Quantitative single-molecule microscopy reveals that CENP-A(Cnp1) deposition occurs during G2 in fission yeast.
Quantitative single-molecule microscopy reveals that CENP-A(Cnp1) deposition occurs during G2 in fission yeast.
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DOI：
10.1098/rsob.120078
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发表时间：
2012-07
期刊：
影响因子：
5.8
通讯作者：
Laue ED
中科院分区：
文献类型：
--
作者：
Lando D;Endesfelder U;Berger H;Subramanian L;Dunne PD;McColl J;Klenerman D;Carr AM;Sauer M;Allshire RC;Heilemann M;Laue ED
组蛋白H3变异体CENP-A在DNA复制后在着丝粒的核小体中的遗传是由表观遗传机制介导的。为了了解在活的真核细胞中，随着核小体的分解和重组，组蛋白和组蛋白变体的表观遗传或繁殖过程，我们探索了开发光激活定位显微镜(Palm)的可行性。活细胞中的单分子手掌有可能揭示细胞生物学中的新概念，为细胞状态的随机变化提供见解。然而，到目前为止，它的使用仅限于细菌研究或发生在真核细胞表面附近的过程。使用Palm，人们可以逐个观察和计数细胞中的单个分子，这使得记录图像的分辨率高于由光的衍射确定的分辨率(所谓的超分辨率显微镜)。在这里，我们研究了不同荧光团的使用，并开发了在裂殖酵母(裂殖酵母)中对着丝粒特异性组蛋白H3变体CENP-ACnp1进行单分子敏感性计数的程序。所得结果得到了CHIP-SEQ分析的验证，并与之进行了比较。利用这种方法，跟踪了分裂酵母(S.pombe)着丝粒上的CENP-ACnp1水平在细胞周期中的变化。我们的测量表明，CENP-ACnp1仅在细胞周期的G2期沉积。
The inheritance of the histone H3 variant CENP-A in nucleosomes at centromeres following DNA replication is mediated by an epigenetic mechanism. To understand the process of epigenetic inheritance, or propagation of histones and histone variants, as nucleosomes are disassembled and reassembled in living eukaryotic cells, we have explored the feasibility of exploiting photo-activated localization microscopy (PALM). PALM of single molecules in living cells has the potential to reveal new concepts in cell biology, providing insights into stochastic variation in cellular states. However, thus far, its use has been limited to studies in bacteria or to processes occurring near the surface of eukaryotic cells. With PALM, one literally observes and ‘counts’ individual molecules in cells one-by-one and this allows the recording of images with a resolution higher than that determined by the diffraction of light (the so-called super-resolution microscopy). Here, we investigate the use of different fluorophores and develop procedures to count the centromere-specific histone H3 variant CENP-ACnp1 with single-molecule sensitivity in fission yeast (Schizosaccharomyces pombe). The results obtained are validated by and compared with ChIP-seq analyses. Using this approach, CENP-ACnp1 levels at fission yeast (S. pombe) centromeres were followed as they change during the cell cycle. Our measurements show that CENP-ACnp1 is deposited solely during the G2 phase of the cell cycle.
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影响因子：
48
作者：
Sengupta, Prabuddha;Jovanovic-Talisman, Tijana;Skoko, Dunja;Renz, Malte;Veatch, Sarah L.;Lippincott-Schwartz, Jennifer
通讯作者：
Lippincott-Schwartz, Jennifer
影响因子：
48
作者：
Fuchs, Jochen;Boehme, Susan;Nienhaus, G. Ulrich
通讯作者：
Nienhaus, G. Ulrich
DOI：
10.1073/pnas.251525598
发表时间：
2001-12-04
影响因子：
11.1
作者：
Garcia-Parajo, MF;Koopman, M;van Hulst, NF
通讯作者：
van Hulst, NF
裂殖酵母中着丝粒和端粒的细胞周期依赖性特定定位和聚类。
影响因子：
7.8
作者：
Funabiki, H;Hagan, I;Uzawa, S;Yanagida, M
通讯作者：
Yanagida, M
影响因子：
5.7
作者：
Annibale, P.;Scarselli, M.;Radenovic, A.
通讯作者：
Radenovic, A.