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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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Pharmacological inhibition of EGFR kinase activity increases the meanp-EGFR amount per endosomes.(A) Representative images of HeLa EGFR BAC cells aftercontinuous stimulation with 10 ngl/ml EGF for 30 min. Inhibitors were added10 min after stimulation with EGF and remained in the medium throughout thetime course. Scale bars, 10 μm. (B) Time course of themean p-EGFR integral intensity per endosome in AG1478 (red), Gefitinib(blue), Lapatinib (green), or DMSO-treated cells (black). All curves werenormalized by the intensity value at 10 min for the mock sample.Experimental points were fitted as in Figure1. Measurements for AG1478 were done in three independentexperiments; measurements from Gefitinib and Lapatinib show a representativeexperiment with a total of ∼150 cells per time point andcondition.DOI:http://dx.doi.org/10.7554/eLife.06156.021
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fig2s7: Pharmacological inhibition of EGFR kinase activity increases the meanp-EGFR amount per endosomes.(A) Representative images of HeLa EGFR BAC cells aftercontinuous stimulation with 10 ngl/ml EGF for 30 min. Inhibitors were added10 min after stimulation with EGF and remained in the medium throughout thetime course. Scale bars, 10 μm. (B) Time course of themean p-EGFR integral intensity per endosome in AG1478 (red), Gefitinib(blue), Lapatinib (green), or DMSO-treated cells (black). All curves werenormalized by the intensity value at 10 min for the mock sample.Experimental points were fitted as in Figure1. Measurements for AG1478 were done in three independentexperiments; measurements from Gefitinib and Lapatinib show a representativeexperiment with a total of ∼150 cells per time point andcondition.DOI:http://dx.doi.org/10.7554/eLife.06156.021

Mentions: Upon internalization, EGF enters the early endosomal network and, similar to LDL (Rink et al., 2005), following endosome homotypicfusion and fission reactions, accumulates in few large endosomes prior to transfer tolate endosomes. A mechanism must exist that prevents the continuous accretion of p-EGFRupon endosome fusion. A simple mechanism could be that the de-phosphorylation rateincreases with the increase in p-EGFR per endosome. When two endosomes fuse, theresulting endosome should contain the sum of EGFR and p-EGFR of the original endosomes.However, given such de-phosphorylation rate dependency, the amount of p-EGFR wouldreturn to the level prior to fusion, thus stabilizing the mean amount of p-EGFR perendosome. A prediction of this hypothesis is that the kinase activity of EGFR inendosomes controls its own dephosphorylation. To test this, we inhibited the EGFR kinaseactivity pharmacologically with AG1478, lapatinib or gefitinib 10 min after EGFstimulation (to prevent alterations on receptor internalization) and determined theeffects on the receptors already internalized and phosphorylated. We compared low withhigh concentrations of EGF, that is, under conditions of saturation of p-EGFR packagingin endosomes (Figure 1—figure supplement8). At low EGF concentrations, when the packaging mechanism is not saturated,the total amount of p-EGFR was not significantly reduced by the inhibitors (Figure 2—figure supplement 6 compare blackand green curves). This behaviour argues that the packages of p-EGFR in endosomes areprotected from the phosphatases. In addition, the inhibitors caused a continuousaccumulation of p-EGFR in fewer and larger endosomes over time (Figure 2—figure supplement 7). In contrast, adding theinhibitor after stimulation with high concentrations of EGF caused a sharp reduction inthe total amount of p-EGFR (Figure 2—figuresupplement 6 compare red and blue curves), as observed previously (Kleiman et al., 2011). This means that the kinaseactivity of EGFR is necessary to maintain the levels of p-EGFR in individual endosomes.These results support the idea that the dephosphorylation of p-EGFR in endosomes indeeddepends on the EGFR activity within the endosomal packages.


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)

Pharmacological inhibition of EGFR kinase activity increases the meanp-EGFR amount per endosomes.(A) Representative images of HeLa EGFR BAC cells aftercontinuous stimulation with 10 ngl/ml EGF for 30 min. Inhibitors were added10 min after stimulation with EGF and remained in the medium throughout thetime course. Scale bars, 10 μm. (B) Time course of themean p-EGFR integral intensity per endosome in AG1478 (red), Gefitinib(blue), Lapatinib (green), or DMSO-treated cells (black). All curves werenormalized by the intensity value at 10 min for the mock sample.Experimental points were fitted as in Figure1. Measurements for AG1478 were done in three independentexperiments; measurements from Gefitinib and Lapatinib show a representativeexperiment with a total of ∼150 cells per time point andcondition.DOI:http://dx.doi.org/10.7554/eLife.06156.021
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fig2s7: Pharmacological inhibition of EGFR kinase activity increases the meanp-EGFR amount per endosomes.(A) Representative images of HeLa EGFR BAC cells aftercontinuous stimulation with 10 ngl/ml EGF for 30 min. Inhibitors were added10 min after stimulation with EGF and remained in the medium throughout thetime course. Scale bars, 10 μm. (B) Time course of themean p-EGFR integral intensity per endosome in AG1478 (red), Gefitinib(blue), Lapatinib (green), or DMSO-treated cells (black). All curves werenormalized by the intensity value at 10 min for the mock sample.Experimental points were fitted as in Figure1. Measurements for AG1478 were done in three independentexperiments; measurements from Gefitinib and Lapatinib show a representativeexperiment with a total of ∼150 cells per time point andcondition.DOI:http://dx.doi.org/10.7554/eLife.06156.021
Mentions: Upon internalization, EGF enters the early endosomal network and, similar to LDL (Rink et al., 2005), following endosome homotypicfusion and fission reactions, accumulates in few large endosomes prior to transfer tolate endosomes. A mechanism must exist that prevents the continuous accretion of p-EGFRupon endosome fusion. A simple mechanism could be that the de-phosphorylation rateincreases with the increase in p-EGFR per endosome. When two endosomes fuse, theresulting endosome should contain the sum of EGFR and p-EGFR of the original endosomes.However, given such de-phosphorylation rate dependency, the amount of p-EGFR wouldreturn to the level prior to fusion, thus stabilizing the mean amount of p-EGFR perendosome. A prediction of this hypothesis is that the kinase activity of EGFR inendosomes controls its own dephosphorylation. To test this, we inhibited the EGFR kinaseactivity pharmacologically with AG1478, lapatinib or gefitinib 10 min after EGFstimulation (to prevent alterations on receptor internalization) and determined theeffects on the receptors already internalized and phosphorylated. We compared low withhigh concentrations of EGF, that is, under conditions of saturation of p-EGFR packagingin endosomes (Figure 1—figure supplement8). At low EGF concentrations, when the packaging mechanism is not saturated,the total amount of p-EGFR was not significantly reduced by the inhibitors (Figure 2—figure supplement 6 compare blackand green curves). This behaviour argues that the packages of p-EGFR in endosomes areprotected from the phosphatases. In addition, the inhibitors caused a continuousaccumulation of p-EGFR in fewer and larger endosomes over time (Figure 2—figure supplement 7). In contrast, adding theinhibitor after stimulation with high concentrations of EGF caused a sharp reduction inthe total amount of p-EGFR (Figure 2—figuresupplement 6 compare red and blue curves), as observed previously (Kleiman et al., 2011). This means that the kinaseactivity of EGFR is necessary to maintain the levels of p-EGFR in individual endosomes.These results support the idea that the dephosphorylation of p-EGFR in endosomes indeeddepends on the EGFR activity within the endosomal packages.

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