Two-detector number and brightness analysis reveals spatio-temporal oligomerization of proteins in living cells
Two-detector number and brightness analysis reveals spatio-temporal oligomerization of proteins in living cells
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双检测器数量和亮度分析揭示活细胞中蛋白质的时空寡聚化
DOI：
10.1016/j.ymeth.2018.03.007
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发表时间：
2018
期刊：
影响因子：
4.8
通讯作者：
Masataka Kinjo
中科院分区：
文献类型：
--
作者：
Ryosuke Fukushima;Johtaro Yamamoto;Hideto Ishikawa;Masataka Kinjo
数字和亮度分析（N&B）是一个有用的工具，同时可视化的蛋白质寡聚体和它们的定位，与单分子的灵敏度。N&B测定颗粒亮度（每个颗粒的荧光强度）并通过对使用激光扫描显微镜获得的图像系列进行统计分析来绘制荧光标记蛋白质的空间分布。亮度图揭示了靶蛋白的寡聚体的存在及其在活细胞中的分布。然而，即使在应用校正时，传统N&B也会受到后脉冲、散粒噪声、热噪声、死区时间以及当测量期间荧光颗粒的浓度变化时颗粒亮度的高估的影响。传统N&B的缺点可以通过使用两个检测器来规避，这是一种新颖的方法，我们在下文中称之为双检测器数量和亮度分析（TD-N&B），并引入荧光强度的线性回归。这在统计上消除了来自检测器的噪声的影响，并确保获得正确的粒子亮度。我们的方法进行了理论评估，通过数值模拟和实验验证，使用一系列稀释的纯化增强型绿色荧光蛋白（EGFP），EGFP串联低聚物在细胞裂解液中，和EGFP串联低聚物在活细胞中。此外，这种方法被用来表征复杂的过程中的配体诱导的糖皮质激素受体二聚体和它们的易位到细胞核中的活细胞。我们的方法可以应用于细胞信号传导中的其他寡聚体形成蛋白，或蛋白质的聚集，例如引起神经退行性疾病的蛋白质。
Number and brightness analysis (N&B) is a useful tool for the simultaneous visualization of protein oligomers and their localization, with single-molecule sensitivity. N&B determines particle brightness (fluorescence intensity per particle) and maps the spatial distribution of fluorescently labeled proteins by performing statistical analyses of the image series obtained using laser scanning microscopy. The brightness map reveals presence of the oligomers of the targeted protein and their distribution in living cells. However, even when corrections are applied, conventional N&B is affected by afterpulsing, shot noise, thermal noise, dead time, and overestimation of particle brightness when the concentration of the fluorescent particles changes during measurement.The drawbacks of conventional N&B can be circumvented by using two detectors, a novel approach that we henceforth call two-detector number and brightness analysis (TD-N&B), and introducing a linear regression of fluorescence intensity. This statistically eliminates the effect of noise from the detectors, and ensures that the correct particle brightness is obtained. Our method was theoretically assessed by numerical simulations and experimentally validated using a dilution series of purified enhanced green fluorescent protein (EGFP), EGFP tandem oligomers in cell lysate, and EGFP tandem oligomers in living cells. Furthermore, this method was used to characterize the complex process of ligand-induced glucocorticoid receptor dimerization and their translocation to the cell nucleus in live cells. Our method can be applied to other oligomer-forming proteins in cell signaling, or to aggregations of proteins such as those that cause neurodegenerative diseases.