(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.
DOI:
http://dx.doi.org/10.7554/eLife.06156.025