Neutral lipids restrict the mobility of broken DNA molecules during comet assays

IF 2.4 4区 生物学 Q4 CELL BIOLOGY
Caroline Soulet, Jordi Josa-Castro, María Moriel-Carretero
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Abstract

One widespread technique to assess in relative terms the amount of broken DNA present in the genome of individual cells consists of immobilizing the cell's nucleus under an agarose pad (called the nucleoid) and subjecting the whole genome to electrophoresis to force broken DNA molecules out of it. Since the migrating broken DNA molecules create a tail behind the nucleoid, this technique is named the comet assay. While performing comet assays regularly, we systematically observed circular regions devoid of DNA within the nucleoid region. We characterize here that these correspond to clusters of neutral (apolar) lipids, since they could be labeled with neutral lipid-dying molecules, increased when cells were fed with oleic acid, and were irresponsive to the electrophoretic field. Of relevance, de-lipidation assays, either in vivo, or in vitro using acetone, show that these neutral lipids (NL) within the nucleoid limit the ability of broken DNA molecules to migrate into the comet tail. From a technical point of view, we show that de-lipidation permits a wider range for the detection of broken DNA molecules. Biologically, we put forward the notion that NL in contact with DNA may locally exert regulatory functions within the cell's nucleus.

Abstract Image

中性脂质在彗星分析中限制了断裂DNA分子的流动性。
一种广泛应用的评估单个细胞基因组中断裂DNA相对数量的技术包括将细胞核固定在琼脂糖垫(称为类核)下,并对整个基因组进行电泳以迫使断裂的DNA分子脱离。由于迁移的断裂DNA分子在类核后面形成一条尾巴,这项技术被命名为彗星试验。在定期进行彗星分析时,我们系统地观察到在类核区域内缺乏DNA的圆形区域。我们在这里描述了这些与中性(极性)脂质簇相对应的特征,因为它们可以被中性脂质死亡分子标记,当细胞被喂以油酸时增加,并且对电泳场无反应。与此相关的是,在体内或体外使用丙酮进行的去脂化试验表明,类核内的中性脂质(NL)限制了断裂DNA分子迁移到彗星尾部的能力。从技术的角度来看,我们表明,去脂化允许更广泛的范围内检测破碎的DNA分子。在生物学上,我们提出了与DNA接触的NL可能在细胞核内局部发挥调节功能的概念。
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来源期刊
Biology of the Cell
Biology of the Cell 生物-细胞生物学
CiteScore
5.30
自引率
0.00%
发文量
53
审稿时长
>12 weeks
期刊介绍: The journal publishes original research articles and reviews on all aspects of cellular, molecular and structural biology, developmental biology, cell physiology and evolution. It will publish articles or reviews contributing to the understanding of the elementary biochemical and biophysical principles of live matter organization from the molecular, cellular and tissues scales and organisms. This includes contributions directed towards understanding biochemical and biophysical mechanisms, structure-function relationships with respect to basic cell and tissue functions, development, development/evolution relationship, morphogenesis, stem cell biology, cell biology of disease, plant cell biology, as well as contributions directed toward understanding integrated processes at the organelles, cell and tissue levels. Contributions using approaches such as high resolution imaging, live imaging, quantitative cell biology and integrated biology; as well as those using innovative genetic and epigenetic technologies, ex-vivo tissue engineering, cellular, tissue and integrated functional analysis, and quantitative biology and modeling to demonstrate original biological principles are encouraged.
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