Single-molecule localization microscopy as a promising tool for γH2AX/53BP1 foci exploration

Depeš, Daniel; Lee, Jinho; Bobkova, Elizaveta; Ježková, Lucie; Falková, Iva; Bestvater, Felix; Pagáčová, Eva; Kopečná, Olga; Zadneprianetc, M. G.; Bačı́ková, Alena; Kulikova, E.; Смирнова, Е. В.; Bulanova, Tatiana; Boreyko, A. V.; Krasavin, E. A.; Hausmann, Michael; Falk, Martin

doi:10.1140/epjd/e2018-90148-1

Cited by 21 publications

(46 citation statements)

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“…Using an external alpha irradiation device with an Am-241 source, Reindl et al observed large 53BP1 clusters (super-foci) along damage tracks in the µm range [66]. The 53BP1 nano-foci located within the γ-H2AX environment showed only partial co-localization, compatible with our findings for heavy N ions [28] and the results after C ion irradiation [31]. The super-foci (cluster) sizes observed for γ-H2AX and 53BP1 in our alpha particle investigations are in agreement with the view that both proteins are part of the broader chromatin environment of DSBs, likely facilitating DSB repair [24,27,34].…”

Section: Discussionsupporting

confidence: 90%

“…After high LET particle irradiation and immunofluorescent demarcation of the DNA damage tracks, the structural nature and the distribution of DSB sites and associated DNA repair complexes along high-LET-induced damage tracks over a large range of LETs (LET > 100-1800 keV µm −1 ) remains largely obscure [28,29]. However, substructures (clusters) of DNA damage-associated and repair proteins (which often serve as DSB surrogate markers) have been revealed for high and low LET irradiation by super-resolution light microscopy [30][31][32][33] and transmission electron microscopy (TEM) [3,34].…”

Section: Introductionmentioning

confidence: 99%

“…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.…”

Section: Introductionmentioning

confidence: 99%

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

Scherthan

Lee

Maus

et al. 2019

Cancers

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Background: Cancer patients are increasingly treated with alpha-particle-emitting radiopharmaceuticals. At the subcellular level, alpha particles induce densely spaced ionizations and molecular damage. Induction of DNA lesions, especially clustered DNA double-strand breaks (DSBs), threatens a cell’s survival. Currently, it is under debate to what extent the spatial topology of the damaged chromatin regions and the repair protein arrangements are contributing. Methods: Super-resolution light microscopy (SMLM) in combination with cluster analysis of single molecule signal-point density regions of DSB repair markers was applied to investigate the nano-structure of DNA damage foci tracks of Ra-223 in-solution irradiated leukocytes. Results: Alpha-damaged chromatin tracks were efficiently outlined by γ-H2AX that formed large (super) foci composed of numerous 60–80 nm-sized nano-foci. Alpha damage tracks contained 60–70% of all γ-H2AX point signals in a nucleus, while less than 30% of 53BP1, MRE11 or p-ATM signals were located inside γ-H2AX damage tracks. MRE11 and p-ATM protein fluorescent tags formed focal nano-clusters of about 20 nm peak size. There were, on average, 12 (±9) MRE11 nanoclusters in a typical γ-H2AX-marked alpha track, suggesting a minimal number of MRE11-processed DSBs per track. Our SMLM data suggest regularly arranged nano-structures during DNA repair in the damaged chromatin domain.

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Section: Discussionsupporting

confidence: 90%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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

Scherthan

Lee

Maus

et al. 2019

Cancers

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“…Nevertheless, an increase in the size of 53BP1 repair foci was observed in tumors coming from patients exposed to C-ions compared to X-rays [ 49 ]. High-resolution microscopy, such as single-molecule localization microscopy (nanoscopy, resolution ~10 nm), is necessary to carefully describe radiation-induced foci and their clusters [ 50 ]. Recently evidence of clusters of the DNA repair protein 53BP1 has been reported following exposure to 15 N-ions (very similar to carbon ions), and these study also show that the number and relaxation of clusters is cell-type dependent [ 51 ].…”

Section: Dna Damagementioning

confidence: 99%

Carbon Ion Radiobiology

Tinganelli

Durante

2020

Cancers

156

134

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Radiotherapy using accelerated charged particles is rapidly growing worldwide. About 85% of the cancer patients receiving particle therapy are irradiated with protons, which have physical advantages compared to X-rays but a similar biological response. In addition to the ballistic advantages, heavy ions present specific radiobiological features that can make them attractive for treating radioresistant, hypoxic tumors. An ideal heavy ion should have lower toxicity in the entrance channel (normal tissue) and be exquisitely effective in the target region (tumor). Carbon ions have been chosen because they represent the best combination in this direction. Normal tissue toxicities and second cancer risk are similar to those observed in conventional radiotherapy. In the target region, they have increased relative biological effectiveness and a reduced oxygen enhancement ratio compared to X-rays. Some radiobiological properties of densely ionizing carbon ions are so distinct from X-rays and protons that they can be considered as a different “drug” in oncology, and may elicit favorable responses such as an increased immune response and reduced angiogenesis and metastatic potential. The radiobiological properties of carbon ions should guide patient selection and treatment protocols to achieve optimal clinical results.

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“…DSB damage induction and repair monitoring thus opens the doors to personalized medicine [3,4,[13][14][15], for instance, coupled with direct radiotherapy effect monitoring [13,16], and rational development of new DNA damaging [17][18][19][20] or protecting drugs [21][22][23][24] needed in medicine [25,13,16] and radiation protection [26,27]. Precise and automated DSB damage monitoring is also irreplaceable in biodosimetry [26][27][28][29][30], for instance in situations related to mass radiation accidents (terrorist attacks with radioactive materials) or space exploration where astronauts would be exposed to a mixed field of different radiation types [31][32][33][34][35][36][37].…”

Section: Introductionmentioning

confidence: 99%

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

Vičar

Gumulec

Kolář

et al. 2020

Preprint

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DNA double-strand breaks, marked by Ionizing Radiation-Induced (Repair) Foci (IRIF), are the most serious DNA lesions, dangerous to human health. IRIF quantification based on confocal microscopy represents the most sensitive and gold standard method in radiation biodosimetry and allows research of DSB induction and repair at the molecular and a single cell level. In this study, we introduce DeepFoci - a deep learning-based fully-automatic method for IRIF counting and its morphometric analysis. DeepFoci is designed to work with 3D multichannel data (trained for 53BP1 and γH2AX) and uses U-Net for the nucleus segmentation and IRIF detection, together with maximally stable extremal region-based IRIF segmentation.The proposed method was trained and tested on challenging datasets consisting of mixtures of non-irradiated and irradiated cells of different types and IRIF characteristics - permanent cell lines (NHDF, U-87) and cell primary cultures prepared from tumors and adjacent normal tissues of head and neck cancer patients. The cells were dosed with 1-4 Gy gamma-rays and fixed at multiple (0-24 h) post-irradiation times. Upon all circumstances, DeepFoci was able to quantify the number of IRIF foci with the highest accuracy among current advanced algorithms. Moreover, while the detection error of DeepFoci remained comparable to the variability between two experienced experts, the software kept its sensitivity and fidelity across dramatically different IRIF counts per nucleus. In addition, information was extracted on IRIF 3D morphometric features and repair protein colocalization within IRIFs. This allowed multiparameter IRIF categorization, thereby refining the analysis of DSB repair processes and classification of patient tumors with a potential to identify specific cell subclones.The developed software improves IRIF quantification for various practical applications (radiotherapy monitoring, biodosimetry, etc.) and opens the door to an advanced DSB focus analysis and, in turn, a better understanding of (radiation) DNA damaging and repair.HighlightsNew method for DSB repair focus (IRIF) detection and multi-parameter analysisTrainable deep learning-based methodFully automated analysis of multichannel 3D datasetsTrained and tested on extremely challenging datasets (tumor primary cultures)Comparable to an expert analysis and superb to available methodsGraphical Abstract

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Single-molecule localization microscopy as a promising tool for γH2AX/53BP1 foci exploration

Cited by 21 publications

References 50 publications

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

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

Carbon Ion Radiobiology

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

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