Mapping eGFP oligomer mobility in living cell nuclei.
Mapping eGFP oligomer mobility in living cell nuclei.
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DOI：
10.1371/journal.pone.0005041
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发表时间：
2009
期刊：
影响因子：
3.7
通讯作者：
Langowski J
中科院分区：
文献类型：
--
作者：
Dross N;Spriet C;Zwerger M;Müller G;Waldeck W;Langowski J
细胞核中颗粒的运动可能受到粘度、定向流动、主动运输或染色质网络等障碍的影响。在这里，我们研究小荧光蛋白的迁移是否受到染色质密度的影响。利用荧光相关光谱（FCS）和双色共聚焦扫描检测系统研究了惰性荧光蛋白在活细胞核中的扩散。我们首先展示了在活细胞研究中遇到的fcs特异性伪像的实验，以及防止它们的策略，特别是那些因选择用于校准焦体积的荧光团而产生的伪像，以及用于荧光波动测量的温度和采集条件。在确定最佳获取条件后，我们展示了各种人类细胞系GFP的流动性在细胞核内显著变化，但与染色质密度无关。核内扩散迁移率很大程度上取决于蛋白质的大小：在一系列gfp低聚物中，作为游离惰性荧光示踪剂，扩散系数从单体到四聚体的下降幅度远远超过水溶液中游离分子的预期。尽管如此，无论染色质密度或细胞系如何，整个核内染色质网络都可以自由地接触到egfp四聚体大小的小蛋白质。即使是最密集的染色质区域也不排除游离的egfp单体或多聚体。
Movement of particles in cell nuclei can be affected by viscosity, directed flows, active transport, or the presence of obstacles such as the chromatin network. Here we investigate whether the mobility of small fluorescent proteins is affected by the chromatin density. Diffusion of inert fluorescent proteins was studied in living cell nuclei using fluorescence correlation spectroscopy (FCS) with a two-color confocal scanning detection system. We first present experiments exposing FCS-specific artifacts encountered in live cell studies as well as strategies to prevent them, in particular those arising from the choice of the fluorophore used for calibration of the focal volume, as well as temperature and acquisition conditions used for fluorescence fluctuation measurements. After defining the best acquisition conditions, we show for various human cell lines that the mobility of GFP varies significantly within the cell nucleus, but does not correlate with chromatin density. The intranuclear diffusional mobility strongly depends on protein size: in a series of GFP-oligomers, used as free inert fluorescent tracers, the diffusion coefficient decreased from the monomer to the tetramer much more than expected for molecules free in aqueous solution. Still, the entire intranuclear chromatin network is freely accessible for small proteins up to the size of eGFP-tetramers, regardless of the chromatin density or cell line. Even the densest chromatin regions do not exclude free eGFP-monomers or multimers.
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