Localization Microscopy Analyses of MRE11 Clusters in 3D-Conserved Cell Nuclei of Different Cell Lines

Abstract

In radiation biophysics, it is a subject of nowadays research to investigate DNA strand break repair in detail after damage induction by ionizing radiation. It is a subject of debate as to what makes up the cell’s decision to use a certain repair pathway and how the repair machinery recruited in repair foci is spatially and temporarily organized. Single-molecule localization microscopy (SMLM) allows super-resolution analysis by precise localization of single fluorescent molecule tags, resulting in nuclear structure analysis with a spatial resolution in the 10 nm regime. Here, we used SMLM to study MRE11 foci. MRE11 is one of three proteins involved in the MRN-complex (MRE11-RAD50-NBS1 complex), a prominent DNA strand resection and broken end bridging component involved in homologous recombination repair (HRR) and alternative non-homologous end joining (a-NHEJ). We analyzed the spatial arrangements of antibody-labelled MRE11 proteins in the nuclei of a breast cancer and a skin fibroblast cell line along a time-course of repair (up to 48 h) after irradiation with a dose of 2 Gy. Different kinetics for cluster formation and relaxation were determined. Changes in the internal nano-scaled structure of the clusters were quantified and compared between the two cell types. The results indicate a cell type-dependent DNA damage response concerning MRE11 recruitment and cluster formation. The MRE11 data were compared to H2AX phosphorylation detected by γH2AX molecule distribution. These data suggested modulations of MRE11 signal frequencies that were not directly correlated to DNA damage induction. The application of SMLM in radiation biophysics offers new possibilities to investigate spatial foci organization after DNA damaging and during subsequent repair.

Using Persistent Homology as a New Approach for Super-Resolution Localization Microscopy Data Analysis and Classification of γH2AX Foci/Clusters

“…It has been shown that such foci can be separated into functionally relevant sub-foci or clusters of about the same size independent from the dose applied [23,28,29]. This has also been found for the cell nuclei analyzed here (see Figure S2).…”

Nanostructure of Clustered DNA Damage in Leukocytes after In-Solution Irradiation with the Alpha Emitter Ra-223

“…After data analysis, artificial "pointillist" super-resolution images can be generated, in which the results of data analysis can additionally be encoded [40]. SMLM was applied to investigate the irradiation effects on chromatin nano-architecture [28,36,37] and protein organization at the nano scale [41][42][43], revealing systematic dose- [40,44] and locus-dependent [45] chromatin conformation changes. For multi-color localization microscopy of DNA damage markers along alpha tracks, we here applied molecular labelling with specific antibodies against γ-H2AX, MRE11, and 53BP1 in alpha-traversed cell nuclei and studied their distribution within γ-H2AX foci domains (super-foci) that demarcate the particle track.…”

Quantification of DNA damage induced γH2AX focus formation via super-resolution dSTORM localization microscopy

“…These steps allow the efficient recruitment of the repair factors to the damaged DNA regions and implicate in the choice between the DNA repair pathways. For examining the DSB-induced chromatin changes, confocal microscopy-based techniques are used in most of the studies, although in the last few years high-throughput chromosome conformation capture technique (4C) and single cell microscopy were utilized to gain detailed insights about the protein interactions and cascades involved in the different repair pathways 31,53–57 . A more detailed overview has raised more questions, which could be answered only at a single-cell level: how the different DNA repair pathways are chosen and how individual repair proteins are regulated to access the DNA repair site.…”

“…However, the limitation of the technique is that it shows the average of a given focus by combining the data obtained from a large population of cells. Recent applications of electron-microscopy and super-resolution light microscopy 24,30,31,55–57 have demonstrated that it is feasible to study single molecular arrangements within a repair focus. By improving the resolution of microscopy and data evaluation of structures in meso-and nano-scale level, the searching for the best-suited analysis parameters and potentially useful classification criteria of repair foci and damaged chromatin sites have become to be important.…”