dSTORM microscopy evidences in HeLa cells clustered and scattered γH2AX nanofoci sensitive to ATM, DNA-PK, and ATR kinase inhibitors

Liddle, Pablo; Jara‐Wilde, Jorge; Lafon-Hughes, Laura; Castro, Iván Galindo; Härtel, Steffen; Folle, Gustavo A.

doi:10.1007/s11010-020-03809-4

Cited by 9 publications

(4 citation statements)

References 63 publications

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“…The response function of the imaging system (the average number of localizations belonging to a single labeled histone molecule) depends on the lifetime of the fluorescence ON state, the labeling density and the number of reactivation cycles of the applied dye molecules. These parameters can be determined by a rigorous calibration process as was discussed earlier [25][26][27].…”

Section: Introductionmentioning

confidence: 99%

Analysis of Ionizing Radiation Induced DNA Damage by Superresolution dSTORM Microscopy

Brunner¹,

Varga²,

Bozó³

et al. 2021

Pathol. Oncol. Res.

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The quantitative detection of radiation caused DNA double-strand breaks (DSB) by immunostained γ-H2AX foci using direct stochastic optical reconstruction microscopy (dSTORM) provides a deeper insight into the DNA repair process at nanoscale in a time-dependent manner. Glioblastoma (U251) cells were irradiated with 250 keV X-ray at 0, 2, 5, 8 Gy dose levels. Cell cycle phase distribution and apoptosis of U251 cells upon irradiation was assayed by flow cytometry. We studied the density, topology and volume of the γ-H2AX foci with 3D confocal microscopy and the dSTORM superresolution method. A pronounced increase in γ-H2AX foci and cluster density was detected by 3D confocal microscopy after 2 Gy, at 30 min postirradiation, but both returned to the control level at 24 h. Meanwhile, at 24 h a considerable amount of residual foci could be measured from 5 Gy, which returned to the normal level 48 h later. The dSTORM based γ-H2AX analysis revealed that the micron-sized γ-H2AX foci are composed of distinct smaller units with a few tens of nanometers. The density of these clusters, the epitope number and the dynamics of γ-H2AX foci loss could be analyzed. Our findings suggest a discrete level of repair enzyme capacity and the restart of the repair process for the residual DSBs, even beyond 24 h. The dSTORM superresolution technique provides a higher precision over 3D confocal microscopy to study radiation induced γ-H2AX foci and molecular rearrangements during the repair process, opening a novel perspective for radiation research.

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Section: Introductionmentioning

confidence: 99%

Analysis of Ionizing Radiation Induced DNA Damage by Superresolution dSTORM Microscopy

Brunner¹,

Varga²,

Bozó³

et al. 2021

Pathol. Oncol. Res.

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“…In this study, we used 3D STED microscopy, which has a lateral resolution of 105 nm, well above the measured structure sizes of interest here. Other studies using STORM imaging have even identified substructures in the radiation-induced γH2AX foci, called nanofoci, with smaller sizes down to tens of nanometers [ 51 , 52 , 53 , 54 ]. Although we were not able to resolve these nanofoci in this study, we believe that their existence points to another level of spatial arrangement, which is related to the way H2AX is incorporated into nucleosomes [ 52 ] and to the phosphorylation status upon damage induction [ 53 , 54 ], which we were not aiming to investigate here.…”

Section: Discussionmentioning

confidence: 99%

Chromatin Organization after High-LET Irradiation Revealed by Super-Resolution STED Microscopy

Schwarz,

Matejka,

Rudigkeit

et al. 2024

IJMS

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Ion-radiation-induced DNA double-strand breaks can lead to severe cellular damage ranging from mutations up to direct cell death. The interplay between the chromatin surrounding the damage and the proteins responsible for damage recognition and repair determines the efficiency and outcome of DNA repair. The chromatin is organized in three major functional compartments throughout the interphase: the chromatin territories, the interchromatin compartment, and the perichromatin lying in between. In this study, we perform correlation analysis using super-resolution STED images of chromatin; splicing factor SC35, as an interchromatin marker; and the DNA repair factors 53BP1, Rad51, and γH2AX in carbon-ion-irradiated human HeLa cells. Chromatin and interchromatin overlap only in protruding chromatin branches, which is the same for the correlation between chromatin and 53BP1. In contrast, between interchromatin and 53BP1, a gap of (270 ± 40) nm is visible. Rad51 shows overlap with decondensed euchromatic regions located at the borders of condensed heterochromatin with further correlation with γH2AX. We conclude that the DNA damage is repaired in decondensed DNA loops in the perichromatin, located in the periphery of the DNA-dense chromatin compartments containing the heterochromatin. Proteins like γH2AX and 53BP1 serve as supporters of the chromatin structure.

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“…Direct STORM (dSTORM) was implemented by Varga et al to reach a resolution of 20 nm and characterize the nucleosome density around γH2AX break sites [111], by Hofmann et al to visualize the topology of γH2AX foci clusters according to their distance to heterochromatin [112], and by Liddle et al to visualize γH2AX nanofoci induced by the radiomimetic drug bleomycin and compare their size and density with endogenous γH2AX foci [113].…”

Section: Super Resolution: Stochastic Techniques For Single Molecule mentioning

confidence: 99%

In Situ Detection of Complex DNA Damage Using Microscopy: A Rough Road Ahead

Nikitaki

Pariset

Sudar

et al. 2020

Cancers

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Complexity of DNA damage is considered currently one if not the primary instigator of biological responses and determinant of short and long-term effects in organisms and their offspring. In this review, we focus on the detection of complex (clustered) DNA damage (CDD) induced for example by ionizing radiation (IR) and in some cases by high oxidative stress. We perform a short historical perspective in the field, emphasizing the microscopy-based techniques and methodologies for the detection of CDD at the cellular level. We extend this analysis on the pertaining methodology of surrogate protein markers of CDD (foci) colocalization and provide a unique synthesis of imaging parameters, software, and different types of microscopy used. Last but not least, we critically discuss the main advances and necessary future direction for the better detection of CDD, with important outcomes in biological and clinical setups.

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dSTORM microscopy evidences in HeLa cells clustered and scattered γH2AX nanofoci sensitive to ATM, DNA-PK, and ATR kinase inhibitors

Cited by 9 publications

References 63 publications

Analysis of Ionizing Radiation Induced DNA Damage by Superresolution dSTORM Microscopy

Analysis of Ionizing Radiation Induced DNA Damage by Superresolution dSTORM Microscopy

Chromatin Organization after High-LET Irradiation Revealed by Super-Resolution STED Microscopy

In Situ Detection of Complex DNA Damage Using Microscopy: A Rough Road Ahead

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