Location, location, location: controlling growth factor receptor signalling at the endosome

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Growth factor receptors such as EGFR initiate signalling pathways controlling key cellular processes such as growth, differentiation and migration. The activated receptors are rapidly taken up from the cell surface, and then a critical decision is reached in controlling the lifetime of the signalling response: whether the receptor returns to the cell surface so that signalling can continue, or enters the lysosomal pathway and is degraded. The decision to enter recycling or degradative pathways occurs at the endosome, where the receptor will enter neighbouring domains within the endosome that recruit molecular machineries to drive recycling or transit towards the lysosome, respectively. Hence, providing a quantitative measure of how EGFR distributes between these domains under different signalling conditions will be critical for understanding how EGFR fate is determined.This project has two major components:1. Using a range of imaging techniques to determine at nanometer-scale resolution and at a quantitative level the sub-endosomal localisation of EGFR: what proportion enters endosomal domains that drive receptor recycling vs degradation, and how is this balance influenced by EGFR-dependent signalling pathways. Imaging techniques will include:i. Live cell imaging, including fast two-colour imagingii. Conventional epifluorescence imaging combined with using bespoke quantitative colocalisation algorithmsiii. Super-resolution fluorescence microscopy (PALM/STORM), with the prospect of performing it in combination with electron microscopy2. Using biochemical approaches to measure the interaction of EGFR with previously identified switching molecules, and with components of recycling and degradative machineries; understanding how these interactions are controlled by EGFR signalling pathways. This will use state-of-the-art biochemical techniques including mass spectroscopy, and APEX-based proximity probes, to provide dynamic information about the transit of EGFR through the endocytic system and the role of critical switching molecules in determining how each pathway is engaged.