Counting nucleosomes in living cells with a combination of fluorescence correlation spectroscopy and confocal imaging
Counting nucleosomes in living cells with a combination of fluorescence correlation spectroscopy and confocal imaging
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DOI：
10.1016/j.jmb.2003.08.063
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发表时间：
2003-11-21
影响因子：
5.6
通讯作者：
Langowski, J
中科院分区：
文献类型：
--
作者：
Weidemann, T;Wachsmuth, M;Langowski, J
尽管染色质光学显微镜检查方法已经很成熟，但没有体内核小体浓度的定量数据。为了建立这样的方法，我们使用了表达与增强型黄色荧光蛋白（H2B-EYFP）融合的核心组蛋白 H2B 的 HeLa 克隆。分离的寡核小体的定量凝胶电泳和荧光相关光谱 (FCS) 显示，总 H2B 的 5% 携带荧光标签，并且荧光细胞的核小体重复长度增加了 204 个。在体内，结合 FCS 和共焦成像研究了 H2B-EYFP 的迁移率和分布。使用FCS，测量细胞质中新生分子的浓度和亮度，而在核质中，通过连续光漂白确定移动荧光组蛋白的背景。结合这些结果可以将细胞核的共焦荧光图像转换为校准的核小体密度图。局部间期的绝对核小体浓度高达 250 μM，平均值为 140(+/-28) μM，表明位点可及性的凝结控制调节发生在远低于 200 nm 的长度尺度上。 (C) 2003 Elsevier Ltd. 保留所有权利。
Although methods for light microscopy of chromatin are well established, there are no quantitative data for nucleosome concentrations in vivo. To establish such a method we used a HeLa clone expressing the core histone H2B fused to the enhanced yellow fluorescent protein (H2B-EYFP). Quantitative gel electrophoresis and fluorescence correlation spectroscopy (FCS) of isolated oligonucleosomes show that 5% of the total H2Bs carry the fluorescent tag and an increased nucleosome repeat length of 204 by for the fluorescent cells. In vivo, the mobility and distribution of H2B-EYFP were studied with a combination of FCS and confocal imaging. With FCS, concentration and brightness of nascent molecules were measured in the cytoplasm, while in the nucleoplasm a background of mobile fluorescent histones was determined by continuous photobleaching. Combining these results allows converting confocal fluorescence images of nuclei into calibrated nucleosome density maps. Absolute nucleosome concentrations in interphase amount up to 250 muM locally, with mean values of 140(+/-28) muM, suggesting that a condensation-controlled regulation of site accessibility takes place at length scales well below 200 nm. (C) 2003 Elsevier Ltd. All rights reserved.