In vivo pair correlation analysis of EGFP intranuclear diffusion reveals DNA-dependent molecular flow
In vivo pair correlation analysis of EGFP intranuclear diffusion reveals DNA-dependent molecular flow
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DOI：
10.1073/pnas.1006731107
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发表时间：
2010-09-21
影响因子：
11.1
通讯作者：
Gratton, Enrico
中科院分区：
文献类型：
--
作者：
Hinde, Elizabeth;Cardarelli, Francesco;Gratton, Enrico
迄今为止，还没有提出的方法能够测量活细胞核中的总体分子流。在这里，我们应用对相关函数分析（pCF）来测量分子各向异性扩散的间期核的活细胞。在pCF方法中，我们在核内的几个距离和位置交叉关联波动，使我们能够定义迁移路径和扩散障碍。我们使用单体EGFP作为原型惰性分子，并测量不同核环境中和之间的流动。我们的研究结果表明，有两个断开的分子流在整个细胞核与高和低DNA密度区。我们发现，DNA的不同密度区域形成了一个网络通道，允许EGFP自由扩散，但穿越通道的能力有限。我们还观察到罕见的和突然爆发的分子穿越DNA密度区域的特征时间约为300毫秒，这表明内在的局部变化的染色质结构。这是一个独特的复杂的染色质网络显示通道定向扩散的惰性分子与高空间和时间分辨率的体内演示。
No methods proposed thus far have the capability to measure overall molecular flow in the nucleus of living cells. Here, we apply the pair correlation function analysis (pCF) to measure molecular anisotropic diffusion in the interphase nucleus of live cells. In the pCF method, we cross-correlate fluctuations at several distances and locations within the nucleus, enabling us to define migration paths and barriers to diffusion. We use monomeric EGFP as a prototypical inert molecule and measure flow in and between different nuclear environments. Our results suggest that there are two disconnect molecular flows throughout the nucleus associated with high and low DNA density regions. We show that different density regions of DNA form a networked channel that allows EGFP to diffuse freely throughout, however with restricted ability to traverse the channel. We also observe rare and sudden bursts of molecules traveling across DNA density regions with characteristic time of approximate to 300 ms, suggesting intrinsic localized change in chromatin structure. This is a unique in vivo demonstration of the intricate chromatin network showing channel directed diffusion of an inert molecule with high spatial and temporal resolution.