Department of Life Sciences, Ben-Gurion University of the Negev, Beer Sheva 84105, Israel.
Nanoscale. 2020 Feb 6;12(5):3236-3248. doi: 10.1039/c9nr08594g.
Tracking the localization and mobility of individual proteins in live cells is key for understanding how they mediate their function. Such information can be obtained from single molecule imaging techniques including as Single Particle Tracking (SPT) and Single Molecule Localization Microscopy (SMLM). Genetic code expansion (GCE) combined with bioorthogonal chemistry offers an elegant approach for direct labeling of proteins with fluorescent dyes, holding great potential for improving protein labeling in single molecule applications. Here we calibrated conditions for performing SPT and live-SMLM of bioorthogonally labeled plasma membrane proteins in live mammalian cells. Using SPT, the diffusion of bioorthogonally labeled EGF receptor and the prototypical Shaker voltage-activated potassium channel (Kv) was measured and characterized. Applying live-SMLM to bioorthogonally labeled Shaker Kv channels enabled visualizing the plasma membrane distribution of the channel over time with ∼30 nm accuracy. Finally, by competitive labeling with two Fl-dyes, SPT and live-SMLM were performed in a single cell and both the density and dynamics of the EGF receptor were measured at single molecule resolution in subregions of the cell. We conclude that GCE and bioorthogonal chemistry is a highly suitable, flexible approach for protein labeling in quantitative single molecule applications that outperforms current protein live-cell labeling approaches.
追踪活细胞中单个蛋白质的定位和迁移对于理解它们如何介导其功能至关重要。这种信息可以通过单分子成像技术获得，包括单颗粒跟踪（SPT）和单分子定位显微镜（SMLM）。遗传密码扩展（GCE）与生物正交化学相结合，为用荧光染料直接标记蛋白质提供了一种优雅的方法，在单分子应用中提高蛋白质标记的潜力巨大。在这里，我们对活哺乳动物细胞中生物正交标记的质膜蛋白进行 SPT 和活 SMLM 的条件进行了校准。使用 SPT 测量和表征了生物正交标记的表皮生长因子受体（EGFR）和原型 Shaker 电压激活钾通道（Kv）的扩散。将活 SMLM 应用于生物正交标记的 Shaker Kv 通道，能够以约 30nm 的精度实时可视化通道在质膜上的分布。最后，通过与两种 Fl-染料的竞争性标记，在单个细胞中进行了 SPT 和活 SMLM，并在细胞的亚区以单分子分辨率测量了 EGF 受体的密度和动力学。我们得出结论，GCE 和生物正交化学是一种高度合适、灵活的定量单分子应用中的蛋白质标记方法，优于当前的蛋白质活细胞标记方法。
