Single-molecule tracking tools to study plasma membrane receptor dynamics

Abstract

In this thesis we developed tools to utilize single-molecule tracking microscopy to study the signalling mechanism of the ErbB family of receptors, and in particular of the epidermal growth factor receptor (EGFR). This receptor family is prototypical of receptor tyrosine kinases (RTK) and is implicated in the development and progression of various human cancers, and therefore form attractive targets for drug discovery. We related the diffusion speed of EGFR to its dimerization state, which is an essential step in its central role of transducing extracellular signals into cellular outcomes. Malignancies therein and drug-induced dimerizations are therefore important research topics for therapeutic application. Our single-molecule tracking data of EGFR and ErbB3 revealed particular molecular interactions in tumour cells, and detected altered behaviour when drugs are applied which are especially designed to prevent such molecular interactions. Before we were able to record trajectories of EGFR, and analyse these in term of dimerization and interactions with other cellular structures upon addition of ligand and antagonists (chapter 5), we realized a microscope for this purpose (chapter 2), devised a framework to analyse trajectories in term of different diffusion populations (chapter 3), and further advanced an existing protein labelling system – SNAP-tag – to the single molecule level (chapter 4). The research described in this thesis offers desirable advancements to this proteintag labelling system for application in single-molecule imaging and tracking, and to the postmeasurement analysis of protein trajectories recorded by introducing a classification framework for multiple populations of diffusion, to be able to investigate dynamic protein motion and interactions in live cells. We concluded our thesis with a proof of principle that locally induced stimulation of receptor by ligand functionalized AFM tips can provide additional insight in molecular interactions when combined with single-molecule tracking (chapter 6).