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Abstract

Quantitative Fuorescence microscopy is increasingly promoting the understanding of cellular processes on a molecular level. With many of these processes happening on short timescales, low frequencies and in chemical equilibrium, single-molecule techniques provide the necessary resolution and sensitivity to unravel molecular dynamics. To determine protein diffusion as well as protein-protein interactions in live cells, single-molecule tracking is one method of choice. In this study, I have established a two-color single-molecule tracking system to detect and quantify receptor-receptor interactions in the plasma membrane of live cells based on SNAPf-tag and HaloTag labeling. As a proof-of-function, I could verify the well-described ligand-induced heterodimerization of the type I Interferon receptor and determine its interaction dynamics at physiological conditions. In the clinically relevant setting of lung cancer therapy, I could directly proof the prediction of a mathematical model, upon which the direct interaction of epidermal growth factor receptor (EGFR) and hepatocyte growth factor receptor (c-Met) critically tunes cell signaling and sensitivity against tyrosine kinase inhibitors (TKIs) in non-small cell lung cancer (NSCLC) cells. This has led to the proposal of the EGFR/c-Met expression ratio as a relevant biological marker for TKI responsiveness in advanced NSCLC patients. To benchmark the two-color single-molecule tracking system and facilitate the quantifcation of microscopy data in general, I have developed a modular artifcial protein (gSEP) that could be used as a monomer and dimer control in tracking experiments, as well as to determine the degree of labeling (DOL) of protein tags and Fuorescent proteins. Applying 40 different staining conditions, I found that at most 40 % of SNAPf-tags and 50 % of HaloTags could be fuorescently labeled. As the DOL is a crucial, yet hard-to-determine, correction factor for quantitative single-molecule microscopy, the artifcial gSEP protein represents a valuable and versatile tool for the quantifcation of microscopy data within a cellular system and on a single-molecule level.