School of Chemistry & Chemical Engineering, State Key Laboratory of Metal Matrix Composites, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai, 200240, People's Republic of China.
Methods Appl Fluoresc. 2017 Apr 28;5(2):024008. doi: 10.1088/2050-6120/aa6b07.
PTEN (phosphatase and tensin homolog on chromosome 10) is one of the most important tumor-suppressor proteins, which plays a key role in negative regulation of the PI3K/AKT pathway, and governs many cellular processes including growth, proliferation, survival and migration. The dynamics of PTEN proteins in single living cells is as yet unclear owing to a shortage of suitable in vivo approaches. Here, we report a single-molecule method for in vivo study of the dynamics of PTEN proteins in living cells using fluorescence correlation spectroscopy (FCS). First, we established a monoclonal H1299 stable cell line expressing enhanced green fluorescent protein (EGFP) and PTEN (EGFP-PTEN) fusion proteins; we then developed an in vivo FCS method to study the dynamics of EGFP-PTEN both in the nucleus and the cytoplasm. We investigated the diffusion behaviors of EGFP and EGFP-PTEN in solution, nucleus and cytosol, and observed that the motion of PTEN in living cells was restricted compared with EGFP. Finally, we investigated the protein dynamics in living cells under oxidative stress stimulation and a cellular ATP depletion treatment. Under oxidative stress stimulation, the EGFP-PTEN concentration increased in the nucleus, but slightly decreased in the cytoplasm. The diffusion coefficient and alpha value of EGFP-PTEN reduced significantly both in the nucleus and cytoplasm; the significantly decreased alpha parameter indicates a more restricted Brownian diffusion behavior. Under the cellular ATP depletion treatment, the concentration of EGFP-PTEN remained unchanged in the nucleus and decreased significantly in cytosol. The diffusion coefficient of EGFP-PTEN decreased significantly in cytosol, but showed no significant change in the nucleus; the alpha value decreased significantly in both the nucleus and cytoplasm. These results suggest that the concentration and mobility of PTEN in the nucleus and cytoplasm can be regulated by stimulation methods. Our approach provides a unique method for real-time monitoring of protein dynamics in different subcellular compartments under different stimulation treatments.
PTEN（10 号染色体磷酸酶及张力蛋白同源物）是最重要的肿瘤抑制蛋白之一，在负调控 PI3K/AKT 通路中发挥关键作用，调控包括生长、增殖、存活和迁移在内的多种细胞过程。由于缺乏合适的体内方法，PTEN 蛋白在单个活细胞中的动态尚不清楚。在这里，我们使用荧光相关光谱（FCS）报告了一种用于体内研究活细胞中 PTEN 蛋白动力学的单分子方法。首先，我们建立了表达增强型绿色荧光蛋白（EGFP）和 PTEN（EGFP-PTEN）融合蛋白的 H1299 稳定细胞系；然后开发了一种体内 FCS 方法来研究 EGFP-PTEN 在核和细胞质中的动力学。我们研究了 EGFP 和 EGFP-PTEN 在溶液、核和细胞质中的扩散行为，观察到 PTEN 在活细胞中的运动受到限制。最后，我们研究了氧化应激刺激和细胞内 ATP 耗竭处理下活细胞中的蛋白质动力学。在氧化应激刺激下，EGFP-PTEN 浓度在核中增加，但在细胞质中略有减少。EGFP-PTEN 的扩散系数和 alpha 值在核和细胞质中均显著降低；alpha 值的显著降低表明更受限的布朗扩散行为。在细胞内 ATP 耗竭处理下，EGFP-PTEN 浓度在核中保持不变，而在细胞质中显著减少。EGFP-PTEN 的扩散系数在细胞质中显著降低，但在核中无明显变化；alpha 值在核和细胞质中均显著降低。这些结果表明，PTEN 在核和细胞质中的浓度和迁移率可以通过刺激方法进行调节。我们的方法为不同刺激处理下不同亚细胞区室中蛋白质动力学的实时监测提供了一种独特的方法。
