Determining Omics Spatiotemporal Dimensions Using Exciting New Nanoscopy Techniques to Assess Complex Cell Responses to DNA Damage: Part - Structuromics

Abstract

Recent groundbreaking developments in Omics and bioinformatics have generated new hope for overcoming the complexity and variability of (radio)biological systems while simultaneously shedding more light on fundamental radiobiological questions that have remained unanswered for decades. In the era of Omics, our knowledge of how genes and dozens of proteins interact in the frame of complex signaling and repair pathways (or, rather, networks) to preserve the integrity of the genome has been rapidly expanding. Nevertheless, these functional networks must be observed with strong correspondence to the cell nucleus, which is the main target of ionizing radiation. Information regarding these intricate processes cannot be achieved using high-throughput Omics approaches alone; it requires sophisticated structural probing and imaging. In the first part of this review, the article "Giving Omics Spatiotemporal Dimensions Using Exciting New Nanoscopy Techniques to Assess Complex Cell Responses to DNA Damage: Part A--Radiomics," we showed the development of different Omics solutions and how they are contributing to a better understanding of cellular radiation response. In this Part B we show how high-resolution confocal microscopy as well as novel approaches of molecular localization nanoscopy fill the gaps to successfully place Omics data in the context of space and time. The dynamics of double-strand breaks during repair processes and chromosomal rearrangements at the microscale correlated to aberration induction are explained. For the first time we visualize pan-nuclear nucleosomal rearrangements and clustering at the nanoscale during repair processes. Finally, we introduce a novel method of specific chromatin nanotargeting based on a computer database search of uniquely binding oligonucleotide combinations (COMBO-FISH). With these challenging techniques on hand, we speculate future perspectives that may combine specific COMBO-FISH nanoprobing and structural nanoscopy to observe structure-function correlations in living cells in real-time. Thus, the Omics networks obtained from function analyses may be enriched by real-time visualization of Structuromics.

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

“…After having induced a DSB by ionizing radiation, chromatin re-arranges and H2AX phosphorylation occurs in the damage environment [1,2,28] within a few minutes accompanied by the recruitment of proteins specific for a certain repair mechanism. During the last decades modern techniques applied in radiation biology and radiation biophysics, have offered detailed insights into the protein interactions and cascades along the different repair pathways [8,9]. These investigations have completed our understanding about repair processes and boundary conditions that favor repair towards either end-joining processes such as NHEJ or recombination processes such as HRR.…”

“…One of the first steps of chromatin modification after DSB induction is phosphorylation of the histone variant H2AX [4] to γH2AX within a given neighborhood of the damaged site [5,6,7]. Such foci seem to “tag” the locations of damaged DNA for the recruitment of proteins that are starting and processing the follow-up repair [8,9]. At that point, a decision about the next procedure has to be made by the cell [10].…”

“…This mechanism should work at each damaged side individually. This means that physical as well as topological parameters of the DNA strand break environment may determine the repair pathway choice together with epigenetic conditions [8,9]. This assumption has been recently supported by investigations showing that radio-sensitivity can be modulated by chromatin remodeling in daughter cells of irradiated samples [20].…”

Combining Low Temperature Fluorescence DNA-Hybridization, Immunostaining, and Super-Resolution Localization Microscopy for Nano-Structure Analysis of ALU Elements and Their Influence on Chromatin Structure

“…At this point, we were able to microscopically confirm the hypothesis that heterochromatin and ALU do not co-localize, but may associate functionally in a certain way. We also gained some information about the structural connection of ALU and heterochromatin, which could contribute to a better understanding of chromatin interaction mechanisms after irradiation and during repair [52,59,60]. Since our major goal was methodological development, we used the SkBr3 cell line as an easy-to-handle but well-established cell line in breast cancer research [61].…”

DeepFoci: Deep Learning-Based Algorithm for Fast Automatic Analysis of DNA Double Strand Break Ionizing Radiation-Induced Foci

“…The ability to precisely and rapidly monitor DNA double-strand break (DSBs) induction and repair undermines numerous fields of biological, medical and space research (reviewed e.g., in [1][2][3][4]). DNA double-strand breaks (DSBs) are permanently introduced into the DNA molecule by ionizing radiation, radiomimetic chemicals and vital cell processes [5][6][7].…”