RT Journal Article
SR Electronic
T1 Harnessing Molecular Motors for Nanoscale Pulldown in Live Cells
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 053744
DO 10.1101/053744
A1 Jonathan E. Bird
A1 Melanie Barzik
A1 Meghan C. Drummond
A1 Daniel C. Sutton
A1 Spencer M. Goodman
A1 Eva L. Morozko
A1 Stacey M. Cole
A1 Jennifer Skidmore
A1 Diana Syam
A1 Elizabeth A. Wilson
A1 Tracy Fitzgerald
A1 Atteeq U. Rehman
A1 Donna M. Martin
A1 Erich T. Boger
A1 Inna A. Belyantseva
A1 Thomas B. Friedman
YR 2016
UL http://biorxiv.org/content/early/2016/06/06/053744.abstract
AB 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.