A visual immunoprecipitation assay for live-cell profiling of cytosolic protein complexes on micropatterned substrates

Analysis of protein-protein interactions in living cells by protein micropatterning is currently limited to the spatial arrangement of transmembrane proteins (‘bait’, e.g. receptor) and their corresponding downstream adaptor molecules (‘prey’). Here we present a method for visual immunoprecipitation of cytosolic protein complexes by use of an artificial transmembrane bait construct in combination with micropatterned antibody arrays on cyclic olefin polymer (COP) substrates. The method was used to characterize initial signalling complexes of the Ras-Raf-MEK and PI3K-Akt pathway downstream the epidermal growth factor receptor (EGFR) on a single cell level. We found prominent differences in EGFR-mediated cytosolic protein complex formation. Interaction dynamics of cytosolic effector proteins were unambiguously quantified by fluorescence recovery after photobleaching as well as by the use of Grb2-related protein domain inhibitors. Furthermore, implementation of specific Grb2-mutants confirmed the role of SH2/SH3 domains in controlling binding events with a variety of adaptor molecules. In general, the assay sheds light on the importance of in-depth characterization of cytosolic protein-protein interactions as regulatory mechanisms for coordinated cellular downstream signalling.