%0 Generic
%A Hänselmann, Siegfried
%D 2018
%F heidok:23619
%R 10.11588/heidok.00023619
%T Quantitative Microscopy:  Measuring membrane  receptor interactions in live  cells
%U https://archiv.ub.uni-heidelberg.de/volltextserver/23619/
%X 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.