The connection between chromatin motion on the 100 nm length scale and core histone dynamics in live XTC-2 cells and isolated nuclei
The connection between chromatin motion on the 100 nm length scale and core histone dynamics in live XTC-2 cells and isolated nuclei
复制标题
DOI：
10.1016/s0006-3495(04)74134-x
复制
发表时间：
2004-01-01
影响因子：
3.4
通讯作者：
Bardeen, CJ
中科院分区：
文献类型：
--
作者：
Davis, SK;Bardeen, CJ
用双光子驻波荧光漂白实验研究了含DNA染色质在活细胞和离体细胞核中的扩散运动，空间分辨率为100 nm。使用小沟结合染料Hoechst 33342标记染色质。在活细胞中，平均扩散速率为5x 10（-4）mum（2）/s，细胞间差异相当大。这种扩散受到高度限制，并且在光漂白实验后的标准单光束荧光恢复中无法观察到。为了确定扩散的化学起源，我们研究了孤立的细胞核中的运动，并通过改变离子强度和使用化学和光交联实验来改变组蛋白-DNA相互作用的强度。在较高的NaCl浓度下，我们看到染色质扩散增加，因为组蛋白-DNA相互作用由于离子屏蔽而减弱，而核心组蛋白与DNA的光交联导致完全不存在扩散运动。这些趋势与100 nm尺度的运动是一致的，与组蛋白与DNA的相互作用相关。如果染色质扩散与核小体动力学在小得多的长度尺度上相关联，则这可以提供一种基于通过荧光显微镜观察到的较大尺度大分子动力学来测定体内生化活性的方法。
The diffusive motion of DNA-containing chromatin in live cells and isolated nuclei is investigated using a two-photon standing wave fluorescence photobleaching experiment with 100 nm spatial resolution. The chromatin is labeled using the minor groove binding dye Hoechst 33342. In live cells, the mean diffusion rate is 5x10(-4) mum(2)/s, with considerable cell-to-cell variation. This diffusion is highly constrained and cannot be observed in a standard, single beam fluorescence recovery after photobleaching experiment. To determine the chemical origin of the diffusion, we study motion in isolated nuclei and vary the strength of the histone-DNA interactions by changing the ionic strength and using chemical and photocross-linking experiments. At higher NaCl concentrations, we see increased chromatin diffusion as the histone-DNA interaction is weakened due to ionic screening, whereas photocross-linking the core histones to the DNA results in a complete absence of diffusive motion. These trends are consistent with the 100 nm scale motion being correlated with the interactions of histone proteins with the DNA. If chromatin diffusion is connected to the nucleosomal dynamics on much smaller length scales, this may provide a way to assay biochemical activity in vivo based on larger scale macromolecular dynamics observed via fluorescence microscopy.