Nanoscopy and NIC@IIT, Istituto Italiano di Tecnologia, Genoa, Italy.
Nanoscopy and NIC@IIT, Istituto Italiano di Tecnologia, Genoa, Italy; Laboratory for Fluorescence Dynamics, Department of Biomedical Engineering, University of California, Irvine, California.
Biophys J. 2019 Dec 3;117(11):2054-2065. doi: 10.1016/j.bpj.2019.10.036. Epub 2019 Nov 2.
Deciphering the spatiotemporal coordination between nuclear functions is important to understand its role in the maintenance of human genome. In this context, super-resolution microscopy has gained considerable interest because it can be used to probe the spatial organization of functional sites in intact single-cell nuclei in the 20-250 nm range. Among the methods that quantify colocalization from multicolor images, image cross-correlation spectroscopy (ICCS) offers several advantages, namely it does not require a presegmentation of the image into objects and can be used to detect dynamic interactions. However, the combination of ICCS with super-resolution microscopy has not been explored yet. Here, we combine dual-color stimulated emission depletion (STED) nanoscopy with ICCS (STED-ICCS) to quantify the nanoscale distribution of functional nuclear sites. We show that super-resolved ICCS provides not only a value of the colocalized fraction but also the characteristic distances associated to correlated nuclear sites. As a validation, we quantify the nanoscale spatial distribution of three different pairs of functional nuclear sites in MCF10A cells. As expected, transcription foci and a transcriptionally repressive histone marker (H3K9me3) are not correlated. Conversely, nascent DNA replication foci and the proliferating cell nuclear antigen(PCNA) protein have a high level of proximity and are correlated at a nanometer distance scale that is close to the limit of our experimental approach. Finally, transcription foci are found at a distance of 130 nm from replication foci, indicating a spatial segregation at the nanoscale. Overall, our data demonstrate that STED-ICCS can be a powerful tool for the analysis of the nanoscale distribution of functional sites in the nucleus.
解析核功能的时空协调对于理解其在人类基因组维持中的作用至关重要。在这种情况下，超分辨率显微镜技术引起了相当大的兴趣，因为它可以用于探测完整单细胞核中功能位点的空间组织，其范围在 20-250nm 之间。在定量多色图像共定位的方法中，图像互相关光谱学（ICCS）具有几个优势，即它不需要将图像预先分割成对象，并且可以用于检测动态相互作用。然而，ICCS 与超分辨率显微镜的结合尚未得到探索。在这里，我们将双颜色受激发射耗尽（STED）纳米显微镜与 ICCS（STED-ICCS）相结合，以定量功能核位点的纳米尺度分布。我们表明，超分辨 ICCS 不仅提供共定位分数的值，还提供与相关核位点相关的特征距离。作为验证，我们在 MCF10A 细胞中定量了三种不同的功能核位点对的纳米尺度空间分布。正如预期的那样，转录焦点和转录抑制组蛋白标记（H3K9me3）没有相关性。相反，新生 DNA 复制焦点和增殖细胞核抗原（PCNA）蛋白具有高度的接近度，并且在接近我们实验方法极限的纳米距离尺度上相关。最后，转录焦点与复制焦点的距离为 130nm，表明在纳米尺度上存在空间隔离。总体而言，我们的数据表明，STED-ICCS 可以成为分析核中功能位点纳米尺度分布的有力工具。
