Harnessing molecular motors for nanoscale pulldown in live cells
Harnessing molecular motors for nanoscale pulldown in live cells
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DOI：
10.1091/mbc.e16-08-0583
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发表时间：
2017-02-01
影响因子：
3.3
通讯作者：
Friedman, Thomas B.
中科院分区：
文献类型：
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作者：
Bird, Jonathan E.;Barzik, Melanie;Friedman, Thomas B.
蛋白质-蛋白质相互作用（PPIs）调节大分子复合物的组装，但在其通常发挥生物学作用的天然细胞质中进行研究仍然具有挑战性。在这里，我们将亲和下拉（一种金标准的体外PPI询问技术）的概念小型化，以在活细胞内执行纳米级下拉（NanoSPDs）。NanoSPD通过肌凝蛋白马达劫持细胞内运输的正常过程，强行沿着丝状肌动蛋白丝拉荧光标记的蛋白质复合物。使用双色全内反射荧光显微镜，我们通过显示诱饵和猎物分子同时被运输并积极集中到丝状足尖端的纳米级体积来展示复杂的形成。在丝状尖端产生的分子交通堵塞放大了荧光强度，并允许使用标准的荧光显微镜对ppi进行询问。提供了一个严格的量化框架和软件工具来统计评估NanoSPD数据集。除了使用结构域作图和诱变实验解剖这些相互作用外，我们还展示了NanoSPD对与人类耳聋有关的一系列核和细胞质PPIs的能力。除了蛋白质外，NanoSPD方法还可扩展用于其他荧光分子，并且该平台可以轻松扩展以用于高通量应用。
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.