Hong Yi-Ru, Lam Chak Hin, Tan Kui-Thong
Department of Chemistry and ‡Frontier Research Center on Fundamental and Applied Sciences of Matters, National Tsing Hua University , 101 Sec. 2, Kuang Fu Rd, Hsinchu 30013, Taiwan (ROC).
Bioconjug Chem. 2017 Nov 15;28(11):2895-2902. doi: 10.1021/acs.bioconjchem.7b00611. Epub 2017 Nov 3.
Although many protein labeling probes have been developed to elucidate the trafficking and turnover processes of cell surface proteins, real-time tracking of intracellular proteins remains a challenging task. Herein, we describe a new design to construct a cell-permeable, photostable, and far-red fluorescent turn-on probe to enable no-wash, organelle-specific, and long-term visualization of intracellular SNAP-tagged proteins in living cells. When the probe was used in dual-color pulse chase labeling experiments to differentiate between preLamin and mature Lamin, our results reveal that the shape of mature Lamin can be altered by the newly synthesized preLamin and that this alteration is progressive, cumulative, and due to a concentration-dependent dominant-negative effect of preLamin. We believe that this probe can also be applied to other intracellular proteins whose cellular localization and synthesis changes dynamically in response to external stimuli.
尽管已经开发了许多蛋白质标记探针来阐明细胞表面蛋白的运输和周转过程，但对细胞内蛋白质进行实时追踪仍然是一项具有挑战性的任务。在此，我们描述了一种新的设计，用于构建一种可穿透细胞、光稳定且远红光荧光开启型探针，以实现对活细胞中细胞内SNAP标签蛋白的免洗、细胞器特异性和长期可视化。当该探针用于双色脉冲追踪标记实验以区分前层粘连蛋白和成熟层粘连蛋白时，我们的结果表明，新合成的前层粘连蛋白可改变成熟层粘连蛋白的形状，并且这种改变是渐进的、累积的，并且是由于前层粘连蛋白浓度依赖性的显性负效应所致。我们相信，该探针也可应用于其他细胞内蛋白质，其细胞定位和合成会根据外部刺激而动态变化。
