Studying the Dynamics of Chromatin-Binding Proteins in Mammalian Cells Using Single-Molecule Localisation Microscopy
Studying the Dynamics of Chromatin-Binding Proteins in Mammalian Cells Using Single-Molecule Localisation Microscopy
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DOI：
10.1007/978-1-4939-3631-1_17
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发表时间：
2016-01-01
期刊：
影响因子：
--
通讯作者：
Lee, Steven F.
中科院分区：
文献类型：
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作者：
Basu, Srinjan;Tan, Yi Lei;Lee, Steven F.
单分子定位显微镜（SMLM）允许对哺乳动物细胞核内的蛋白质进行超分辨率成像，其空间分辨率与核小体本身相当（类似于20纳米）。因此，该技术非常适合于研究染色质结构。固定细胞SMLM已经允许对哺乳动物细胞核内蛋白质在染色质上的排列方式进行短暂的“快照”。在本章中，我们将重点关注最近的发展，例如在选择性平面照明和蛋白质标记方面，如何导致一系列活细胞SMLM研究。我们描述了如何对单个蛋白质进行单粒子跟踪（SPT），并通过分析它们的扩散参数，如何确定蛋白质是与染色质相互作用，自由扩散还是两者兼有。我们可以研究与染色质相互作用的蛋白质的数量，也可以确定它们在染色质上的停留时间。我们可以确定这些蛋白质是否在细胞核内形成功能簇，以及它们是否形成特定的核结构。
Single-molecule localisation microscopy (SMLM) allows the super-resolved imaging of proteins within mammalian nuclei at spatial resolutions comparable to that of a nucleosome itself (similar to 20 nm). The technique is therefore well suited to the study of chromatin structure. Fixed-cell SMLM has already allowed temporal 'napshots' of how proteins are arranged on chromatin within mammalian nuclei. In this chapter, we focus on how recent developments, for example in selective plane illumination and protein labelling, have led to a range of live-cell SMLM studies. We describe how to carry out single-particle tracking (SPT) of single proteins and, by analysing their diffusion parameters, how to determine whether proteins interact with chromatin, diffuse freely or do both. We can study the numbers of proteins that interact with chromatin and also determine their residence time on chromatin. We can determine whether these proteins form functional clusters within the nucleus as well as whether they form specific nuclear structures.