Analysis of the Trypanosoma brucei cell cycle by quantitative DAPI imaging
Analysis of the Trypanosoma brucei cell cycle by quantitative DAPI imaging
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DOI：
10.1016/j.molbiopara.2008.04.004
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发表时间：
2008-08-01
影响因子：
1.5
通讯作者：
Cross, George A. M.
中科院分区：
文献类型：
--
作者：
Siegel, T. Nicolai;Hekstra, Doeke R.;Cross, George A. M.
布氏锥虫有两个DNA区室：细胞核和动基体。这两个区室的DNA复制仅部分重合。伍德沃德和海鸥[伍德沃德R，海鸥K。布氏锥虫细胞周期中细胞核和动质体DNA复制的时间和早期形态学事件。J Cell Sci 1990; 95：49-57]全面研究了核DNA（nDNA）和动质体DNA（kDNA）的复制和分离的相对时间。其他人则认为，细胞周期阶段的形态指标在不同菌株和条件下是一致的。我们报告使用定量DAPI成像来确定单个前循环细胞的细胞周期阶段。使用这种方法，我们发现，动基体伸长主要发生在核S期，而不是在G2，如先前假设的。我们通过DNA含量对细胞进行分类，然后用荧光显微镜检查证实了这一发现。此外，在两个波长下同时定量成像可用于确定细胞周期期间细胞周期调节蛋白的丰度。我们证明了这种技术的非乙酰化状态的组蛋白H4（H4 K4），这是在核S期富集的赖氨酸4共染色。(C)2008 Elsevier B. V.保留所有权利。
Trypanosoma brucei has two DNA compartments: the nucleus and the kinetoplast. DNA replication of these two compartments only partially coincides. Woodward and Gull [Woodward R, Gull K. Timing of nuclear and kinetoplast DNA replication and early morphological events in the cell cycle of Trypanosoma brucei. J Cell Sci 1990;95:49-57] comprehensively studied the relative timing of the replication and segregation of nuclear DNA (nDNA) and kinetoplast DNA (kDNA). Others have since assumed the consistency of morphological indicators of cell-cycle stage among strains and conditions. We report the use of quantitative DAPI imaging to determine the cell-cycle stage of individual procyclic cells. Using this approach, we found that kinetoplast elongation occurs mainly during nuclear S phase and not during G2, as previously assumed. We confirmed this finding by sorting cells by DNA content, followed by fluorescence microscopy. In addition, simultaneous quantitative imaging at two wavelengths can be used to determine the abundance of cell-cycle-regulated proteins during the cell cycle. We demonstrate this technique by co-staining for the non-acetylated state of lysine 4 of histone H4 (H4K4), which is enriched during nuclear S phase. (C) 2008 Elsevier B.V. All rights reserved.