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First published online 9 December 2008
doi: 10.1242/jcs.031062

Journal of Cell Science 122, 83-91 (2009)
Published by The Company of Biologists 2009
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Spatial organization of nucleotide excision repair proteins after UV-induced DNA damage in the human cell nucleus

Liliana Solimando1,*,{ddagger}, Martijn S. Luijsterburg2,*,§, Lorella Vecchio1,*, Wim Vermeulen3, Roel van Driel2,¶ and Stanislav Fakan1

1 Centre of Electron Microscopy, University of Lausanne, 27 Bugnon, CH-1005 Lausanne, Switzerland
2 Swammerdam Institute for Life Sciences, University of Amsterdam, Kruislaan 318, 1098 SM Amsterdam, The Netherlands
3 Department of Cell Biology and Genetics, Medical Genetics Center, Erasmus Medical Center, P.O. Box 1738, 3000 DR Rotterdam, The Netherlands


Figure 1
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  		Fig. 1. Immunofluorescence and live-cell microscopy. (A,B) Example of XPC-EGFP (A) and EGFP-XPA (B) cells immunofluorescently labeled against CPDs in non-irradiated control cells (left panels) or 10 minutes after global UV-C irradiation at 60 J.m–2 (right panels). The EGFP signal is shown in green (upper panels) and the anti-CPD staining of the same cells in red (lower panels). (C) Confocal slice of living XPC-EGFP cells either non-irradiated (upper panel) or globally irradiated at 60 J.m–2 (lower panel). (D) Confocal slice of XPC-EGFP cells immunofluorescently labeled against EGFP in cells 10 minutes after global UV-C irradiation at 60 J.m–2. The intrinsic EGFP signal is shown in green (upper panel) and the anti-EGFP staining of the same cell in red (lower panel). (E) Confocal slice of living EGFP-XPA cells either non-irradiated (upper panel) or globally irradiated at 60 J.m–2 (lower panel). (F) Confocal slice of EGFP-XPA cells immunofluorescently labeled against EGFP in cells 10 minutes after global UV-C irradiation at 60 J.m–2. The intrinsic EGFP signal is shown in green (upper panel) and the anti-EGFP staining of the same cell in red (lower panel). (G) Line scan of the arrows depicted in the upper and lower panels of D, reflecting intrinsic XPC-GFP fluorescence (green line) and anti-EGFP signal (red line) in the same cell. (H) Line scan of the arrows depicted in the upper and lower panels of F, reflecting intrinsic EGFP-XPA fluorescence (green line) and anti-EGFP signal (red line) in the same cell. (I) FRAP analysis of XPC-EGFP in non-irradiated cells (blue line, n=9) or globally irradiated cells at 60 J.m–2 (red line, n=9). (J) FRAP analysis of EGFP-XPA in non-irradiated cells (blue line, n=9) or globally irradiated cells at 60 J.m–2 (red line, n=9).

 

Figure 2
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  		Fig. 2. Ultrastructural localization of CPDs. Example of a human XP2OS-SV fibroblast, immunolabeled using a specific antibody against CPDs, that has been (A) mock treated and (B) fixed 10 minutes after global UV-C irradiation at 60 J.m–2. Samples were contrasted with the EDTA regressive technique in order to reduce the contrast of chromatin. Arrowheads indicate condensed chromatin; arrows designate the perichromatin region. Scale bars: 0.5 µm.

 

Figure 3
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  		Fig. 3. Ultrastructural localization of EGFP-XPA and XPC-EGFP using anti-EGFP antibodies in non-irradiated and UV-irradiated cells. (A) An example of an XP2OS-SV EGFP-XPA-expressing fibroblast that has been mock treated. (B) A nucleus globally irradiated with UV-C at 60 J.m–2. (C,D) Similarly, an XP4PA-SV XPC-EGFP-expressing cell that was mock treated (C) and a UV-C irradiated cell nucleus (D) are shown. The shown examples were fixed 10 minutes following UV irradiation. Cell monolayers were fixed in paraformaldehyde and thin sections were subsequently immunolabeled with antibodies specific for EGFP. Samples were contrasted with the EDTA regressive technique in order to reduce the contrast of chromatin. The arrows designate the perichromatin region and the arrowheads designate condensed chromatin domains. N, nucleolus. Scale bars: 0.5 µm.

 

Figure 4
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  		Fig. 4. Increase in the amount of dense chromatin areas upon global UV-C irradiation. XP2OS-SV EGFP-XPA-expressing cells were (A,C) mock treated or (B,D) fixed 10 minutes after global UV irradiation at 60 J.m–2, and subsequently stained with osmium ammine to specifically visualize DNA (dark color). The analyzed sections were collected at the middle nuclear plane of the cells. C and D represent close-ups of the boxed areas in A and B, respectively. IC, interchromatin compartment. Scale bars: 1.25 µm.

 

Figure 5
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  		Fig. 5. Quantitative evaluations of ultrastructural data. (A) A quantitative evaluation of CPD labeling density in irradiated cells either 10 minutes (n=10) or 1 hour (n=10) following irradiation in condensed chromatin domains (orange bars) and the perichromatin region (blue bars). Values were corrected for residual labeling in non-irradiated cells (n=20). The values represent the average gold-grain number per µm2 nuclear surface area ± s.e.m. (B,C) A quantitative evaluation of EGFP-XPA (blue bars) and XPC-EGFP (orange bars) labeling density (gold particles per µm2 nuclear area) in the perichromatin region (B) or in condensed chromatin domains (C). The labeling density was determined in non-irradiated cells (n=20 for XPC and XPA; NoDa), and in nuclei 10 minutes (n=15 for XPC and XPA) and 1 hour (n=15 for XPC and XPA) following irradiation. Values represent the average ± s.e.m. (D) A quantitative evaluation of the fraction of the nucleus occupied by condensed chromatin domains (%) before (n=29; NoDa) and after global UV irradiation at 60 J.m–2 determined 10 minutes (n=15) or 1 hour (n=15) following UV exposure. The values represent the mean ± s.e.m. NoDa, no UV irradiation; GloDa, after global UV irradiation.

 

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