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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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Redistribution of endosomal EGF is sufficient to increase MAPKactivation in PC12 cells.(A–D) Analysis of MAPK activation in PC12cells after partial protein depletion of either EEA1, Rabenosyn5, and Vps45or EEA1, Syntaxin-6, and Syntaxin-13, or mock treatment and stimulation with100 ng/ml EGF or 50 ng/ml NGF for 30 min (A) Representativeimages of Erk1/2 activation by immunofluorescence in PC12 cells.phospho-Erk1/2 is shown in green and nuclei are shown in blue. Scale bars,10 μm. (B) Increase in phospho-Erk1/2 intensity comparedto EGF-treated control cells. The total intensity was normalized by thefraction of the area covered by cells. (C) Representativeimages of c-Fos phosphorylation by immunofluorescence in PC12 cells.phospho-c-Fos is shown in green. Scale bar, 25 μm. (D)Increase in nuclear phospho-c-Fos intensity compared to EGF-treated controlcells. In all cases, data show mean ± SEM. For each parameter,pair-wise comparisons were done against EGF-stimulated mock-treated cells.*p < 0.05, **p < 0.005 by Fisher'sLSD test. All measurements were done in three independent experiments with atotal of ∼500 cells per condition.DOI:http://dx.doi.org/10.7554/eLife.06156.036
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fig7s2: Redistribution of endosomal EGF is sufficient to increase MAPKactivation in PC12 cells.(A–D) Analysis of MAPK activation in PC12cells after partial protein depletion of either EEA1, Rabenosyn5, and Vps45or EEA1, Syntaxin-6, and Syntaxin-13, or mock treatment and stimulation with100 ng/ml EGF or 50 ng/ml NGF for 30 min (A) Representativeimages of Erk1/2 activation by immunofluorescence in PC12 cells.phospho-Erk1/2 is shown in green and nuclei are shown in blue. Scale bars,10 μm. (B) Increase in phospho-Erk1/2 intensity comparedto EGF-treated control cells. The total intensity was normalized by thefraction of the area covered by cells. (C) Representativeimages of c-Fos phosphorylation by immunofluorescence in PC12 cells.phospho-c-Fos is shown in green. Scale bar, 25 μm. (D)Increase in nuclear phospho-c-Fos intensity compared to EGF-treated controlcells. In all cases, data show mean ± SEM. For each parameter,pair-wise comparisons were done against EGF-stimulated mock-treated cells.*p < 0.05, **p < 0.005 by Fisher'sLSD test. All measurements were done in three independent experiments with atotal of ∼500 cells per condition.DOI:http://dx.doi.org/10.7554/eLife.06156.036

Mentions: Finally, we tested whether the differences in endosomal distribution can be, at least inpart, causative of the different cell-fates triggered by EGF and NGF in PC12 cells. Ifso, we would predict that redistributing EGF to a larger number of small endosomes asseen in PC12 stimulated with NGF would be sufficient to switch signalling specificityand induce differentiation of PC12 cells. Therefore, we applied the same protocol ofpartial protein depletion previously used for HeLa cells (Figure 4) in PC12 cells and consistently observed a mildredistribution of EGF into smaller endosomes (Figure7—figure supplement 1A,C,D). Also in this case, the partial depletiondid not result in major changes in EGF transport kinetics in PC12 cells (Figure 7—figure supplement 1B), butincreased the phosphorylation of both Erk (Figure7—figure supplement 2A,B) and c-Fos (Figure 7—figure supplement 2C,D) upon stimulation with EGF. Next, westimulated PC12 cells with EGF or NGF for 24 hr and analysed for neurite formation andβ-III tubulin expression as markers of differentiation (Ohuchi et al., 2002) and for EdU incorporation as a measure ofproliferation (Figure 7A). Stimulation with NGFincreased the number of cells with neurites (Figure7B, quantification in Figure 7C) andpositive for β-III tubulin (Figure 7B,quantification in Figure 7D), and reduced cellproliferation (Figure 7A, quantification in Figure 7E), the opposite of the stimulation withEGF. Remarkably, upon redistribution of endosomes, EGF increased process formation(Figure 7B, quantification in Figure 7C), β-III tubulin expression (Figure 7B, quantification in Figure 7D), and reduced cell proliferation (Figure 7A, quantification in Figure7E). The type of response was therefore similar to that of NGF, although theefficacy was lower. Nevertheless, these results show that a mild reduction of homotypicearly endosome fusion was sufficient to modify cell fate and induce neuronaldifferentiation of PC12 cells.10.7554/eLife.06156.034Figure 7.Redistribution of endosomal EGF is sufficient to trigger neuronaldifferentiation in PC12 cells.


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)

Redistribution of endosomal EGF is sufficient to increase MAPKactivation in PC12 cells.(A–D) Analysis of MAPK activation in PC12cells after partial protein depletion of either EEA1, Rabenosyn5, and Vps45or EEA1, Syntaxin-6, and Syntaxin-13, or mock treatment and stimulation with100 ng/ml EGF or 50 ng/ml NGF for 30 min (A) Representativeimages of Erk1/2 activation by immunofluorescence in PC12 cells.phospho-Erk1/2 is shown in green and nuclei are shown in blue. Scale bars,10 μm. (B) Increase in phospho-Erk1/2 intensity comparedto EGF-treated control cells. The total intensity was normalized by thefraction of the area covered by cells. (C) Representativeimages of c-Fos phosphorylation by immunofluorescence in PC12 cells.phospho-c-Fos is shown in green. Scale bar, 25 μm. (D)Increase in nuclear phospho-c-Fos intensity compared to EGF-treated controlcells. In all cases, data show mean ± SEM. For each parameter,pair-wise comparisons were done against EGF-stimulated mock-treated cells.*p < 0.05, **p < 0.005 by Fisher'sLSD test. All measurements were done in three independent experiments with atotal of ∼500 cells per condition.DOI:http://dx.doi.org/10.7554/eLife.06156.036
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Related In: Results  -  Collection

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fig7s2: Redistribution of endosomal EGF is sufficient to increase MAPKactivation in PC12 cells.(A–D) Analysis of MAPK activation in PC12cells after partial protein depletion of either EEA1, Rabenosyn5, and Vps45or EEA1, Syntaxin-6, and Syntaxin-13, or mock treatment and stimulation with100 ng/ml EGF or 50 ng/ml NGF for 30 min (A) Representativeimages of Erk1/2 activation by immunofluorescence in PC12 cells.phospho-Erk1/2 is shown in green and nuclei are shown in blue. Scale bars,10 μm. (B) Increase in phospho-Erk1/2 intensity comparedto EGF-treated control cells. The total intensity was normalized by thefraction of the area covered by cells. (C) Representativeimages of c-Fos phosphorylation by immunofluorescence in PC12 cells.phospho-c-Fos is shown in green. Scale bar, 25 μm. (D)Increase in nuclear phospho-c-Fos intensity compared to EGF-treated controlcells. In all cases, data show mean ± SEM. For each parameter,pair-wise comparisons were done against EGF-stimulated mock-treated cells.*p < 0.05, **p < 0.005 by Fisher'sLSD test. All measurements were done in three independent experiments with atotal of ∼500 cells per condition.DOI:http://dx.doi.org/10.7554/eLife.06156.036
Mentions: Finally, we tested whether the differences in endosomal distribution can be, at least inpart, causative of the different cell-fates triggered by EGF and NGF in PC12 cells. Ifso, we would predict that redistributing EGF to a larger number of small endosomes asseen in PC12 stimulated with NGF would be sufficient to switch signalling specificityand induce differentiation of PC12 cells. Therefore, we applied the same protocol ofpartial protein depletion previously used for HeLa cells (Figure 4) in PC12 cells and consistently observed a mildredistribution of EGF into smaller endosomes (Figure7—figure supplement 1A,C,D). Also in this case, the partial depletiondid not result in major changes in EGF transport kinetics in PC12 cells (Figure 7—figure supplement 1B), butincreased the phosphorylation of both Erk (Figure7—figure supplement 2A,B) and c-Fos (Figure 7—figure supplement 2C,D) upon stimulation with EGF. Next, westimulated PC12 cells with EGF or NGF for 24 hr and analysed for neurite formation andβ-III tubulin expression as markers of differentiation (Ohuchi et al., 2002) and for EdU incorporation as a measure ofproliferation (Figure 7A). Stimulation with NGFincreased the number of cells with neurites (Figure7B, quantification in Figure 7C) andpositive for β-III tubulin (Figure 7B,quantification in Figure 7D), and reduced cellproliferation (Figure 7A, quantification in Figure 7E), the opposite of the stimulation withEGF. Remarkably, upon redistribution of endosomes, EGF increased process formation(Figure 7B, quantification in Figure 7C), β-III tubulin expression (Figure 7B, quantification in Figure 7D), and reduced cell proliferation (Figure 7A, quantification in Figure7E). The type of response was therefore similar to that of NGF, although theefficacy was lower. Nevertheless, these results show that a mild reduction of homotypicearly endosome fusion was sufficient to modify cell fate and induce neuronaldifferentiation of PC12 cells.10.7554/eLife.06156.034Figure 7.Redistribution of endosomal EGF is sufficient to trigger neuronaldifferentiation in PC12 cells.

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