Figure 1.

EGFR Phosphorylation and Vesicular Dynamics

(A) Quantifying ectopic EGFR-mTFP expression in MCF7 cells. Average EGF-Alexa647 versus EGFR-mTFP fluorescence in single MCF7 (green) or MCF10A cells without EGFR-mTFP (black). Histograms (left) reflect that levels of EGF-Alexa647 binding to MCF7 with ectopic EGFR-mTFP expression (green) and MCF10A with endogenous EGFR (black) are similar.

(B) EGFR Y₁₀₆₈ phosphorylation (left) and Akt phosphorylation (right) in MCF7 cells ectopically expressing EGFR-mTFP (solid lines) and for endogenous EGFR in MCF10A cells (dashed lines), following 5-min pulsed (5P-EGF, 200 ng/mL, blue) or sustained EGF stimulation (S-EGF, 200 ng/mL, red), determined by in-cell western assay. Data are normalized to the maximum response in each respective condition (means ± SEM, N = 3).

(C) Representative fluorescence image series of EGF-Alexa647, EGFR-mTFP, PTB-mCherry, and PTB-mCherry (magenta)/EGFR-mTFP (green) overlay from single-cell dose-response experiment. Cells were stimulated every ∼1.5 min with increasing EGF-Alexa647 doses (2.5–600 ng/mL). Scale bar, 20 μm.

(D) Fraction of phosphorylated versus ligand-bound EGFR-mTFP (n = 21, N = 10; Figures S1B–S1D). Dashed lines: moving averages from single cells; shaded bounds: SDs; dash-dotted lines: estimated contribution of ligandless to the fraction of phosphorylated EGFR.

(E) Live cell fluorescence anisotropy microscopy measurements of EGFR-QG-mCitrine dimerization state as a function of the fraction of ligand-bound receptor (mean ± SEM, n = 30, N = 3, Figures S1F and S1G).

(F–H) Average spatial-temporal maps of the estimated fraction of ligand-bound EGFR (F, EGF-Alexa647/EGFR-mCitrine), ligandless EGFR (G, 1 − [EGF-Alexa647/EGFR-mCitrine]), and the fraction of phosphorylated EGFR-mCitrine estimated by PTB-mCherry translocation (H, PTB-mCherry/EGFR-mCitrine). Data were acquired at 1-min intervals in live MCF7 cells following 200 ng/mL S-EGF (top, n = 16, N = 3; Figures S1I and S1J) or 5P-EGF (n = 14, N = 2; Figures S1I and S1J) stimulation. White dotted lines: trajectories representing the change in distribution of ligand-bound (F) and ligandless (G) EGFR. PM, plasma membrane; NM, nuclear membrane.

(I) The respective plasma membrane fractions of ligand-bound (EGF-Alexa647/EGFR-mCitrine, red) and phosphorylated EGFR (PTB-mCherry/EGFR-mCitrine, blue) derived from (F) and (H) (median ± AMD). Extracellular EGF-Alexa647 fluorescence is shown in gray.

(J) Dimerization state measured by anisotropy (black) and the fraction of ligand-bound EGFR-QG-mCitrine (red) at the plasma membrane for live cells following 200 ng/mL S-EGF (top, n = 5, N = 3) or 5P-EGF (bottom, n = 5, N = 3) stimulation (means ± SEM).

(K) The dose response of EGFR-mTFP phosphorylation (red, control) is significantly altered upon ectopic Rab11^S25N expression (green; p = 0.02; n = 12, N = 4). Lines are the same as in (D).

(L) EGFR trafficking dynamics: ligandless EGFR recycles via early (EE) and recycling endosomes (RE) to the plasma membrane (red arrows) whereas upon EGF binding (thin green arrow), ubiquitinated EGF-EGFR_Ub unidirectionally traffics via the early to the late endosomes (LE, green arrow) to be degraded in lysosomes (∅). Causal links are denoted by solid black lines.