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

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Abstract

In response to DNA double-strand breaks (DSB), histone H2AX is phosphorylated around the lesion by a feed forward signal amplification loop, originating γH2AX foci detectable by immunofluorescence and confocal microscopy as elliptical areas of uniform intensity. We exploited the significant increase in resolution (~ × 10) provided by single-molecule localization microscopy (SMLM) to investigate at nanometer scale the distribution of γH2AX signals either endogenous (controls) or induced by the radiomimetic bleomycin (BLEO) in HeLa cells. In both conditions, clustered substructures (nanofoci) confined to γH2AX foci and scattered nanofoci throughout the remnant nuclear area were detected. SR-Tesseler software (Voronoï tessellation-based segmentation) was combined with a custom Python script to first separate clustered nanofoci inside γH2AX foci from scattered nanofoci, and then to perform a cluster analysis upon each nanofoci type. Compared to controls, γH2AX foci in BLEO-treated nuclei presented on average larger areas (0.41 versus 0.19 µm2), more nanofoci per focus (22.7 versus 13.2) and comparable nanofoci densities (~ 60 nanofoci/µm2). Scattered γH2AX nanofoci were equally present (~ 3 nanofoci/µm2), suggesting an endogenous origin. BLEO-treated cells were challenged with specific inhibitors of canonical H2AX kinases, namely: KU-55933, VE-821 and NU-7026 for ATM, ATR and DNA-PK, respectively. Under treatment with pooled inhibitors, clustered nanofoci vanished from super-resolution images while scattered nanofoci decreased (~ 50%) in density. Residual scattered nanofoci could reflect, among other alternatives, H2AX phosphorylation mediated by VRK1, a recently described non-canonical H2AX kinase. In addition to H2AX findings, an analytical approach to quantify clusters of highly differing density from SMLM data is put forward.

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Abbreviations

γH2AX:

Histone H2AX phosphorylated on serine 139

ATM:

Ataxia telangiectasia mutated

ATR:

ATM and Rad3-related

BLEO:

Bleomycin

CLSM:

Confocal laser scanning microscopy

DDR:

DNA damage response

DNA-PK:

DNA-dependent protein kinase

DSB:

Double-strand break

dSTORM:

Direct stochastic optical reconstruction microscopy

IR:

Ionizing radiation

LET:

Linear energy transfer

MAPK:

Mitogen-activated protein kinase

PI3K:

Phosphatidylinositol 3-kinase

ROI:

Region of interest

SIM:

Structured illumination microscopy

SMLM:

Single-molecule localization microscopy

STED:

Stimulated emission depletion microscopy

TSS:

Transcription start site

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Acknowledgements

We wish to express our gratitude to Markus Sauer for the invitation to perform dSTORM experiments at the Department of Biotechnology and Biophysics (University of Würzburg) as well as to Pablo Mateos-Gil, Sebastian Letschert and Fabian Zwettler for training and advice in dSTORM methodology (PL). We are also indebted to ANII (National Agency for Research and Innovation, Uruguay) for PhD Scholarship (POS_NAC_2014_1_102214) to PL as well as research support to LL-H and GF, and to PEDECIBA (Program for the Development of Basic Sciences, Uruguay). Research in SCIAN-Lab is funded by the Chilean Millennium Scientific Initiative P09-015-F to IC, JJ-W, SH; FONDECYT 11170475 to IC; FONDECYT 1181823 to IC, SH; FONDECYT 1161274, FONDECYT Ring Initiative ACT-1402, DAAD 57220037 and 57168868, CORFO 16CTTS-66390 to SH; CONICYT PhD Scholarship to JJ-W.

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Authors and Affiliations

  1. Departamento de Genética, Instituto de Investigaciones Biológicas Clemente Estable, Montevideo, Uruguay

    Pablo Liddle, Laura Lafon-Hughes & Gustavo Folle

  2. SCIAN-Lab, Biomedical Neuroscience Institute (BNI), Santiago, Chile

    Jorge Jara-Wilde, Iván Castro & Steffen Härtel

  3. Departamento de Ciencias de la Computación, Universidad de Chile, Santiago, Chile

    Jorge Jara-Wilde

  4. Instituto de Ciencias Biomédicas, Facultad de Medicina, Universidad de Chile, Santiago, Chile

    Steffen Härtel

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  1. Pablo Liddle
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  3. Laura Lafon-Hughes
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Contributions

PL: conceived and designed studies, performed research, analyzed data and wrote the draft manuscript. JJ-W co-wrote data analysis section in Materials and Methods and revised the manuscript. JJ-W, IC and SH developed the scripts for processing the SR-Tesseler files and contributed to data analysis. LL-H: contributed to design experiments, data interpretation and revised the manuscript. GF: revised the manuscript.

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Correspondence to Pablo Liddle.

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11010_2020_3809_MOESM1_ESM.tif

Supplementary file1 (TIF 10820 kb) Supplemental Fig. 1 Selection of the density factor threshold (α) to segment γH2AX foci regions from control and BLEO20 nuclei. In each case, the visual inspection of segmentation results was considered to set an optimal α value. Results are exemplified in a BLEO20 nucleus (a–i). From the localization file image (a), output images harboring the obtained segmented γH2AX foci regions after increasing values of α (0.1–10) are shown (b–i). The corresponding localizations associated, in each case, to the segmented γH2AX foci regions after applying the distinct α are depicted in blue. The nuclear ROI (r1) is outlined by the red line. Bar: 3 µm. As seen on the image, α values ≤ 0.25 overestimated the number of nuclear areas assigned as foci (b, c), while applying α values within the range [0.5, 2] (d–f) matched the foci observed in the original image (a). The use of α ≥ 3 resulted in a growing number of foci not being segmented (g–i), since α values became so demanding that most localization belonging to γH2AX foci regions were incorrectly linked to non-foci areas. In BLEO20 nuclei, α = 1.0 was the parameter value that best equaled the obtained foci segmentation with γH2AX foci regions. Since the mean localization density in the entire nucleus was lower in controls (307 localizations/μm2) than in BLEO-treated nuclei (620 localizations/μm2) a higher α value was needed in these cells to achieve a proper segmentation. α = 2.0 was found the value that best matched foci segmentation with endogenous γH2AX foci in control cells

11010_2020_3809_MOESM2_ESM.tif

Supplementary file2 (TIF 13890 kb) Supplemental Fig. 2 Adjustment of distance parameter d. A Python script was implemented to calculate extended convex hull polygons for each γH2AX focus, parameterized by a distance d, with the purpose of producing two new localization files from each original image (control and BLEO20 nuclei with foci and non-foci localizations) (Fig. 2). Since the parameters for foci segmentation provided in SR-Tesseler led to convex hull polygons (foci regions) with neighboring localizations that could not be included by any set of parameter values, a post-processing step was implemented within the Python script to include these localizations. d is an extra border distance from each convex hull that defines a neighborhood to include localizations from outside the convex hull polygons, reassigning them as part of the segmented foci regions. a–l Output images of a BLEO20 nucleus obtained with increasing values of d (0–1000 nm). Sets of localizations linked to non-foci areas (a, b, e, f, i, j) and the corresponding remaining localizations associated to γH2AX foci (c, d, g, h, k, l) are depicted. The nuclear ROI (r1) is outlined by the red line. Bar: 3 µm. As shown in a, using only the convex hull polygons with no additional distance (d = 0) for each object (γH2AX focus) resulted in a halo of points (localizations) surrounding each substracted γH2AX focus. d = 20 yielded a modest impact on the halos disappearance (b), while d = 50 nm was found to successfully remove them (e), reassigning halo localizations to the pooled foci areas (g). Larger neighborhoods (d ≥ 200 nm) resulted in oversegmentation, with localizations well outside γH2AX foci allocated to foci regions (h–l)

11010_2020_3809_MOESM3_ESM.tif

Supplementary file3 (TIF 228251 kb) Supplemental Fig. 3 dSTORM super-resolution imaging of γH2AX nuclear signal under distinct conditions of BLEO-exposure and post-damage recovery time. Cells were untreated (Control) or exposed to different doses of BLEO: 5 µg/mL (BLEO5), 20 µg/mL (BLEO20), 80 µg/mL (BLEO80) and 160 µg/mL (BLEO160) for 45 min before fixation (PFA 3.7%). In addition, cells exposed to BLEO20 for 45 min were allowed to recover in fresh culture medium for different post-damage times (0.5 h, 1 h and 2 h) before being fixed and immunolabeled. a and b Average foci number per cell (mean ± standard error) in relation to BLEO dose (no recovery time) (a) or in cultures treated with BLEO20 and subjected to different post-damage recovery times (0–2 h) (b). For each condition 11 < n < 32 nuclei, coming from at least two independent experiments, were analyzed (**p < 0.01, ***p < 0.001 with respect to control by one-way ANOVA with Bonferroni correction). An appropriate level of γH2AX foci induction was obtained over the entire dose range (5–160 µg/mL) (a). An increase in foci number was detected only for BLEO160 in comparison with the other doses of BLEO (p < 0.05). Otherwise, a similar outcome was found between cultures exposed to BLEO20 but recovered at different post-damage times (0–2 h) (b). c–m Representative dSTORM images of HeLa nuclei for each condition are shown. Bar: 3 µm. Immunolabeling was performed either with Alexa Fluor 647 (c–k) or Alexa Fluor 532 (l, m) secondary antibodies. Upper left insets: corresponding γH2AX images for each nucleus by standard wide-field fluorescence microscopy. In all cases γH2AX foci were observed to be composed of smaller nanometric subunits when samples were incubated with the primary anti-γH2AX antibody (c–l)

11010_2020_3809_MOESM4_ESM.tif

Supplementary file4 (TIF 55769 kb) Supplemental Fig. 4 Preclusion of γH2AX foci formation was maximized under simultaneous treatment with ATM, ATR and DNA-PK inhibitors. HeLa cells were pretreated for 1 h with 20 µM of ATM, ATR and/or DNA-PK inhibitors (denoted ATMi, ATRi and DNA-PKi, respectively), either alone or in combinations, and then co-exposed for 45 min to BLEO20 and the inhibitor(s) before being fixed in 3.7% PFA and immunostained for γH2AX (Alexa Fluor 532 or Alexa Fluor 647). In addition, untreated cell cultures (control), exposed only to BLEO20 or to the solvent of inhibitors (vehicle control; 1.2% DMSO) were considered. a Foci number per cell (mean ± standard error) for each condition. 10–20 CLSM z-stacks having several nuclei per field, from three independent experiments, were analyzed. 250–650 cells were considered in each case. A diminution of foci number was detected only in BLEO-treated cultures exposed either to ATMi alone or in combination with the other inhibitors when compared to BLEO20 or BLEO20 cultures exposed to ATRi and/or DNA-PKi (**p < 0.01 respect to BLEO20 by one-way ANOVA with Bonferroni correction). b–j Representative slices from cultures exposed to each condition. γH2AX immunostaining is shown in grey. Bar: 15 µm. Occasionally, γH2AX pan-nuclear cells were detected in BLEO-treatment cultures even under simultaneous treatment with the three kinase inhibitors

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Supplementary file5 (TIF 4314 kb) Supplemental Fig. 5 Distribution of γH2AX foci areas for control and BLEO20 nuclei. The histograms of the segmented object areas (μm2) obtained by combining SR-Tesseler with a custom script (Fig. 2) are shown. Altogether, n = 135 (control) and n = 638 (BLEO20) γH2AX foci coming from n = 11 (control) and n = 21 (BLEO20) cells were considered (data from three independent experiments). For each condition, mean foci areas (± standard error) are depicted on the plot. As evidenced by the histogram, BLEO-induced γH2AX foci exhibited a broad range of size, covering all histogram bins. Conversely, endogenous foci in controls were mainly restricted to areas among 0.025 µm2 (the minimum area considered) and 0.3 µm2. On average, BLEO-induced foci areas were approximately two times bigger than endogenous foci areas

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Supplementary file6 (TIF 4318 kb) Supplemental Fig. 6 Presence of γH2AX signal outside nuclei. Images from localization files of a Control, b BLEO20, c BLEO20 + 3i and d Ab control nuclei in SR-Tesseler are shown. Nuclear ROI are outlined by red lines. Bar: 3 µm. As seen on images, in all conditions γH2AX signals were also observed external to nuclear ROI (that is, probably in cytoplasmic areas). By drawing ROI in these regions (not shown) we verified that this signal could be well segmented with the same parameters as the scattered nuclear γH2AX mark. Nevertheless, signal densities were not exhaustively calculated since cytoplasm boundaries could not be precisely defined. In any case, for all conditions the extranuclear density was considerably lower than the nuclear γH2AX density inside ROI (rough estimation)

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Liddle, P., Jara-Wilde, J., Lafon-Hughes, L. et al. dSTORM microscopy evidences in HeLa cells clustered and scattered γH2AX nanofoci sensitive to ATM, DNA-PK, and ATR kinase inhibitors. Mol Cell Biochem 473, 77–91 (2020). https://doi.org/10.1007/s11010-020-03809-4

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