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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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HGF triggers sustained Erk1/2 activation in primary mousehepatoblasts.(A–B) Time course of Erk1/2 phosphorylationafter continuous stimulation with 10 ng/ml HGF or EGF for the indicatedtimes in mouse primary hepatoblasts. (A) Representativephospho-Erk1/2 and Erk1/2 Western blots and (B) itsquantification for HGF (red curve) or EGF (black curve) stimulation.DOI:http://dx.doi.org/10.7554/eLife.06156.031
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fig6s1: HGF triggers sustained Erk1/2 activation in primary mousehepatoblasts.(A–B) Time course of Erk1/2 phosphorylationafter continuous stimulation with 10 ng/ml HGF or EGF for the indicatedtimes in mouse primary hepatoblasts. (A) Representativephospho-Erk1/2 and Erk1/2 Western blots and (B) itsquantification for HGF (red curve) or EGF (black curve) stimulation.DOI:http://dx.doi.org/10.7554/eLife.06156.031

Mentions: The experiments on HeLa cells and the theoretical analysis raise the question of whethermodulation of early endosome homotypic fusion is a general mechanism to regulate signalamplitude and duration. If this were the case, we would predict that growth factors withdifferent signalling outputs (amplitude and duration) differentially modulate theendosomal distribution (i.e., endosome number, size, and cargo content). To test thisprediction, we examined different growth factors and cellular systems. First, we usedprimary mouse hepatoblasts where HGF promotes their proliferation (Tanimizu et al., 2003). In these cells, HGF but not EGF elicits asustained Erk response (Figure 6—figuresupplement 1). Indeed as predicted, stimulation of hepatoblasts with HGFcaused a strong shift in the distribution of early endosomes toward smaller sizes (Figure 6A, red curve Figure 6B), whereas EGF had the opposite effect (Figure 6A, green curve, Figure6B). Second, we turned to an in vitro model of reference for cell-fatedecisions, PC12 cells. In PC12 cells, EGF stimulation leads to transient Erkphosphorylation and cell proliferation, whereas NGF leads to sustained Erkphosphorylation and cell differentiation (Marshall,1995). Consistent with our results in primary mouse hepatoblasts, NGFstimulation in PC12 cells caused a significant shift in the distribution of earlyendosomes toward smaller sizes compared with EGF (Figure6C,D). Moreover, NGF itself was distributed to a larger number of smallerendosomes in comparison with EGF (Figure 6E,F).Altogether, these data argue that the modulation of endosome fusion, reflected by thechanges in endosome number and size, is a general property of growth factors. These datafurther suggest that signalling amplitude and duration can be regulated by changes inthe fusion rate of endosomes (see Table 1).10.7554/eLife.06156.030Figure 6.Growth factors differentially shift the distribution of the number andsize of endosomes.


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)

HGF triggers sustained Erk1/2 activation in primary mousehepatoblasts.(A–B) Time course of Erk1/2 phosphorylationafter continuous stimulation with 10 ng/ml HGF or EGF for the indicatedtimes in mouse primary hepatoblasts. (A) Representativephospho-Erk1/2 and Erk1/2 Western blots and (B) itsquantification for HGF (red curve) or EGF (black curve) stimulation.DOI:http://dx.doi.org/10.7554/eLife.06156.031
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4384751&req=5

fig6s1: HGF triggers sustained Erk1/2 activation in primary mousehepatoblasts.(A–B) Time course of Erk1/2 phosphorylationafter continuous stimulation with 10 ng/ml HGF or EGF for the indicatedtimes in mouse primary hepatoblasts. (A) Representativephospho-Erk1/2 and Erk1/2 Western blots and (B) itsquantification for HGF (red curve) or EGF (black curve) stimulation.DOI:http://dx.doi.org/10.7554/eLife.06156.031
Mentions: The experiments on HeLa cells and the theoretical analysis raise the question of whethermodulation of early endosome homotypic fusion is a general mechanism to regulate signalamplitude and duration. If this were the case, we would predict that growth factors withdifferent signalling outputs (amplitude and duration) differentially modulate theendosomal distribution (i.e., endosome number, size, and cargo content). To test thisprediction, we examined different growth factors and cellular systems. First, we usedprimary mouse hepatoblasts where HGF promotes their proliferation (Tanimizu et al., 2003). In these cells, HGF but not EGF elicits asustained Erk response (Figure 6—figuresupplement 1). Indeed as predicted, stimulation of hepatoblasts with HGFcaused a strong shift in the distribution of early endosomes toward smaller sizes (Figure 6A, red curve Figure 6B), whereas EGF had the opposite effect (Figure 6A, green curve, Figure6B). Second, we turned to an in vitro model of reference for cell-fatedecisions, PC12 cells. In PC12 cells, EGF stimulation leads to transient Erkphosphorylation and cell proliferation, whereas NGF leads to sustained Erkphosphorylation and cell differentiation (Marshall,1995). Consistent with our results in primary mouse hepatoblasts, NGFstimulation in PC12 cells caused a significant shift in the distribution of earlyendosomes toward smaller sizes compared with EGF (Figure6C,D). Moreover, NGF itself was distributed to a larger number of smallerendosomes in comparison with EGF (Figure 6E,F).Altogether, these data argue that the modulation of endosome fusion, reflected by thechanges in endosome number and size, is a general property of growth factors. These datafurther suggest that signalling amplitude and duration can be regulated by changes inthe fusion rate of endosomes (see Table 1).10.7554/eLife.06156.030Figure 6.Growth factors differentially shift the distribution of the number andsize of endosomes.

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