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

生长因子受体如EGFR启动信号通路，控制关键的细胞过程，如生长、分化和迁移。激活的受体被迅速从细胞表面带走，然后在控制信号反应的寿命方面做出关键决定：受体是返回细胞表面以使信号继续，还是进入溶酶体途径并被降解。进入循环或降解途径的决定发生在内小体，在那里受体将进入内小体内的相邻区域，这些区域招募分子机械来分别推动循环或向溶酶体转移。因此，提供EGFR在不同信号条件下如何在这些结构域之间分布的定量测量对于了解EGFR命运是如何决定的至关重要。这个项目有两个主要组成部分：1.使用一系列成像技术在纳米级分辨率和定量水平上确定EGFR的亚内体定位：有多大比例进入内体结构域，驱动受体循环和降解，以及这种平衡是如何受到依赖于EGFR的信号通路的影响。成像技术将包括：i.活细胞成像，包括快速双色成像。传统的荧光成像结合使用定制的定量共定位算法。超分辨率荧光显微镜(Palm/STORM)，并有可能与电子显微镜结合使用。使用生化方法测量EGFR与先前确定的开关分子以及与回收和降解机械的组件的相互作用；了解这些相互作用如何由EGFR信号通路控制。这将使用最先进的生化技术，包括质谱学和基于APEX的接近探针，以提供关于EGFR通过内吞系统的运输以及关键开关分子在决定每条途径如何参与的过程中的作用的动态信息。

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.