Subcellular Dynamic Immunopatterning of Cytosolic Protein Complexes on Microstructured Polymer Substrates

[Image: see text] Analysis of protein–protein interactions in living cells by protein micropatterning is currently limited to the spatial arrangement of transmembrane proteins and their corresponding downstream molecules. Here, we present a robust and straightforward method for dynamic immunopatterning of cytosolic protein complexes by use of an artificial transmembrane bait construct in combination with microstructured antibody arrays on cyclic olefin polymer substrates. As a proof, the method was used to characterize Grb2-mediated signaling pathways downstream of the epidermal growth factor receptor (EGFR). Ternary protein complexes (Shc1:Grb2:SOS1 and Grb2:Gab1:PI3K) were identified, and we found that EGFR downstream signaling is based on constitutively bound (Grb2:SOS1 and Grb2:Gab1) as well as on agonist-dependent protein associations with transient interaction properties (Grb2:Shc1 and Grb2:PI3K). Spatiotemporal analysis further revealed significant differences in stability and exchange kinetics of protein interactions. Furthermore, we could show that this approach is well suited to study the efficacy and specificity of SH2 and SH3 protein domain inhibitors in a live cell context. Altogether, this method represents a significant enhancement of quantitative subcellular micropatterning approaches as an alternative to standard biochemical analyses.