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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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Quantification of number of EGFR and pEGFR molecules perendosome.(A) Distribution histogram of the differences in intensitybetween individual endosomes in consecutive frames during sequentialphoto-bleaching of EGFR-GFP (see ‘Materials and methods’ fordetails). (B) Difference between the number of positive andnegative events in (A) for each ΔIntensity value.(C) Distribution histogram of the differences in intensitybetween individual endosomes in consecutive frames during sequentialphoto-bleaching of p-EGFR (see ‘Materials and methods’ fordetails). (D) Difference between the number of positive andnegative events in (C) for eachΔIntensity value. Thelocal amplitude maxima of the periodic function in (B) and(D) give an estimate of the change in intensity values when1,2,3, …, n number of molecules are bleached. (E)Distribution of the number of molecules of EGFR-GFP and p-EGFR in individualendosomes after stimulation with 10 ng/ml EGF for 10 min.DOI:http://dx.doi.org/10.7554/eLife.06156.017
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fig2s3: Quantification of number of EGFR and pEGFR molecules perendosome.(A) Distribution histogram of the differences in intensitybetween individual endosomes in consecutive frames during sequentialphoto-bleaching of EGFR-GFP (see ‘Materials and methods’ fordetails). (B) Difference between the number of positive andnegative events in (A) for each ΔIntensity value.(C) Distribution histogram of the differences in intensitybetween individual endosomes in consecutive frames during sequentialphoto-bleaching of p-EGFR (see ‘Materials and methods’ fordetails). (D) Difference between the number of positive andnegative events in (C) for eachΔIntensity value. Thelocal amplitude maxima of the periodic function in (B) and(D) give an estimate of the change in intensity values when1,2,3, …, n number of molecules are bleached. (E)Distribution of the number of molecules of EGFR-GFP and p-EGFR in individualendosomes after stimulation with 10 ng/ml EGF for 10 min.DOI:http://dx.doi.org/10.7554/eLife.06156.017

Mentions: Our data suggest the existence of a saturable mechanism adjusting the amount of p-EGFRin each individual endosome. Such a constant mean amount may be due to the formation ofsmall clusters within early endosomes. To test this possibility, we imaged the spatialdistribution of p-EGFR in endosomes using the anti-EGFR phosphoTyr1068 antibody bysuper-resolution microscopy. Using direct Stochastic Optical Reconstruction Microscopy(dSTORM) (Lampe et al., 2012), we could indeedvisualize clusters of p-EGFR (Figure 2D, leftpanel) that decreased in size between 10 and 30 min of EGF internalization, in agreementwith the narrowing of p-EGFR distribution over time (Figure 1D). To determine the number of molecules in the clusters, we used twomethods. First, we developed a new method to estimate the number of fluorescentmolecules in light microscopy images by measuring the intensity fluctuations duringphoto-bleaching over time (for details see ‘Materials and methods’ andFigure 2—figure supplement 3). Basedon the fluorescence signal from the anti-phosphoTyr1068 antibody and EGFR-GFP, weestimated an average of 102 ± 38 and 76 ± 29 (Mean ± SEM) moleculesof EGFR and p-EGFR per endosome 30 min after EGF (10 ng/ml) internalization (Figure 2—figure supplement 3),corresponding to 707 ± 265 and 527 ± 202 molecules perμm3 of endosomal volume (apparent, assessed by light microscopy),respectively. A hundred EGFR molecules would require ∼12 clathrin-coated vesiclesfor delivery to endosomes (see ‘Materials and methods’). We also estimatedthe total number of GFP-EGFR per cell and found values (29,000) well in agreement withprevious estimates for HeLa cells (see ‘Materials and methods’ and Figure 2—figure supplement 1A,B). Second,based on the size of receptor from the PDB database (structure ID: 3NJP), we calculatedthat 83 ± 25 (Mean ± SEM, N = 1456) receptors could fit in theapparent area of p-EGFR visualized by dSTORM, a value which is remarkably in agreementwith the fluorescence intensities estimates.


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 number of EGFR and pEGFR molecules perendosome.(A) Distribution histogram of the differences in intensitybetween individual endosomes in consecutive frames during sequentialphoto-bleaching of EGFR-GFP (see ‘Materials and methods’ fordetails). (B) Difference between the number of positive andnegative events in (A) for each ΔIntensity value.(C) Distribution histogram of the differences in intensitybetween individual endosomes in consecutive frames during sequentialphoto-bleaching of p-EGFR (see ‘Materials and methods’ fordetails). (D) Difference between the number of positive andnegative events in (C) for eachΔIntensity value. Thelocal amplitude maxima of the periodic function in (B) and(D) give an estimate of the change in intensity values when1,2,3, …, n number of molecules are bleached. (E)Distribution of the number of molecules of EGFR-GFP and p-EGFR in individualendosomes after stimulation with 10 ng/ml EGF for 10 min.DOI:http://dx.doi.org/10.7554/eLife.06156.017
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Related In: Results  -  Collection

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

fig2s3: Quantification of number of EGFR and pEGFR molecules perendosome.(A) Distribution histogram of the differences in intensitybetween individual endosomes in consecutive frames during sequentialphoto-bleaching of EGFR-GFP (see ‘Materials and methods’ fordetails). (B) Difference between the number of positive andnegative events in (A) for each ΔIntensity value.(C) Distribution histogram of the differences in intensitybetween individual endosomes in consecutive frames during sequentialphoto-bleaching of p-EGFR (see ‘Materials and methods’ fordetails). (D) Difference between the number of positive andnegative events in (C) for eachΔIntensity value. Thelocal amplitude maxima of the periodic function in (B) and(D) give an estimate of the change in intensity values when1,2,3, …, n number of molecules are bleached. (E)Distribution of the number of molecules of EGFR-GFP and p-EGFR in individualendosomes after stimulation with 10 ng/ml EGF for 10 min.DOI:http://dx.doi.org/10.7554/eLife.06156.017
Mentions: Our data suggest the existence of a saturable mechanism adjusting the amount of p-EGFRin each individual endosome. Such a constant mean amount may be due to the formation ofsmall clusters within early endosomes. To test this possibility, we imaged the spatialdistribution of p-EGFR in endosomes using the anti-EGFR phosphoTyr1068 antibody bysuper-resolution microscopy. Using direct Stochastic Optical Reconstruction Microscopy(dSTORM) (Lampe et al., 2012), we could indeedvisualize clusters of p-EGFR (Figure 2D, leftpanel) that decreased in size between 10 and 30 min of EGF internalization, in agreementwith the narrowing of p-EGFR distribution over time (Figure 1D). To determine the number of molecules in the clusters, we used twomethods. First, we developed a new method to estimate the number of fluorescentmolecules in light microscopy images by measuring the intensity fluctuations duringphoto-bleaching over time (for details see ‘Materials and methods’ andFigure 2—figure supplement 3). Basedon the fluorescence signal from the anti-phosphoTyr1068 antibody and EGFR-GFP, weestimated an average of 102 ± 38 and 76 ± 29 (Mean ± SEM) moleculesof EGFR and p-EGFR per endosome 30 min after EGF (10 ng/ml) internalization (Figure 2—figure supplement 3),corresponding to 707 ± 265 and 527 ± 202 molecules perμm3 of endosomal volume (apparent, assessed by light microscopy),respectively. A hundred EGFR molecules would require ∼12 clathrin-coated vesiclesfor delivery to endosomes (see ‘Materials and methods’). We also estimatedthe total number of GFP-EGFR per cell and found values (29,000) well in agreement withprevious estimates for HeLa cells (see ‘Materials and methods’ and Figure 2—figure supplement 1A,B). Second,based on the size of receptor from the PDB database (structure ID: 3NJP), we calculatedthat 83 ± 25 (Mean ± SEM, N = 1456) receptors could fit in theapparent area of p-EGFR visualized by dSTORM, a value which is remarkably in agreementwith the fluorescence intensities estimates.

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