As predicted by the model, EGFR also forms oligomers on cells treated with 200 nM 9G8-NB (Supplementary Fig. 11a, b). f Two-colour SPT on live cells at 37 °C showing the fraction of tracks where two particles labelled with Alexa 488-Affibody and CF640R-Affibody spent ≥5 frames (250 ms) together within <1 pixel (pairwise particle colocalisation fraction) for cells expressing ΔC-EGFR, wtEGFR and I942E-EGFR. Horizontal spreads separate data points (~5000) within each condition. g Distribution of the duration of pairwise interactions (τON) in f. Horizontal lines mark mean and SD. Coincidental colocalisation statistics were accounted for12. p-Values (two-tailed Kolmogorov–Smirnov test) are in Supplementary Fig. 12. h Front view of a ligand-free oligomer illustrating the separation between non-interacting ICM units predicted by extracellular head-to-head interactions. All panels: DI, green; DII, red; DIII, blue; DIV, dark grey; plasma membrane, yellow; TM, grey; TKD silver
