Multiscale Research Institute of Complex Systems, Fudan University, Shanghai 200433, China.
Department of Chemistry, University of California, Berkeley, CA 94720.
Proc Natl Acad Sci U S A. 2021 Feb 2;118(5). doi: 10.1073/pnas.2012229118.
Multicolor single-molecule tracking (SMT) provides a powerful tool to mechanistically probe molecular interactions in living cells. However, because of the limitations in the optical and chemical properties of currently available fluorophores and the multiprotein labeling strategies, intracellular multicolor SMT remains challenging for general research studies. Here, we introduce a practical method employing a nanopore-electroporation (NanoEP) technique to deliver multiple organic dye-labeled proteins into living cells for imaging. It can be easily expanded to three channels in commercial microscopes or be combined with other in situ labeling methods. Utilizing NanoEP, we demonstrate three-color SMT for both cytosolic and membrane proteins. Specifically, we simultaneously monitored single-molecule events downstream of EGFR signaling pathways in living cells. The results provide detailed resolution of the spatial localization and dynamics of Grb2 and SOS recruitment to activated EGFR along with the resultant Ras activation.
多色单分子追踪 (SMT) 为在活细胞中机械地探测分子相互作用提供了有力的工具。然而，由于目前可用的荧光染料的光学和化学性质以及多蛋白标记策略的限制，细胞内多色 SMT 对于一般的研究仍然具有挑战性。在这里，我们介绍了一种实用的方法，该方法采用纳米孔电穿孔 (NanoEP) 技术将多个有机染料标记的蛋白质递送到活细胞中进行成像。它可以很容易地在商业显微镜中扩展到三个通道，或者与其他原位标记方法结合使用。利用 NanoEP，我们展示了用于细胞质和膜蛋白的三色 SMT。具体来说，我们同时监测了活细胞中 EGFR 信号通路下游的单分子事件。结果提供了 Grb2 和 SOS 募集到激活的 EGFR 以及由此产生的 Ras 激活的空间定位和动力学的详细分辨率。
