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

生长因子受体（如EGFR）启动控制关键细胞过程（如生长、分化和迁移）的信号通路。被激活的受体迅速从细胞表面被吸收，然后在控制信号反应的生命周期中做出一个关键的决定：受体是返回细胞表面使信号传导继续，还是进入溶酶体途径并被降解。进入再循环或降解途径的决定发生在核内体，受体将进入核内体内的邻近区域，分别招募分子机制来驱动再循环或转运到溶酶体。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理解EGFR的命运是如何决定的至关重要。本项目有两个主要组成部分：1。使用一系列成像技术在纳米尺度分辨率和定量水平上确定EGFR的亚内体定位：进入驱动受体再循环与降解的内体结构域的比例，以及这种平衡如何受到EGFR依赖的信号通路的影响。成像技术将包括：1。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），与电子显微镜结合使用的前景2。使用生化方法测量EGFR与先前确定的开关分子的相互作用，以及与回收和降解机器的组件；了解这些相互作用是如何由EGFR信号通路控制的。这将使用最先进的生化技术，包括质谱和基于apex的接近探针，提供关于EGFR通过内吞系统转运的动态信息，以及关键开关分子在确定每个途径如何参与中的作用。

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.