Abstract
Protein-protein interactions (PPIs) regulate assembly of macromolecular complexes, yet remain challenging to study within the native cytoplasm where they normally exert their biological effect. Here we miniaturize the concept of affinity pulldown, a gold-standard in vitro PPI interrogation technique, to perform nanoscale pulldowns (NanoSPDs) within living cells. NanoSPD hijacks the normal process of intracellular trafficking by myosin motors to forcibly pull fluorescently tagged protein complexes along filopodial actin filaments. Using dual-color total internal reflection fluorescence microscopy, we demonstrate complex formation by showing that bait and prey molecules are simultaneously trafficked and actively concentrated into a nanoscopic volume at the tips of filopodia. The resulting molecular traffic jams at filopodial tips amplify fluorescence intensities and allow PPIs to be interrogated using standard epifluorescence microscopy. A rigorous quantification framework and software tool are provided to statistically evaluate NanoSPD data sets. We demonstrate the capabilities of NanoSPD for a range of nuclear and cytoplasmic PPIs implicated in human deafness, in addition to dissecting these interactions using domain mapping and mutagenesis experiments. The NanoSPD methodology is extensible for use with other fluorescent molecules, in addition to proteins, and the platform can be easily scaled for high-throughput applications.