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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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EGFR and p-EGFR measurements by FRET microscopy.(A) Representative images of HeLa EGFR-GFP BAC cells aftercontinuous stimulation with 10 ng/ml EGF for the indicated time points.EGFR-GFP fluorescence is shown in green, the corrected p-EGFR intensity isshown in red, and DAPI-stained nuclei are shown in blue. Measurements fromeach individual endosome were used for all quantifications. Scale bars, 10μm. (B–C) Time course of histogramdistributions of the total p-EGFR (B) or EGFR (C)integral intensity per endosome upon EGF stimulation as in Figure 1. The histogram shows the numberof vesicles per 1000 μm2 of the area covered by cells.Intensity values were scaled by the mode of the histogram at 10 min. In allgraphs, experimental points were fitted with a log-normal distribution.Points show the mean from three independent experiments with a total of∼150 cells per time point or condition.DOI:http://dx.doi.org/10.7554/eLife.06156.006
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fig1s2: EGFR and p-EGFR measurements by FRET microscopy.(A) Representative images of HeLa EGFR-GFP BAC cells aftercontinuous stimulation with 10 ng/ml EGF for the indicated time points.EGFR-GFP fluorescence is shown in green, the corrected p-EGFR intensity isshown in red, and DAPI-stained nuclei are shown in blue. Measurements fromeach individual endosome were used for all quantifications. Scale bars, 10μm. (B–C) Time course of histogramdistributions of the total p-EGFR (B) or EGFR (C)integral intensity per endosome upon EGF stimulation as in Figure 1. The histogram shows the numberof vesicles per 1000 μm2 of the area covered by cells.Intensity values were scaled by the mode of the histogram at 10 min. In allgraphs, experimental points were fitted with a log-normal distribution.Points show the mean from three independent experiments with a total of∼150 cells per time point or condition.DOI:http://dx.doi.org/10.7554/eLife.06156.006

Mentions: To measure the content of p-EGFR in individual endosomes, we used two independent assays(for a detailed description see ‘Materials and methods’ and Figure 1—figure supplement 1). First, wemodified a FRET-FLIM microscopy assay previously used to measure the spatialdistribution of p-EGFR at the plasma membrane (Woutersand Bastiaens, 1999; Verveer et al.,2000). The assay measured the FRET signal between EGFR-GFP and ananti-phospho-tyrosine antibody (p-Tyr-ab) labelled with AlexaFluor 555. Since FLIMmicroscopy lacks the spatial resolution to analyse the receptor activation at asub-cellular level, we modified the assay into a high-resolution FRET microscopy assay.However, instead of the total cell signal, we measured the distribution of EGFR andp-EGFR at the level of individual endosomes resolved by high-resolution confocalmicroscopy and quantitative automated image analysis (Rink et al., 2005; Collinet et al.,2010) (Figure 1—figure supplement2). To avoid artefacts of overexpression, we used HeLa cells transfected witha bacterial artificial chromosome (BAC) transgene stably expressing EGFR-GFP under itsendogenous promoter (Poser et al., 2008). Inthese cells (Figure 1—figure supplement3A), the uptake of EGF was only ∼twofold higher compared to endogenous(Figure 1—figure supplement 3B).However, the transport kinetics were similar (Figure1—figure supplement 3C). Second, we measured p-EGFR with an antibodyagainst a specific phospho-tyrosine residue (Tyr1068). Both assays gave very similarresults (Figure 1—figure supplement 4).As the FRET assay is not restricted to a single phosphorylation site that can changeover time (Morandell et al., 2008), we used itas a primary assay in further experiments. Under the fixation conditions used, weobserved no significant difference in the morphology (Figure 1—figure supplement 5A) or area (Figure 1—figure supplement 5B) of EGFR-positive endosomes(Video 1). For every time point,∼15,000 endosomes from over 200 cells were analysed.Video 1.Live-cell imaging of EGFR endocytosis.


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)

EGFR and p-EGFR measurements by FRET microscopy.(A) Representative images of HeLa EGFR-GFP BAC cells aftercontinuous stimulation with 10 ng/ml EGF for the indicated time points.EGFR-GFP fluorescence is shown in green, the corrected p-EGFR intensity isshown in red, and DAPI-stained nuclei are shown in blue. Measurements fromeach individual endosome were used for all quantifications. Scale bars, 10μm. (B–C) Time course of histogramdistributions of the total p-EGFR (B) or EGFR (C)integral intensity per endosome upon EGF stimulation as in Figure 1. The histogram shows the numberof vesicles per 1000 μm2 of the area covered by cells.Intensity values were scaled by the mode of the histogram at 10 min. In allgraphs, experimental points were fitted with a log-normal distribution.Points show the mean from three independent experiments with a total of∼150 cells per time point or condition.DOI:http://dx.doi.org/10.7554/eLife.06156.006
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4384751&req=5

fig1s2: EGFR and p-EGFR measurements by FRET microscopy.(A) Representative images of HeLa EGFR-GFP BAC cells aftercontinuous stimulation with 10 ng/ml EGF for the indicated time points.EGFR-GFP fluorescence is shown in green, the corrected p-EGFR intensity isshown in red, and DAPI-stained nuclei are shown in blue. Measurements fromeach individual endosome were used for all quantifications. Scale bars, 10μm. (B–C) Time course of histogramdistributions of the total p-EGFR (B) or EGFR (C)integral intensity per endosome upon EGF stimulation as in Figure 1. The histogram shows the numberof vesicles per 1000 μm2 of the area covered by cells.Intensity values were scaled by the mode of the histogram at 10 min. In allgraphs, experimental points were fitted with a log-normal distribution.Points show the mean from three independent experiments with a total of∼150 cells per time point or condition.DOI:http://dx.doi.org/10.7554/eLife.06156.006
Mentions: To measure the content of p-EGFR in individual endosomes, we used two independent assays(for a detailed description see ‘Materials and methods’ and Figure 1—figure supplement 1). First, wemodified a FRET-FLIM microscopy assay previously used to measure the spatialdistribution of p-EGFR at the plasma membrane (Woutersand Bastiaens, 1999; Verveer et al.,2000). The assay measured the FRET signal between EGFR-GFP and ananti-phospho-tyrosine antibody (p-Tyr-ab) labelled with AlexaFluor 555. Since FLIMmicroscopy lacks the spatial resolution to analyse the receptor activation at asub-cellular level, we modified the assay into a high-resolution FRET microscopy assay.However, instead of the total cell signal, we measured the distribution of EGFR andp-EGFR at the level of individual endosomes resolved by high-resolution confocalmicroscopy and quantitative automated image analysis (Rink et al., 2005; Collinet et al.,2010) (Figure 1—figure supplement2). To avoid artefacts of overexpression, we used HeLa cells transfected witha bacterial artificial chromosome (BAC) transgene stably expressing EGFR-GFP under itsendogenous promoter (Poser et al., 2008). Inthese cells (Figure 1—figure supplement3A), the uptake of EGF was only ∼twofold higher compared to endogenous(Figure 1—figure supplement 3B).However, the transport kinetics were similar (Figure1—figure supplement 3C). Second, we measured p-EGFR with an antibodyagainst a specific phospho-tyrosine residue (Tyr1068). Both assays gave very similarresults (Figure 1—figure supplement 4).As the FRET assay is not restricted to a single phosphorylation site that can changeover time (Morandell et al., 2008), we used itas a primary assay in further experiments. Under the fixation conditions used, weobserved no significant difference in the morphology (Figure 1—figure supplement 5A) or area (Figure 1—figure supplement 5B) of EGFR-positive endosomes(Video 1). For every time point,∼15,000 endosomes from over 200 cells were analysed.Video 1.Live-cell imaging of EGFR endocytosis.

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