Harnessing molecular motors for nanoscale pulldown in live cells
| Autor: | Melanie Barzik, Elizabeth A. Wilson, Spencer M. Goodman, Jonathan E. Bird, Atteeq U. Rehman, Diana Syam, Meghan C. Drummond, Eva L. Morozko, Inna A. Belyantseva, Stacey M. Cole, Tracy S. Fitzgerald, Jennifer M. Skidmore, Alexandra K. Boukhvalova, Erich T. Boger, Donna M. Martin, Thomas B. Friedman, Daniel C. Sutton |
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| Rok vydání: | 2017 |
| Předmět: |
0301 basic medicine
Green Fluorescent Proteins macromolecular substances Myosins Biology 03 medical and health sciences 0302 clinical medicine Single-cell analysis Cell Movement Methods Fluorescence microscope Molecular motor Protein Interaction Domains and Motifs Pseudopodia Molecular Biology Total internal reflection fluorescence microscope Molecular Motor Proteins Articles Cell Biology Actin cytoskeleton Molecular Imaging Cell biology Actin Cytoskeleton Protein Transport 030104 developmental biology Microscopy Fluorescence Cytoplasm Single-Cell Analysis Filopodia 030217 neurology & neurosurgery |
| Zdroj: | Molecular Biology of the Cell |
| ISSN: | 1939-4586 1059-1524 |
| DOI: | 10.1091/mbc.e16-08-0583 |
| Popis: | Nanoscale pulldown (NanoSPD) miniaturizes the concept of affinity pulldown to detect protein–protein interactions in live cells. NanoSPD hijacks the myosin-based intracellular trafficking machinery to assess interactions under physiological buffer conditions and is microscopy-based, allowing for sensitive detection and quantification. 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. |
| Databáze: | OpenAIRE |
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