Figure 4. Increasing the number and life-time of p-EGFR endosomes results in prolonged EGFR activation.
(A) Protein down-regulation of EEA1 and Rabenosyn5 72 hr after siRNA transfection. RT-PCR showed an 80% reduction in Vps45 mRNA levels (data not shown). (B) Time course of EGFR integral intensity in endosomes after partial protein depletion of EEA1, Rabenosyn5, and Vps45 (red curve) or mock treatment (black curve). Cells were given a 1-min pulse of 10 ng/ml EGF, washed and chased for the indicated time points before fixation. (C) Representative images of HeLa EGFR BAC cells after EEA1, Rabenosyn5, and Vps45 knock-down or treatment with transfection reagent only (mock). Scale bars, 10 μm. (D) Shift in the EGFR-endosome area distribution toward smaller endosomes after EEA1, Rabenosyn5, and Vps45 knock-down. The values of the histograms of endosome area distribution for the control and knock-down conditions were normalized and subtracted. The curve shows the relative increase (above zero) or reduction (below zero) in the number of endosomes for each area bin (in logarithmic scale) (for details see ‘Materials and methods’ and Figure 6—figure supplement 2). Experimental points were fitted with two log-normal distributions. (E–G) Changes in p-EGFR endosomes in EEA1, Rabenosyn5, and Vps45 knock-down (red curve) or mock-treated (black curve) cells after continuous stimulation with 10 ng/ml EGF. Time courses of the mean integral intensity of p-EGFR per endosome (E), mean number of p-EGFR endosomes determined experimentally (squares) or predicted by the mathematical model (solid curves) for a 37% endosomes fusion rate (red curve) compared to control (black curve) (F), and total p-EGFR integral intensity in endosomes (G) measured as in Figure 1. Intensity curves were normalized to the intensity value at 10 min for mock-treated cells. Experimental points show mean ± SEM. All measurements were done in three independent experiments with a total of ∼150 cells per time point or condition. Time courses were fitted as in Figure 1.
Figure 4—figure supplement 1. Knock-down of fusion machinery does not change EGFR distribution at the plasma membrane in HeLa cells.
Figure 4—figure supplement 2. Knock-down of fusion machinery does not change EGFR degradation in HeLa cells.
Figure 4—figure supplement 3. Blocking endosome acidification with Bafilomycin increases both total EGFR and p-EGFR, but not the mean amount of p-EGFR per endosome.
