Fig 1.
Single molecule tracking of EGFR activity.
(A) Detection scheme for tracking EGFR activity at a single molecule level with TIRF. Single Cy3-SNAP-EGFR particles can be observed in the TIRF illumination field. Cytosolic EGFP-PTB contributes to a blurred background but does not interfere with the detection of EGFP-PTB particles that bind to the basal plasma membrane. (B) A single frame of a dual-color time-series acquired in an unstimulated MCF-7 cell expressing EGFP-PTB (green) and SNAP-EGFR labeled with Cy3 (red). (C) A single frame of a dual-color time-series acquired after 10 minutes of stimulation with 16 nM EGF in a MCF-7 cell expressing EGFP-PTB (green) and SNAP-EGFR labeled with Cy3 (red). Scale bar is 5 μm.
Fig 2.
Characterization of the mobility states of EGFR.
(A) Classified tracks of Cy3-SNAP-EGFR. The track segments are color-coded according to their mobility state (blue: free; green: confined; red: immobile). Shown is a 5×5 μm region of interest in an MCF-7 cell after 10 minutes of stimulation with 16 nM EGF (see also S4 Fig). (B) MSD analysis by state, after 10 minutes of stimulation with 16 nM EGF. The linear curves (blue symbols) are typical for Brownian motion, indicating free diffusion on this time-scale. The green symbols show a flattening of the curve due to confinement of the particles within a limited area. The red symbols show only minor displacement on this time-scale, indicating that the particles are immobile. Lines: linear fits to the beginning of each curve. Legend: fitted diffusion coefficients (D, μm2s-1). Error bars denote SEM.
Fig 3.
vbSPT analysis of Cy3-SNAP-EGFR tracks.
(A) Results of the vbSPT algorithm after 10 minutes of stimulation with 16 nM EGF. Circles: percentages of particles in the state (state occupations). Arrows: probabilities to switch to another state between frames (transition probabilities). Dashed arrows indicate transition probabilities < 0.01. (B) Lifetime of the mobility states, as a function of EGF stimulation, calculated from the classified track segments. (C) State occupations as a function of EGF stimulation. (D) The ratio of the forward to the backward probabilities, for the transitions between the free and the confined states, and for the transitions between the confined and the immobile states. ***P < 0.001, t test. n = 43 cells per time point. Error bars denote SEM.
Fig 4.
Quantification of EGFR aggregation.
(A) Probability of colocalization between Cy3-SNAP-EGFR and Alexa488-SNAP-EGFR. (B) Normalized intensity histograms of the Cy3-SNAP-EGFR intensity, for all particles (continuous lines) and for the particles that colocalized with Alexa488-SNAP-EGFR (dashed lines) after 10 minutes of EGF stimulation. n = 43 cells per time point. Error bars denote SEM.
Fig 5.
EGFR phosphorylation is amplified in the immobile state.
(A) Probability of colocalization between Cy3-SNAP-EGFR and EGFP-PTB. (B) Normalized intensity histograms of the Cy3-SNAP-EGFR intensity, for all particles (continuous lines) and for the particles that colocalized with EGFP-PTB (dashed lines) after 10 minutes of EGF stimulation. (C) Probability of colocalization between Cy3-SNAP-EGFR and EGF-Alexa488. (D) Fold-increase of the colocalization probability in the immobile versus the confined state, for EGFP-PTB colocalization (see A), and for EGF-Alexa488 colocalization (see C). A, B: n = 43 cells; C, D: n = 36 cells. ns: P > 0.05, **P < 0.01, ***P < 0.001, t test. Error bars denote SEM.
Fig 6.
Recruitment to clathrin-coated pits is required for robust EGFR activation.
(A) Overlay of the locations of immobile Cy3-SNAP-EGFR (red circles) and the EGFP-clathrin fluorescence (grey-scale image) in MCF-7 cells after 10 minutes of stimulation with 16 nM EGF. (B) Quantification of the colocalization of Cy3-SNAP-EGFR particles with EGFP-clathrin. (C) Colocalization between Cy3-SNAP-EGFR and EGFP-clathrin, after 30 minutes of incubation with 80 μM dynasore. (D) Colocalization probability between Cy3-SNAP-EGFR and EGFP-PTB, after 30 minutes of incubation with 80 μM dynasore. (E) Colocalization between Cy3-SNAP-EGFR-Y1045F and EGFP-clathrin. (F) Colocalization probability between Cy3-SNAP-EGFR-Y1045F and EGFP-PTB. A–D: n = 16 cells per time point, E, F: n = 20 cells per time point. C–F, grey bars: reproduction of the results for wild-type SNAP-EGFR in the absence of dynasore (Figs 5A and 6B). Error bars denote SEM.
Fig 7.
Cross-phosphorylation of EGFR establishes phosphorylation gradients.
(A) Lifetime of the mobility states, after selecting Cy3-SNAP-EGFR particles that colocalized with EGFP-PTB (B) Quantification of the distribution of confined particles around immobile points (Kconfined) calculated for all particles in unstimulated and stimulated cells and for particles that colocalized with PTB after stimulation with EGF. The dashed line represents the Kconfined function in unstimulated cells after randomization of the particle locations. n = 43 cells per time point. *P < 0.05, **P < 0.01, ***P < 0.001, t tests comparing the Kconfined values of particles that colocalize with PTB after 10 minutes (red line) with the corresponding Kconfined values of all particles before stimulation (continuous black line). Error bars denote SEM.
Fig 8.
A new role for clathrin-coated pits in the early phases of EGFR signaling.
Upon stimulation with ligand, epidermal growth-factor receptors are recruited to clathrin-coated pits, where their phosphorylation is amplified by clustering. Phosphorylated receptors are able to escape the clathrin-coated pits leading to an amplified EGFR signal in surrounding plasma membrane.