Abstract
Protein micropatterning is a powerful tool for spatial arrangement of transmembrane and intracellular proteins in living cells. The restriction of one interaction partner (the bait, e.g., the receptor) in regular micropatterns within the plasma membrane and the monitoring of the lateral distribution of the bait’s interaction partner (the prey, e.g., the cytosolic downstream molecule) enables the in-depth examination of protein-protein interactions in a live cell context. This study reports on potential pitfalls and difficulties in data interpretation based on the enrichment of clathrin, which is a protein essential for clathrin-mediated receptor endocytosis. Using a highly modular micropatterning approach based on large-area micro-contact printing and streptavidin-biotin-mediated surface functionalization, clathrin was found to form internalization hotspots within the patterned areas, which, potentially, leads to unspecific bait/prey protein co-recruitment. We discuss the consequences of clathrin-coated pit formation on the quantitative analysis of relevant protein-protein interactions, describe controls and strategies to prevent the misinterpretation of data, and show that the use of DNA-based linker systems can lead to the improvement of the technical platform.
Highlights
Simple and robust quantitation of protein-protein interactions (PPIs) in living cells remains challenging
We recently investigated the downstream signaling events of different receptor families, including G protein-coupled receptors (GPCRs) such as the β2-adrenergic receptor [15], tyrosine kinase receptors (TKRs) such as the insulin-like growth factor (IGF), insulin receptor [16], and epidermal growth factor receptor (EGFR) [12,17]
Protein micropatterning in combination with single-cell analysis has evolved into a powerful tool for basic and advanced research, as proteins and related interactions, as well as signal transduction processes, can be investigated in a native protein and lipid environment and in a live cell context
Summary
Simple and robust quantitation of protein-protein interactions (PPIs) in living cells remains challenging. Current biophysical techniques for quantitative protein interaction analysis in live cells include fluorescence resonance energy transfer (FRET) [5], fluorescence lifetime imaging (FLIM) [6], bimolecular fluorescence complementation (BiFC) [7], fluorescence cross-correlation spectroscopy (FCS) [8], and single molecule techniques [9]. Most of these techniques require special equipment and are costly, laborious, and rather demanding in living cells. We and others have used protein micropatterning on solid substrates to induce the reorganization of target proteins distributed in the plasma membrane of living cells for the investigation of distinctive PPIs [10,11,12,13,14,15,16,17,18,19,20,21]
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