title: Harnessing molecular motors for nanoscale pulldown in live cells.
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.
authors.affiliation: Laboratory of Molecular Genetics, National Institutes of Health, Bethesda, MD 20814 jonathan.bird@nih.gov.
authors.forename: Jonathan E
authors.initials: JE
authors.lastname: Bird
authors.affiliation: Laboratory of Molecular Genetics, National Institutes of Health, Bethesda, MD 20814.
authors.forename: Melanie
authors.initials: M
authors.lastname: Barzik
authors.affiliation: Laboratory of Molecular Genetics, National Institutes of Health, Bethesda, MD 20814.
authors.forename: Meghan C
authors.initials: MC
authors.lastname: Drummond
authors.affiliation: Laboratory of Molecular Genetics, National Institutes of Health, Bethesda, MD 20814.
authors.forename: Daniel C
authors.initials: DC
authors.lastname: Sutton
authors.affiliation: Laboratory of Molecular Genetics, National Institutes of Health, Bethesda, MD 20814.
authors.forename: Spencer M
authors.initials: SM
authors.lastname: Goodman
authors.affiliation: Laboratory of Molecular Genetics, National Institutes of Health, Bethesda, MD 20814.
authors.forename: Eva L
authors.initials: EL
authors.lastname: Morozko
authors.affiliation: Laboratory of Molecular Genetics, National Institutes of Health, Bethesda, MD 20814.
authors.forename: Stacey M
authors.initials: SM
authors.lastname: Cole
authors.affiliation: Laboratory of Molecular Genetics, National Institutes of Health, Bethesda, MD 20814.
authors.forename: Alexandra K
authors.initials: AK
authors.lastname: Boukhvalova
authors.affiliation: Department of Pediatrics, University of Michigan, Ann Arbor, MI 48109.
authors.forename: Jennifer
authors.initials: J
authors.lastname: Skidmore
authors.affiliation: Department of Pediatrics, University of Michigan, Ann Arbor, MI 48109.
authors.forename: Diana
authors.initials: D
authors.lastname: Syam
authors.affiliation: Laboratory of Molecular Genetics, National Institutes of Health, Bethesda, MD 20814.
authors.forename: Elizabeth A
authors.initials: EA
authors.lastname: Wilson
authors.affiliation: Mouse Auditory Testing Core Facility, National Institute on Deafness and Other Communication Disorders, National Institutes of Health, Bethesda, MD 20814.
authors.forename: Tracy
authors.initials: T
authors.lastname: Fitzgerald
authors.affiliation: Laboratory of Molecular Genetics, National Institutes of Health, Bethesda, MD 20814.
authors.forename: Atteeq U
authors.initials: AU
authors.lastname: Rehman
authors.affiliation: Department of Pediatrics, University of Michigan, Ann Arbor, MI 48109.|Department of Human Genetics, University of Michigan, Ann Arbor, MI 48109.
authors.forename: Donna M
authors.initials: DM
authors.lastname: Martin
authors.affiliation: Laboratory of Molecular Genetics, National Institutes of Health, Bethesda, MD 20814.
authors.forename: Erich T
authors.initials: ET
authors.lastname: Boger
authors.affiliation: Laboratory of Molecular Genetics, National Institutes of Health, Bethesda, MD 20814.
authors.forename: Inna A
authors.initials: IA
authors.lastname: Belyantseva
authors.affiliation: Laboratory of Molecular Genetics, National Institutes of Health, Bethesda, MD 20814.
authors.forename: Thomas B
authors.initials: TB
authors.lastname: Friedman
mesh_terms: D008841:Actin Cytoskeleton / Q000378:metabolism; D002465:Cell Movement; D049452:Green Fluorescent Proteins / Q000378:metabolism; D008856:Microscopy, Fluorescence / Q000379:methods*; D057054:Molecular Imaging / Q000379:methods*; D020409:Molecular Motor Proteins; D009218:Myosins / Q000378:metabolism; D054730:Protein Interaction Domains and Motifs / Q000502:physiology; D021381:Protein Transport; D011554:Pseudopodia / Q000378:metabolism; D059010:Single-Cell Analysis / Q000379:methods*
chemical_list: D020409:Molecular Motor Proteins; D049452:Green Fluorescent Proteins; D009218:Myosins