Regulation of EGFR signal transduction by analogue-to-digital conversion in endosomes.
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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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Affiliation: Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany.
ABSTRACT
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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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Mentions: What are the consequences of such mechanism for signal transduction? To address thesequestions and generate testable predictions, we developed a mathematical model thatdescribes the amount of total intracellular p-EGFR over time. Previously, excellentmodels have been developed that quantitatively describe EGFR endocytosis and signalling(Felder et al., 1992; French et al., 1994; Kholodenkoet al., 1999; Kholodenko, 2002; Resat et al., 2003). However, although all thesemodels described in detail the dynamics of ligand binding, dimer formation andendocytosis, recycling and degradation of the receptor, they did not consider thetrafficking dynamics of the phosphorylated receptors with respect to the dynamics of theendosomal network because these data were not available. Our new experimental databrought two new concepts. First, dephosphorylation and degradation of p-EGFR occursequentially but are uncoupled. Second, the amount of p-EGFR is controlled at the levelof individual endosomes. These new concepts require further development of the existingEGFR mathematical models. Our model was formulated as a set of ordinary differentialequations (ODE, see ‘Materials and methods’ and Figure 3) describing (1) the total amount of EGFR and p-EGFR at theplasma membrane as a function of ligand binding, (2) endocytosis of p-EGFR and itsindirect effects on EGFR endocytosis, and (3) distribution of cargo between earlyendosomes at different stages of maturation (e.g., formation of MVB). For this, weconsidered the processes of receptor internalization, dephosphorylation, degradation,recycling, endosome fusion and fission. As in previous models (French et al., 1994; Resat etal., 2003), we described time course kinetics of total cellular p-EGFR,surface and endosomal EGFR and p-EGFR. Importantly, our model also describes the totalnumber of p-EGFR-positive endosomes and mean amount of p-EGFR per endosome (see‘Materials and methods’ for details). To account for the observedstabilization of the mean amount of p-EGFR per endosome over time (Figure 1), the dependency of p-EGFR dephosphorylation on EGFRkinase activity (Figure 2—figuresupplement 6,7) and the fact that the mechanism is saturable (Figure 1—figure supplement 8), we includeda sigmoidal dependency of the p-EGFR dephosphorylation rate on the amount of p-EGFR perendosome. The model was then fitted to the experimental data from the p-EGFR time course(Figure 3A,B). Figure 3C shows that this simple theoretical model can reproduce ourobservations of a constant mean amount of p-EGFR per endosome in a wide range of EGFconcentrations when fitted to the experimental data. Importantly, a model without thisnon-linear dephosphorylation dependency could correctly describe the total amount ofEGFR and p-EGFR in endosomes (Figure 3—figuresupplement 1A,B) but did not agree with the measurements for the mean amountof p-EGFR per endosome (Figure 3—figuresupplement 1C), thus supporting the sigmoidal dependency of the p-EGFRde-phosphorylation rate on the amount of p-EGFR per endosome (Figure 3). Previous models did not include this non-linear termbecause data on the distribution of p-EGFR in individual endosomes was not available.10.7554/eLife.06156.023Figure 3.Mathematical model of p-EGFR predicts signalling amplitude and durationdepends on early endosome fusion/fission rate. |
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Affiliation: Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany.