Nanoscale mapping of nuclear phosphatidylinositol phosphate landscape by dual-color dSTORM
Nanoscale mapping of nuclear phosphatidylinositol phosphate landscape by dual-color dSTORM
复制标题
DOI：
10.1016/j.bbalip.2021.158890
复制
发表时间：
2021-02-13
影响因子：
4.8
通讯作者：
Hozak, Pavel
中科院分区：
文献类型：
--
作者：
Hoboth, Peter;Sztacho, Martin;Hozak, Pavel
当前的基因表达模型基于单分子定位显微镜，承认蛋白质聚类和转录凝聚物的形成是基因表达的驱动力。然而，这些模型很大程度上忽略了核脂质的作用，特别是核磷脂酰肌醇磷酸盐（PIP）。此外，核 PIP 在功能性亚核域中的精确分布仍然难以捉摸。直接随机光学重建显微镜（dSTORM）为生物成像提供了前所未有的分辨率。因此，将其用于密集的细胞核成像是人们所期望的，但也具有挑战性。在这里，我们提出了核 PI (4,5)P2、PI(3,4)P2 和 PI(4)P 分布的双色 dSTORM 成像和图像分析，同时保留了核结构的背景。在核质中，PI(4,5)P2 和 PI(3,4)P2 共模式与 RNA 聚合酶 II 焦点子集非常接近。 PI(4,5)P2 被核仁中的纤维蛋白包围，并且所有三个 PIP 都分散在由核斑点蛋白 SON 形成的基质内。 PI(4,5)P2是最丰富的核PIP，而PI(4)P是PI(4,5)P2和PI(3,4)P2生物合成的前体。因此，我们的数据有助于理解核 PIP 的作用，并为核 PIP 代表细胞核中脂核糖核蛋白中心形成的定位信号的模型提供进一步的证据。所讨论的实验流程适用于其他核 PIP 在基因表达调控等方面的作用的进一步功能研究。
Current models of gene expression, which are based on single-molecule localization microscopy, acknowledge protein clustering and the formation of transcriptional condensates as a driving force of gene expression. However, these models largely omit the role of nuclear lipids and amongst them nuclear phosphatidylinositol phosphates (PIPs) in particular. Moreover, the precise distribution of nuclear PIPs in the functional sub-nuclear domains remains elusive. The direct stochastic optical reconstruction microscopy (dSTORM) provides an unprecedented resolution in biological imaging. Therefore, its use for imaging in the densely crowded cell nucleus is desired but also challenging. Here we present a dual-color dSTORM imaging and image analysis of nuclear PI (4,5)P2, PI(3,4)P2 and PI(4)P distribution while preserving the context of nuclear architecture. In the nucleoplasm, PI(4,5)P2 and PI(3,4)P2 co-pattern in close proximity with the subset of RNA polymerase II foci. PI(4,5)P2 is surrounded by fibrillarin in the nucleoli and all three PIPs are dispersed within the matrix formed by the nuclear speckle protein SON. PI(4,5)P2 is the most abundant nuclear PIP, while PI(4)P is a precursor for the biosynthesis of PI(4,5)P2 and PI(3,4)P2. Therefore, our data are relevant for the understanding the roles of nuclear PIPs and provide further evidence for the model in which nuclear PIPs represent a localization signal for the formation of lipo-ribonucleoprotein hubs in the nucleus. The discussed experimental pipeline is applicable for further functional studies on the role of other nuclear PIPs in the regulation of gene expression and beyond.