Harnessing molecular motors for nanoscale pulldown in live cells.

Autor:	Bird JE; Laboratory of Molecular Genetics, National Institutes of Health, Bethesda, MD 20814 jonathan.bird@nih.gov., Barzik M; Laboratory of Molecular Genetics, National Institutes of Health, Bethesda, MD 20814., Drummond MC; Laboratory of Molecular Genetics, National Institutes of Health, Bethesda, MD 20814., Sutton DC; Laboratory of Molecular Genetics, National Institutes of Health, Bethesda, MD 20814., Goodman SM; Laboratory of Molecular Genetics, National Institutes of Health, Bethesda, MD 20814., Morozko EL; Laboratory of Molecular Genetics, National Institutes of Health, Bethesda, MD 20814., Cole SM; Laboratory of Molecular Genetics, National Institutes of Health, Bethesda, MD 20814., Boukhvalova AK; Laboratory of Molecular Genetics, National Institutes of Health, Bethesda, MD 20814., Skidmore J; Department of Pediatrics, University of Michigan, Ann Arbor, MI 48109., Syam D; Department of Pediatrics, University of Michigan, Ann Arbor, MI 48109., Wilson EA; Laboratory of Molecular Genetics, National Institutes of Health, Bethesda, MD 20814., Fitzgerald T; Mouse Auditory Testing Core Facility, National Institute on Deafness and Other Communication Disorders, National Institutes of Health, Bethesda, MD 20814., Rehman AU; Laboratory of Molecular Genetics, National Institutes of Health, Bethesda, MD 20814., Martin DM; Department of Pediatrics, University of Michigan, Ann Arbor, MI 48109.; Department of Human Genetics, University of Michigan, Ann Arbor, MI 48109., Boger ET; Laboratory of Molecular Genetics, National Institutes of Health, Bethesda, MD 20814., Belyantseva IA; Laboratory of Molecular Genetics, National Institutes of Health, Bethesda, MD 20814., Friedman TB; Laboratory of Molecular Genetics, National Institutes of Health, Bethesda, MD 20814.
Jazyk:	angličtina
Zdroj:	Molecular biology of the cell [Mol Biol Cell] 2017 Feb 01; Vol. 28 (3), pp. 463-475. Date of Electronic Publication: 2016 Dec 08.
DOI:	10.1091/mbc.E16-08-0583
Abstrakt:	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. (© 2017 Bird, Barzik, et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).)
Databáze:	MEDLINE
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