Limits...
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.

Show MeSH

Related in: MedlinePlus

Quantification of the difference between two area distributions.The differences between two endosome area distributions are measured asfollows: (1) The binned histograms of the endosome area are built from themeasurements of individual vesicles with bins linear in a logarithmic scale.(2) The histograms are normalized on their integrals, i.e., histograms arescaled to have the sum of values in all bins equal to one. (3) The histogramfrom the control condition is subtracted from the respective histograms ofinterest. The relative enrichment (red lines) or depletion (black lines) inthe population of vesicles is calculated by the integral over a particulararea interval.DOI:http://dx.doi.org/10.7554/eLife.06156.032
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4384751&req=5

fig6s2: Quantification of the difference between two area distributions.The differences between two endosome area distributions are measured asfollows: (1) The binned histograms of the endosome area are built from themeasurements of individual vesicles with bins linear in a logarithmic scale.(2) The histograms are normalized on their integrals, i.e., histograms arescaled to have the sum of values in all bins equal to one. (3) The histogramfrom the control condition is subtracted from the respective histograms ofinterest. The relative enrichment (red lines) or depletion (black lines) inthe population of vesicles is calculated by the integral over a particulararea interval.DOI:http://dx.doi.org/10.7554/eLife.06156.032

Mentions: An unexpected prediction of our model is that the total de-phosphorylation rate, andthus the total amount of p-EGFR, is dependent on the fusion/fission rate of theendosomes (Figure 3D). If so, could this have aneffect on signal transduction? To test these hypotheses, we reduced early endosomehomotypic fusion by lowering the intracellular concentration of established componentsof the endosome tethering and fusion machinery, EEA1, Rabenosyn5, Vps45 (Christoforidis et al., 1999; Ohya et al., 2010), Syntaxin-6 and Syntaxin-13 (Brandhorst et al., 2006) that play no direct rolein signalling. These genes were down-regulated by RNAi in combinations and onlypartially (∼50–70% depletion for each protein, Figure 4A) to achieve a significant inhibition of endosome fusionand yet prevent or reduce cell toxicity. This procedure caused a mild redistribution ofEGFR to endosomes of smaller size (<0.5 μm2 cross-section area,for details see ‘Materials and methods’ and Figure 6—figure supplement 2) (Figure 4C,D). Similar results were obtained upon depletion of asecond combination of genes (EEA1, Stx13, Stx6, not shown, see below). Note that thesetreatments generated a pattern of endosomes similar to that observed in different celltypes and under different culture conditions (see below, Figure 6) and neither altered the surface levels of EGFR (Figure 4—figure supplement 1) nor itskinetics of uptake (Figure 4B) and exit fromendosomes, that is, recycling and degradation (Figure4—figure supplement 2). We also excluded potential effects on endosomeacidification, because blocking it with bafilomycin did increase both p-EGFR and totalEGFR (Figure 4—figure supplement 3).Remarkably, under our experimental conditions of mild down-regulation of the earlyendosomal fusion machinery the packaging of active receptors was unaffected as shown byboth the time course and the steady-state mean (constant) amount of p-EGFR per endosome(Figure 4E; see above, Figure 1B). In contrast, the total number of endosomes with p-EGFRand their life-time augmented (Figure 4F),resulting in a net increase in the total amount and life-time of p-EGFR (Figure 4G). Notably, reduction of the endosomefusion rate in the mathematical model (∼40%, in line with the depletion oftethering proteins, Figure 4A) is sufficient toreproduce fairly well the experimental increase in p-EGFR endosomes observed (Figure 4F). These results support the hypothesisthat EGFR activation can be modulated by the endosomal system. Since p-EGFRde-phosphorylation precedes EGFR degradation (see above, Figure 1A, Figure 2—figuresupplement 2) and EGFR degradation is unaffected (Figure 4B, Figure 4—figuresupplement 2), we deduce that the effect on the life-time of p-EGFR caused byreduced endosomal fusion is primarily due to reduced de-phosphorylation.10.7554/eLife.06156.025Figure 4.Increasing the number and life-time of p-EGFR endosomes results inprolonged EGFR activation.


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)

Quantification of the difference between two area distributions.The differences between two endosome area distributions are measured asfollows: (1) The binned histograms of the endosome area are built from themeasurements of individual vesicles with bins linear in a logarithmic scale.(2) The histograms are normalized on their integrals, i.e., histograms arescaled to have the sum of values in all bins equal to one. (3) The histogramfrom the control condition is subtracted from the respective histograms ofinterest. The relative enrichment (red lines) or depletion (black lines) inthe population of vesicles is calculated by the integral over a particulararea interval.DOI:http://dx.doi.org/10.7554/eLife.06156.032
© Copyright Policy
Related In: Results  -  Collection

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

fig6s2: Quantification of the difference between two area distributions.The differences between two endosome area distributions are measured asfollows: (1) The binned histograms of the endosome area are built from themeasurements of individual vesicles with bins linear in a logarithmic scale.(2) The histograms are normalized on their integrals, i.e., histograms arescaled to have the sum of values in all bins equal to one. (3) The histogramfrom the control condition is subtracted from the respective histograms ofinterest. The relative enrichment (red lines) or depletion (black lines) inthe population of vesicles is calculated by the integral over a particulararea interval.DOI:http://dx.doi.org/10.7554/eLife.06156.032
Mentions: An unexpected prediction of our model is that the total de-phosphorylation rate, andthus the total amount of p-EGFR, is dependent on the fusion/fission rate of theendosomes (Figure 3D). If so, could this have aneffect on signal transduction? To test these hypotheses, we reduced early endosomehomotypic fusion by lowering the intracellular concentration of established componentsof the endosome tethering and fusion machinery, EEA1, Rabenosyn5, Vps45 (Christoforidis et al., 1999; Ohya et al., 2010), Syntaxin-6 and Syntaxin-13 (Brandhorst et al., 2006) that play no direct rolein signalling. These genes were down-regulated by RNAi in combinations and onlypartially (∼50–70% depletion for each protein, Figure 4A) to achieve a significant inhibition of endosome fusionand yet prevent or reduce cell toxicity. This procedure caused a mild redistribution ofEGFR to endosomes of smaller size (<0.5 μm2 cross-section area,for details see ‘Materials and methods’ and Figure 6—figure supplement 2) (Figure 4C,D). Similar results were obtained upon depletion of asecond combination of genes (EEA1, Stx13, Stx6, not shown, see below). Note that thesetreatments generated a pattern of endosomes similar to that observed in different celltypes and under different culture conditions (see below, Figure 6) and neither altered the surface levels of EGFR (Figure 4—figure supplement 1) nor itskinetics of uptake (Figure 4B) and exit fromendosomes, that is, recycling and degradation (Figure4—figure supplement 2). We also excluded potential effects on endosomeacidification, because blocking it with bafilomycin did increase both p-EGFR and totalEGFR (Figure 4—figure supplement 3).Remarkably, under our experimental conditions of mild down-regulation of the earlyendosomal fusion machinery the packaging of active receptors was unaffected as shown byboth the time course and the steady-state mean (constant) amount of p-EGFR per endosome(Figure 4E; see above, Figure 1B). In contrast, the total number of endosomes with p-EGFRand their life-time augmented (Figure 4F),resulting in a net increase in the total amount and life-time of p-EGFR (Figure 4G). Notably, reduction of the endosomefusion rate in the mathematical model (∼40%, in line with the depletion oftethering proteins, Figure 4A) is sufficient toreproduce fairly well the experimental increase in p-EGFR endosomes observed (Figure 4F). These results support the hypothesisthat EGFR activation can be modulated by the endosomal system. Since p-EGFRde-phosphorylation precedes EGFR degradation (see above, Figure 1A, Figure 2—figuresupplement 2) and EGFR degradation is unaffected (Figure 4B, Figure 4—figuresupplement 2), we deduce that the effect on the life-time of p-EGFR caused byreduced endosomal fusion is primarily due to reduced de-phosphorylation.10.7554/eLife.06156.025Figure 4.Increasing the number and life-time of p-EGFR endosomes results inprolonged EGFR activation.

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