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.
Nanoscale Distribution of Nuclear Sites by Super-Resolved Image Cross-Correlation Spectroscopy / M. Oneto, L. Scipioni, M.J. Sarmento, I. Cainero, S. Pelicci, L. Furia, P.G. Pelicci, G.I. Dellino, P. Bianchini, M. Faretta, E. Gratton, A. Diaspro, L. Lanzano. - In: BIOPHYSICAL JOURNAL. - ISSN 0006-3495. - 117:11(2019 Dec 03), pp. 2054-2065.
|Titolo:||Nanoscale Distribution of Nuclear Sites by Super-Resolved Image Cross-Correlation Spectroscopy|
|Settore Scientifico Disciplinare:||Settore BIO/11 - Biologia Molecolare|
|Data di pubblicazione:||3-dic-2019|
|Digital Object Identifier (DOI):||http://dx.doi.org/10.1016/j.bpj.2019.10.036|
|Appare nelle tipologie:||01 - Articolo su periodico|