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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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The mean p-EGFR amount per endosome does not correlate with endosomearea at late time points after EGF stimulation.(A) Mean p-EGFR integral intensity per endosome as a functionof endosome area upon 10 (black curve) or 30 min (red curve) of EGFstimulation. Both curves were normalized to the intensity value at 1μm2 for 10 min stimulation. (B) Histogramdistribution of endosome area upon 10 (black curve) or 30 min (red curve) ofEGF stimulation. Each histogram was normalized by its respective curveintegral. Points show mean ± SEM. All measurements were done in threeindependent experiments with a total of ∼150 cells per time point orcondition.DOI:http://dx.doi.org/10.7554/eLife.06156.013
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fig1s9: The mean p-EGFR amount per endosome does not correlate with endosomearea at late time points after EGF stimulation.(A) Mean p-EGFR integral intensity per endosome as a functionof endosome area upon 10 (black curve) or 30 min (red curve) of EGFstimulation. Both curves were normalized to the intensity value at 1μm2 for 10 min stimulation. (B) Histogramdistribution of endosome area upon 10 (black curve) or 30 min (red curve) ofEGF stimulation. Each histogram was normalized by its respective curveintegral. Points show mean ± SEM. All measurements were done in threeindependent experiments with a total of ∼150 cells per time point orcondition.DOI:http://dx.doi.org/10.7554/eLife.06156.013

Mentions: The finding that endosomes contain a constant mean level of p-EGFR is striking. Weperformed several control experiments to verify that this is not an artefact caused bythe FRET method or the assay. First, the mean amount of p-EGFR per endosome did increaseat EGF concentrations higher than 10 ng/ml (Figure1—figure supplement 8), indicating that the value measured is notartificially fixed, for example, by limited antigen accessibility. Second, a similarconstant mean value of p-EGFR per endosomes was estimated with an independent methodusing the Tyr1068 antibody (Figure 1—figuresupplement 4). Third, the narrow distribution of p-EGFR per endosome maysimply reflect the sorting into endosomes of regular size. Whereas at 10 min the p-EGFRamount per endosome increased with the endosome area (Figure 1—figure supplement 9A, black curve), at 30 min (steady state,Figure 1B), the same mean amount was presentin small and large endosomes alike (Figure1—figure supplement 9A, red curve). Therefore, the amount of activatedreceptors per endosome is independent of endosome area. Finally, we verified that it isnot a phenomenon peculiar to HeLa cells but also occurring in non-immortalized,non-cancer cell lines. Using the anti-phosphoTyr1068 antibody, we found that in primarymouse hepatocytes upon EGF stimulation the mean amount of p-EGFR per endosome saturatedat ∼20 min whereas the mean amount of EGF continued to grow (data not shown),indicating that the packaging of p-EGFR in endosomes is not peculiar to asignalling-aberrant cancerous cell line.


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)

The mean p-EGFR amount per endosome does not correlate with endosomearea at late time points after EGF stimulation.(A) Mean p-EGFR integral intensity per endosome as a functionof endosome area upon 10 (black curve) or 30 min (red curve) of EGFstimulation. Both curves were normalized to the intensity value at 1μm2 for 10 min stimulation. (B) Histogramdistribution of endosome area upon 10 (black curve) or 30 min (red curve) ofEGF stimulation. Each histogram was normalized by its respective curveintegral. Points show mean ± SEM. All measurements were done in threeindependent experiments with a total of ∼150 cells per time point orcondition.DOI:http://dx.doi.org/10.7554/eLife.06156.013
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4384751&req=5

fig1s9: The mean p-EGFR amount per endosome does not correlate with endosomearea at late time points after EGF stimulation.(A) Mean p-EGFR integral intensity per endosome as a functionof endosome area upon 10 (black curve) or 30 min (red curve) of EGFstimulation. Both curves were normalized to the intensity value at 1μm2 for 10 min stimulation. (B) Histogramdistribution of endosome area upon 10 (black curve) or 30 min (red curve) ofEGF stimulation. Each histogram was normalized by its respective curveintegral. Points show mean ± SEM. All measurements were done in threeindependent experiments with a total of ∼150 cells per time point orcondition.DOI:http://dx.doi.org/10.7554/eLife.06156.013
Mentions: The finding that endosomes contain a constant mean level of p-EGFR is striking. Weperformed several control experiments to verify that this is not an artefact caused bythe FRET method or the assay. First, the mean amount of p-EGFR per endosome did increaseat EGF concentrations higher than 10 ng/ml (Figure1—figure supplement 8), indicating that the value measured is notartificially fixed, for example, by limited antigen accessibility. Second, a similarconstant mean value of p-EGFR per endosomes was estimated with an independent methodusing the Tyr1068 antibody (Figure 1—figuresupplement 4). Third, the narrow distribution of p-EGFR per endosome maysimply reflect the sorting into endosomes of regular size. Whereas at 10 min the p-EGFRamount per endosome increased with the endosome area (Figure 1—figure supplement 9A, black curve), at 30 min (steady state,Figure 1B), the same mean amount was presentin small and large endosomes alike (Figure1—figure supplement 9A, red curve). Therefore, the amount of activatedreceptors per endosome is independent of endosome area. Finally, we verified that it isnot a phenomenon peculiar to HeLa cells but also occurring in non-immortalized,non-cancer cell lines. Using the anti-phosphoTyr1068 antibody, we found that in primarymouse hepatocytes upon EGF stimulation the mean amount of p-EGFR per endosome saturatedat ∼20 min whereas the mean amount of EGF continued to grow (data not shown),indicating that the packaging of p-EGFR in endosomes is not peculiar to asignalling-aberrant cancerous cell line.

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