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bioRxiv · 10.1101/053744

Harnessing Molecular Motors for Nanoscale Pulldown in Live Cells

Jonathan E Bird·Melanie Barzik·Meghan Drummond·Daniel C Sutton·Spencer M Goodman·Eva L Morozko·Stacey M Cole·Jennifer Skidmore·Diana Syam·Elizabeth A Wilson·Tracy Fitzgerald·Atteeq U Rehman·Donna M Martin·Erich T Boger·Inna A Belyantseva·Thomas B Friedman
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Abstract

Protein-protein interactions (PPIs) regulate signal transduction and cellular behavior, yet studying PPIs within live cells remains fundamentally challenging. We have miniaturized the affinity pulldown, a gold-standard PPI interrogation technique, for use within live cells. Our assay hijacks endogenous myosin motors to forcibly traffic, or pulldown, macromolecular complexes within the native cytosolic environment. Macromolecules captured by nanoscale pulldown (NanoSPD) are optically interrogated in situ by tagging individual protein components. Critically, continuous motor trafficking concentrates query complexes into nanoscopic subcellular compartments, providing fluorescence enhancement and allowing nanoscale pulldowns to be visualized and quantified by standard microscopy. Nanoscale pulldown is compatible with nuclear, membrane-associated and cytoplasmic proteins and can investigate functional effects of protein truncations or amino acid substitutions. Moreover, binding hierarchies in larger complexes can be quickly examined within the natural cytosol, making nanoscale pulldown a powerful new optical platform for quantitative high-content screening of known and novel PPIs that act within macromolecular assemblies.

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Jonathan E Bird, Melanie Barzik, Meghan Drummond, Daniel C Sutton, Spencer M Goodman, Eva L Morozko, Stacey M Cole, Jennifer Skidmore, Diana Syam, Elizabeth A Wilson, Tracy Fitzgerald, Atteeq U Rehman, Donna M Martin, Erich T Boger, Inna A Belyantseva, Thomas B Friedman. 2016-06-07. Harnessing Molecular Motors for Nanoscale Pulldown in Live Cells. https://doi.org/10.1101/053744

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