School of Physics and Astronomy, University of Minnesota, MN, 55455, United States.
School of Physics and Astronomy, University of Minnesota, MN, 55455, United States.
Anal Biochem. 2019 Oct 1;582:113359. doi: 10.1016/j.ab.2019.113359. Epub 2019 Jul 4.
Analysis of fluorescence fluctuation data through the time-shifted mean-segmented Q (tsMSQ) analysis method has recently been shown to successfully identify protein oligomerization and mobility in the nuclear envelope by properly accounting for local volume fluctuations of the nuclear envelope within living cells. However, by its nature, tsMSQ produces correlated data which poses unique challenges for applying goodness of fit tests and obtaining parameter uncertainties from individual measurements. In this paper, we overcome these challenges by introducing bootstrap tsMSQ which involves randomly resampling the fluorescence intensity data to eliminate the correlations in the tsMSQ data. This analysis technique was verified in both the cytoplasm and the lumen of the nuclear envelope with well-characterized proteins that served as model systems. Uncertainties and goodness of fit tests of individual measurements were compared to estimates obtained from sampling multiple experiments. We further applied bootstrapping to fluorescence fluctuation data of the luminal domain of the SUN domain-containing protein 2 in order to characterize its self-oligomerization within the nuclear envelope. Analysis of the concentration-dependent brightness suggests a monomer-trimer transition of the protein.
通过时间偏移平均分段 Q（tsMSQ）分析方法对荧光波动数据的分析最近已成功地通过正确考虑活细胞内核膜的局部体积波动来识别核膜中的蛋白质寡聚化和迁移。然而，tsMSQ 的本质是产生相关数据，这对应用拟合优度检验和从单个测量中获得参数不确定性提出了独特的挑战。在本文中，我们通过引入自举 tsMSQ 来克服这些挑战，该方法涉及随机重采样荧光强度数据以消除 tsMSQ 数据中的相关性。该分析技术在细胞质和核膜腔中使用特征明确的蛋白质作为模型系统进行了验证。比较了单个测量的不确定性和拟合优度检验与从多个实验中采样获得的估计值。我们进一步将自举应用于含有 SUN 结构域的蛋白 2 内腔域的荧光波动数据，以表征其在核膜内的自寡聚化。对浓度依赖性亮度的分析表明该蛋白存在单体-三聚体转变。
