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Regulation of EGFR signal transduction by analogue-to-digital conversion in endosomes.

Villaseñor R, Nonaka H, Del Conte-Zerial P, Kalaidzidis Y, Zerial M - Elife (2015)

Bottom Line: By mathematical modelling, we found that this mechanism confers both robustness and regulation to signalling output.Different growth factors caused specific changes in endosome number and size in various cell systems and changing the distribution of p-EGFR between endosomes was sufficient to reprogram cell-fate decision upon EGF stimulation.We propose that the packaging of p-RTKs in endosomes is a general mechanism to ensure the fidelity and specificity of the signalling response.

View Article: PubMed Central - PubMed

Affiliation: Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany.

ABSTRACT
An outstanding question is how receptor tyrosine kinases (RTKs) determine different cell-fate decisions despite sharing the same signalling cascades. Here, we uncovered an unexpected mechanism of RTK trafficking in this process. By quantitative high-resolution FRET microscopy, we found that phosphorylated epidermal growth factor receptor (p-EGFR) is not randomly distributed but packaged at constant mean amounts in endosomes. Cells respond to higher EGF concentrations by increasing the number of endosomes but keeping the mean p-EGFR content per endosome almost constant. By mathematical modelling, we found that this mechanism confers both robustness and regulation to signalling output. Different growth factors caused specific changes in endosome number and size in various cell systems and changing the distribution of p-EGFR between endosomes was sufficient to reprogram cell-fate decision upon EGF stimulation. We propose that the packaging of p-RTKs in endosomes is a general mechanism to ensure the fidelity and specificity of the signalling response.

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ub-EGFR measurements by FRET microscopy.(A) Representative images of HeLa EGFR-GFP BAC cells aftercontinuous stimulation with 10 ng/ml EGF for the indicated time points.EGFR-GFP fluorescence is shown in green, the corrected ub-EGFR intensity isshown in red. Measurements from each individual endosome were used for allquantifications. Scale bars, 10 μm.(B–C) Heat map of the 2D co-distributionof ub-EGFR and EGFR (B) or ub-EGFR and p-EGFR (C)integral intensity per endosome upon EGF stimulation as in Figure 1 after 10, 30, or 60 min.ub-EGFR and EGFR are well correlated, whereas the distribution of p-EGFR issignificantly narrower than ub-EGFR at 30 and 60 min. Heat maps show theresult of one representative experiment.DOI:http://dx.doi.org/10.7554/eLife.06156.016
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fig2s2: ub-EGFR measurements by FRET microscopy.(A) Representative images of HeLa EGFR-GFP BAC cells aftercontinuous stimulation with 10 ng/ml EGF for the indicated time points.EGFR-GFP fluorescence is shown in green, the corrected ub-EGFR intensity isshown in red. Measurements from each individual endosome were used for allquantifications. Scale bars, 10 μm.(B–C) Heat map of the 2D co-distributionof ub-EGFR and EGFR (B) or ub-EGFR and p-EGFR (C)integral intensity per endosome upon EGF stimulation as in Figure 1 after 10, 30, or 60 min.ub-EGFR and EGFR are well correlated, whereas the distribution of p-EGFR issignificantly narrower than ub-EGFR at 30 and 60 min. Heat maps show theresult of one representative experiment.DOI:http://dx.doi.org/10.7554/eLife.06156.016

Mentions: How do the kinetics of p-EGFR endosomal packaging compare with the kinetics of receptordephosphorylation and ubiquitylation? After 10 min of EGF stimulation, the pool ofp-EGFR in EEA1-positive endosomes (Figure2—figure supplement 1A, red curve) decayed faster than total EGFR(Figure 2—figure supplement 1A, greencurve; τdecay EGFR = 56.03 ± 5.72, τdecayp-EGFR = 37.08 ± 4.19), possibly due to de-phosphorylation orpreferential removal of p-EGFR from early endosomes. The latter can be excluded sincethe fraction of p-EGFR in EEA1-positive endosomes remained almost constant throughoutthe time course (Figure 2—figure supplement1B). EGFR ubiquitylation is required for its internalization into endosomes,and this is dependent on EGFR phosphorylation and the recruitment of the c-Cbl E3 ligase(Sigismund et al., 2013). To compare thelevels of ubiquitylated EGFR (ub-EGFR) with those of p-EGFR within the endosomal system,we modified the FRET assay using an anti-ubiquitin antibody (Figure 2—figure supplement 2A). The kinetics of ub-EGFRwere significantly different from those of p-EGFR. Whereas the levels of p-EGFR peakedat 15 min, ub-EGFR reached its maximum at 30 min after stimulation (Figure 2C, compare red with blue curves) and decreased more slowlythan p-EGFR at later times, probably reflecting deubiquitylation prior to receptorsequestration into ILVs (Piper and Katzmann,2007). These results are consistent with the fact that the appearance ofp-EGFR precedes that of ub-EGFR (Umebayashi et al.,2008). Moreover, ub-EGFR had a similar distribution to that of EGFR (Figure 2—figure supplement 2B) butsignificantly wider than p-EGFR (Figure2—figure supplement 2C), suggesting that the mechanisms responsible forstabilizing the mean levels of p-EGFR per endosome are not correlated with receptorubiquitylation.


Regulation of EGFR signal transduction by analogue-to-digital conversion in endosomes.

Villaseñor R, Nonaka H, Del Conte-Zerial P, Kalaidzidis Y, Zerial M - Elife (2015)

ub-EGFR measurements by FRET microscopy.(A) Representative images of HeLa EGFR-GFP BAC cells aftercontinuous stimulation with 10 ng/ml EGF for the indicated time points.EGFR-GFP fluorescence is shown in green, the corrected ub-EGFR intensity isshown in red. Measurements from each individual endosome were used for allquantifications. Scale bars, 10 μm.(B–C) Heat map of the 2D co-distributionof ub-EGFR and EGFR (B) or ub-EGFR and p-EGFR (C)integral intensity per endosome upon EGF stimulation as in Figure 1 after 10, 30, or 60 min.ub-EGFR and EGFR are well correlated, whereas the distribution of p-EGFR issignificantly narrower than ub-EGFR at 30 and 60 min. Heat maps show theresult of one representative experiment.DOI:http://dx.doi.org/10.7554/eLife.06156.016
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4384751&req=5

fig2s2: ub-EGFR measurements by FRET microscopy.(A) Representative images of HeLa EGFR-GFP BAC cells aftercontinuous stimulation with 10 ng/ml EGF for the indicated time points.EGFR-GFP fluorescence is shown in green, the corrected ub-EGFR intensity isshown in red. Measurements from each individual endosome were used for allquantifications. Scale bars, 10 μm.(B–C) Heat map of the 2D co-distributionof ub-EGFR and EGFR (B) or ub-EGFR and p-EGFR (C)integral intensity per endosome upon EGF stimulation as in Figure 1 after 10, 30, or 60 min.ub-EGFR and EGFR are well correlated, whereas the distribution of p-EGFR issignificantly narrower than ub-EGFR at 30 and 60 min. Heat maps show theresult of one representative experiment.DOI:http://dx.doi.org/10.7554/eLife.06156.016
Mentions: How do the kinetics of p-EGFR endosomal packaging compare with the kinetics of receptordephosphorylation and ubiquitylation? After 10 min of EGF stimulation, the pool ofp-EGFR in EEA1-positive endosomes (Figure2—figure supplement 1A, red curve) decayed faster than total EGFR(Figure 2—figure supplement 1A, greencurve; τdecay EGFR = 56.03 ± 5.72, τdecayp-EGFR = 37.08 ± 4.19), possibly due to de-phosphorylation orpreferential removal of p-EGFR from early endosomes. The latter can be excluded sincethe fraction of p-EGFR in EEA1-positive endosomes remained almost constant throughoutthe time course (Figure 2—figure supplement1B). EGFR ubiquitylation is required for its internalization into endosomes,and this is dependent on EGFR phosphorylation and the recruitment of the c-Cbl E3 ligase(Sigismund et al., 2013). To compare thelevels of ubiquitylated EGFR (ub-EGFR) with those of p-EGFR within the endosomal system,we modified the FRET assay using an anti-ubiquitin antibody (Figure 2—figure supplement 2A). The kinetics of ub-EGFRwere significantly different from those of p-EGFR. Whereas the levels of p-EGFR peakedat 15 min, ub-EGFR reached its maximum at 30 min after stimulation (Figure 2C, compare red with blue curves) and decreased more slowlythan p-EGFR at later times, probably reflecting deubiquitylation prior to receptorsequestration into ILVs (Piper and Katzmann,2007). These results are consistent with the fact that the appearance ofp-EGFR precedes that of ub-EGFR (Umebayashi et al.,2008). Moreover, ub-EGFR had a similar distribution to that of EGFR (Figure 2—figure supplement 2B) butsignificantly wider than p-EGFR (Figure2—figure supplement 2C), suggesting that the mechanisms responsible forstabilizing the mean levels of p-EGFR per endosome are not correlated with receptorubiquitylation.

Bottom Line: By mathematical modelling, we found that this mechanism confers both robustness and regulation to signalling output.Different growth factors caused specific changes in endosome number and size in various cell systems and changing the distribution of p-EGFR between endosomes was sufficient to reprogram cell-fate decision upon EGF stimulation.We propose that the packaging of p-RTKs in endosomes is a general mechanism to ensure the fidelity and specificity of the signalling response.

View Article: PubMed Central - PubMed

Affiliation: Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany.

ABSTRACT
An outstanding question is how receptor tyrosine kinases (RTKs) determine different cell-fate decisions despite sharing the same signalling cascades. Here, we uncovered an unexpected mechanism of RTK trafficking in this process. By quantitative high-resolution FRET microscopy, we found that phosphorylated epidermal growth factor receptor (p-EGFR) is not randomly distributed but packaged at constant mean amounts in endosomes. Cells respond to higher EGF concentrations by increasing the number of endosomes but keeping the mean p-EGFR content per endosome almost constant. By mathematical modelling, we found that this mechanism confers both robustness and regulation to signalling output. Different growth factors caused specific changes in endosome number and size in various cell systems and changing the distribution of p-EGFR between endosomes was sufficient to reprogram cell-fate decision upon EGF stimulation. We propose that the packaging of p-RTKs in endosomes is a general mechanism to ensure the fidelity and specificity of the signalling response.

Show MeSH
Related in: MedlinePlus