Isolation of cell nuclei using inert macromolecules to mimic the crowded cytoplasm.
Isolation of cell nuclei using inert macromolecules to mimic the crowded cytoplasm.
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DOI：
10.1371/journal.pone.0007560
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发表时间：
2009-10-23
期刊：
影响因子：
3.7
通讯作者：
Hadj-Sahraoui Y
中科院分区：
文献类型：
--
作者：
Hancock R;Hadj-Sahraoui Y
通常在含有毫摩尔浓度阳离子的介质中分离和研究细胞核，通过筛选染色质上的负电荷并保持其紧凑来保存核体积。然而，有两个因素质疑这些离子条件是否正确地再现了体内细胞核的环境：体内染色质的小规模运动和构象在分离的细胞核中不会再现，并且实验和理论表明细胞质中的小离子在可溶相中不是游离的，而是主要与大分子结合。我们研究了细胞质的另一个但经常被忽视的特性在维持体内细胞核结构和功能方面的可能作用，即由可扩散大分子引起的拥挤或渗透效应，其浓度在几项研究中测得在 130 mg/ml 范围内。在含有惰性聚合物 70 kDa 聚蔗糖 (50% w/v) 或 70 kDa 葡聚糖 (35% w/v) 的培养基中，从 K562 细胞中释放出保留细胞体积和电子显微镜观察到的超微结构的细胞核，以取代用洋地黄皂苷分散在细胞裂解物上的可扩散细胞质分子，并以 100 µM K-Hepes 缓冲液作为唯一离子源。免疫荧光标记和使用表达 GFP 融合蛋白的细胞进行的实验表明，内部区室（核仁、PML 和卷曲体、RNA 聚合酶 II 的焦点）在这些细胞核中是保守的，并且新生的 RNA 转录本可以被延长。我们的观察结果与以下假设一致：可扩散细胞质大分子的拥挤是一个至关重要但被忽视的因素，它通过平衡细胞核中高浓度大分子引起的反向渗透压来支持体内的细胞核，并表明拥挤的介质为研究核结构和功能提供了更多的生理条件。它们还可能有助于解决长期存在的悖论，即体内观察到的染色质的小规模运动和不规则构象无法在传统离子介质中分离的细胞核中重现。
Cell nuclei are commonly isolated and studied in media which include millimolar concentrations of cations, which conserve the nuclear volume by screening the negative charges on chromatin and maintaining its compaction. However, two factors question if these ionic conditions correctly reproduce the environment of nuclei in vivo: the small-scale motion and conformation of chromatin in vivo are not reproduced in isolated nuclei, and experiments and theory suggest that small ions in the cytoplasm are not free in the soluble phase but are predominantly bound to macromolecules. We studied the possible role in maintaining the structure and functions of nuclei in vivo of a further but frequently overlooked property of the cytoplasm, the crowding or osmotic effects caused by diffusible macromolecules whose concentration, measured in several studies, is in the range of 130 mg/ml. Nuclei which conserved their volume in the cell and their ultrastructure seen by electron microscopy were released from K562 cells in media containing the inert polymer 70 kDa Ficoll (50% w/v) or 70 kDa dextran (35% w/v) to replace the diffusible cytoplasmic molecules which were dispersed on cell lysis with digitonin, with 100 µM K-Hepes buffer as the only source of ions. Immunofluorescence labelling and experiments using cells expressing GFP-fusion proteins showed that internal compartments (nucleoli, PML and coiled bodies, foci of RNA polymerase II) were conserved in these nuclei, and nascent RNA transcripts could be elongated. Our observations are consistent with the hypothesis that crowding by diffusible cytoplasmic macromolecules is a crucial but overlooked factor which supports the nucleus in vivo by equilibrating the opposing osmotic pressure cause by the high concentration of macromolecules in the nucleus, and suggest that crowded media provide more physiological conditions to study nuclear structure and functions. They may also help to resolve the long-standing paradox that the small-scale motion and irregular conformation of chromatin seen in vivo are not reproduced in nuclei isolated in conventional ionic media.
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影响因子：
9.2
作者：
Andersen, JS;Lyon, CE;Lamond, AI
通讯作者：
Lamond, AI
影响因子：
4.8
作者：
Galán, A;Sot, B;Muga, A
通讯作者：
Muga, A
影响因子：
3.4
作者：
Davis, SK;Bardeen, CJ
通讯作者：
Bardeen, CJ
影响因子：
5.4
作者：
del Alamo, M;Rivas, G;Mateu, MG
通讯作者：
Mateu, MG
DOI：
10.1007/978-1-60327-461-6_11
发表时间：
2009
期刊：
Methods in molecular biology (Clifton, N.J.)
影响因子：
--
作者：
Cassany, Aurelia;Gerace, Larry
通讯作者：
Gerace, Larry