Laser microirradiation is a useful tool for studies of DNA repair in living cells. A methodological approach for the use of UVA lasers to induce various DNA lesions is shown. We have optimized a method for local microirradiation that maintains the normal cell cycle; thus, irradiated cells proceed through mitosis.
Local microirradiation with lasers represents a useful tool for studies of DNA-repair-related processes in live cells. Here, we describe a methodological approach to analyzing protein kinetics at DNA lesions over time or protein-protein interactions on locally microirradiated chromatin. We also show how to recognize individual phases of the cell cycle using the Fucci cellular system to study cell-cycle-dependent protein kinetics at DNA lesions. A methodological description of the use of two UV lasers (355 nm and 405 nm) to induce different types of DNA damage is also presented. Only the cells microirradiated by the 405-nm diode laser proceeded through mitosis normally and were devoid of cyclobutane pyrimidine dimers (CPDs). We also show how microirradiated cells can be fixed at a given time point to perform immunodetection of the endogenous proteins of interest. For the DNA repair studies, we additionally describe the use of biophysical methods including FRAP (Fluorescence Recovery After Photobleaching) and FLIM (Fluorescence Lifetime Imaging Microscopy) in cells with spontaneously occurring DNA damage foci. We also show an application of FLIM-FRET (Fluorescence Resonance Energy Transfer) in experimental studies of protein-protein interactions.
DNA damage leads to the appearance of DNA lesions consisting of cyclobutane pyrimidine dimers (CPDs), 8-oxo-7,8-dihydro-2'-deoxyguanosine, and single-strand or double-strand breaks1,2. γ-rays are the form of ionizing radiation with the highest energy and high penetrance, thus this source of radiation is widely used in radiotherapy3. On the other hand, experimentally induced DNA damage caused by UV lasers mimics natural exposure to UV light. UVA microirradiation, as a microscopic method, represents an experimental tool for studying DNA damage in individual living cells. Microirradiation was used for the first time 40 years ago in order to reveal the organization of chromosome regions4,5. This technique is highly dependent on either the functional properties of confocal microscopes or the technical limits of modern nanoscopy. To induce DNA lesions, cells can be presensitized by 5' bromodeoxyuridine (BrdU) or Hoechst 33342 prior to UV irradiation. Bártová et al.6 previously described the presensitization step, and recently we optimized this microirradiation technique in order to avoid cell death, or apoptosis. For example, the use of a 405-nm UV laser (without Hoechst 33342 presensitization) leads to the induction of 53BP1-positive double-strand breaks (DSBs) at the expense of cyclobutane pyrimidine dimers (CPDs). On the other hand, presensitization steps combined with UV microirradiation induce very high levels of CPDs and DSBs simultaneously7,8. This methodology is difficult to apply to the study of a single DNA repair pathway.
With microirradiation, it is possible to analyze protein recruitment, kinetics, and interaction at DNA lesions in living cells. An example of this method was published by Luijsterburg et al.9 for heterochromatin protein 1β, and we recently showed for the first time that the pluripotency factor Oct4 and a protein associated with Cajal bodies, coilin, are recruited to UV-induced DNA lesions6,10. Protein kinetics at these DNA lesions can also be studied using the FRAP (Fluorescence Recovery After Photobleaching)11,12,13 or FRET (Fluorescence Resonance Energy Transfer) techniques14,15. These methods have the potential to reveal simple diffusion of proteins at DNA lesions or protein-protein interactions. A useful tool for additional characterization of proteins is FLIM (Fluorescence Lifetime Imaging Microscopy) or its combination with FRET technology (FRET-FLIM)16. These methods enable the study of processes in living cells that are stably or transiently expressing the protein of interest tagged by a fluorescent molecule17. Here, an example of exponential decay time (τ) for GFP-tagged p53 protein and its interaction partner, mCherry-tagged 53BP1, playing an important role in DNA damage response18,19 is shown. The parameter τ, the lifetime of the fluorochrome provided by FLIM calculations, is specific for a given fluorescence dye, its binding abilities, and its cellular environment. Therefore, this method can show us distinctions between protein subpopulations, their binding abilities, and their functional properties after, for example, DNA damage.
Here, an outline of the methodological approaches of the advanced microscopy techniques that is used in our laboratory to study time-specific protein recruitment, kinetics, diffusion, and protein-protein interactions at the site of microirradiated chromatin is presented. The step-by-step methodology for the induction of local DNA lesions in living cells, and a description of methodologies useful for studies of DNA-damage-related events at locally induced DNA lesions caused by UV lasers are provided.
1. Cultivation of Cell Lines
2. Cell Transfection
3. Induction of Local DNA Lesions and Confocal Microscopy
4. Immunofluorescence Staining
5. Fluorescence Lifetime Image (FLIM) Microscopy
6. Donor Lifetime Using a FLIM Script and Calculation of FRET Efficiency
7. FRET Efficiency Using the FLIM-FRET Script
8. FRAP Analysis
Using advanced confocal microscopy, we observed an accumulation of mCherry-tagged 53BP1 and mCherry-PCNA proteins at DNA lesions. Analyses were performed by local microirradiation of living cells. To recognize the nuclear distribution patterns of DNA-repair-related proteins in individual cell cycle phases, we used the Fucci cellular system, by which it is possible to determine the G1, early S, and G2 phases of the cell cycle (Figure 1). The biological application of the Fucci cellular model that we published in Suchankova et al.26 shows that 53BP1 was recruited to locally induced DNA lesions in the G1, S, and G2 phases of the cell cycle, which was associated with the function of this protein during non-homologous end joining (NHEJ), one of the main mechanisms leading to double-strand break repair. On the other hand, this experimental system showed us the individual cellular level that the PCNA protein, which is linked to the homologous recombination repair pathway (HRR), recognizes DSBs in the late S and G2 phases of the cell cycle 27. For studies on DNA repair machinery, we also optimized irradiation conditions in order to ensure that cell cultures continued to undergo mitosis after exposure to UV lasers (Figure 3). Microirradiated cells undergoing mitosis prove that, in these experimental conditions, DNA repair proceeds in a relatively physiological manner and cells have not been injured by the lasers, which at higher intensities induce apoptosis. Here, we also show how to apply FRET-FLIM analysis to the characterization of DNA repair proteins. This advanced technology enables us to study local protein-protein interactions in the cell nucleus or, alternatively, protein interactions in nucleolar regions such as the nucleolus, nuclear lamina, or clusters of heterochromatin. For beginners in this technology, we would like to recommend optimizing this FRET-FLIM technique by using well-known interacting protein partners such as p53 and 53BP1 (Figure 4A-C). In general, a knowledge of protein-protein interaction leads to understanding how protein complexes regulate processes like replication, gene activation, silencing, or DNA repair. In addition, a very useful tool for studies of protein kinetics is the FRAP method, which shows the characteristics of local protein diffusion and mobility (Figure 4D). Taken together, here we provide methodological instructions for applying advanced confocal microscopy techniques in DNA repair studies.
Figure 1: Formation of DNA repair foci can be studied in HeLa-Fucci cells expressing RFP-cdt1 (red) in the G1/early S phases and GFP-geminin (green) in the S/G2 phases of the cell cycle. Spontaneously occurring DNA lesions positive for 53BP1 protein (blue; Alexa 405 staining), were studied in the G1 (red), early S (orange; expression of both RFP-cdt1 and GFP-geminin), and G2 (green) phases of the cell cycle. Please click here to view a larger version of this figure.
Figure 2: Recruitment of mCherry-tagged PCNA at DNA lesions over time. (A) Cells expressing mCherry-PCNA (red), were located on a gridded microscope dish in selected regions (gray). After fixation and immunostaining, irradiated cells (yellow arrows) were located according to registered coordinates (see the gray letter K as an example) (Aa-c). The yellow frame and arrows (Ad) show the cell that was microirradiated and magnified in panels Ae-f. (B) Accumulation of mCherry-PCNA (red) was studied in HeLa cells stably expressing GFP-tagged histone H2B (green). Cells were monitored immediately after local microirradiation for up to 35 min (see Video 1). (C) Microirradiated cells were analyzed by 3D confocal microscopy, and protein accumulation at DNA lesions (e.g., mCherry-53BP1) was found in all three dimensions (x-y, x-z, and y-z) although only the midsection of the cell nucleus was microirradiated (see 3D-cell rotation in Video 2 3D projections in Figure 2C). Please click here to view a larger version of this figure.
Figure 3: Cells microirradiated by a 405-nm laser diode proceed normally through the cell cycle. (A) After irradiation using a 405-nm diode laser, HeLa cells (stably expressing GFP-histone H2B; green) undergo mitosis (see also Video 2). Yellow arrows and frames indicate irradiated ROI in selected cells. White frames show mitosis.(B) Under the same experimental conditions, mitosis in irradiated cells was also observed by time lapse microscopy in HeLa cells stably expressing GFP-H2B (green) and transiently expressing mCherry-PCNA (red). Please click here to view a larger version of this figure.
Figure 4: FLIM and FRET-FLIM analysis of GFP-p53 and mCherry-53BP1. Fluorescence Resonance Energy Transfer (FRET) detected by FLIM and application of Fluorescence Recovery After Photobleaching(FRAP) at various DNA lesions.(A-C) Averaged (n=10) lifetimes of GFP-tagged p53 in the absence and presence of the acceptor (mCherry-53BP1), measured in whole nonreplicating HeLa cell nuclei. (A) Representative fluorescence decay curves and residuals studied in HeLa cells transiently expressing GFP-p53 (donor-only, τD) or GFP-p53 and mCherry-53BP1 (donor – acceptor, τDA). (B) Averaged lifetimes (τ1 -τ3) and amplitudes (A1 – A3) of (a) GFP-p53 and (b) mCherry-53BP1 measured in whole HeLa cell nuclei. χ2 values were also calculated and are shown. (C) Summary of FRET efficiency for the well-known interacting partners GFP-p53 and mCherry-53BP1. Scale bars = 4-5 µm. FRET-FLIM result showing ~35% FRET efficiency for well-known interacting partners GFP-tagged p53 and mCherry-tagged 53BP1. (D)Recovery kinetics of mCherry-53BP1 studied at spontaneously occurring DNA lesions (green curve) and UVA-induced DNA lesions (blue curve). mCherry at DNA lesions was bleached to the level of the background (dispersed form of protein of mCherry-53BP1 protein), and the background fluorescence was subtracted from each value. Data, shown as relative fluorescence intensity of mCherry-53BP1, are presented as the means ± standard error. Student's t-test revealed a statistically significant difference between two types of DNA lesions (* shows p ≤0.05). Please click here to view a larger version of this figure.
Video 1: Recruitment of mCherry-tagged PCNA protein (red fluorescence) to DNA lesions induced by local microirradiation in HeLa cells stably expressing GFP-tagged histone H2B (green fluorescence). Please click here to download this video.
Video 2: Accumulation of mCherry-tagged 53BP1 protein (red fluorescence) at DNA lesions induced by local microirradiation in HeLa cells. The cell nucleus is shown in 3D space using a software mode that enables the visualization of cell rotation in space. Please click here to download this video.
Video 3: Microirradiated HeLa cells stably expressing GFP-tagged histone H2B (green fluorescence) proceed through mitosis. The irradiated region is characterized by the depletion of GFP-H2B (black regions are irradiated strips inside the cell nuclei). Please click here to download this video.
Microscopy techniques represent basic tools in research laboratories. Here, a brief description of the methods used for the study of protein recruitment and kinetics at DNA lesions is presented. We especially noted our experimental experience in the field of local microirradiation of living cells, and we discuss the study of protein kinetics by FRAP and protein-protein interaction at DNA lesions by acceptor-bleaching FRET28 and its advanced modification FRET-FLIM (Figure 4A-D). The methods shown here are essential tools for a true understanding of DNA repair processes, especially protein kinetics in living cellular systems, as inspected by advanced confocal microscopes6,7,8,28. These methods are of immense utility to future medicine in dealing with the effects of radiotherapy on tissues or characterizing tumor cell morphology. To optimize this method, we would like to recommend starting with well-known interacting protein partners, as shown in Figure 4C.
It is well known that protein recruitment to DNA lesions is highly dynamic and time-dependent; thus, the application of time-lapse confocal microscopy after cell irradiation is required not only in the field of basic science but also in clinics26. Locally induced DNA lesions due to laser microirradiation9,28,29 represent genomic regions where it is possible to study protein recruitment, protein-protein, or protein-DNA binding and interaction. The starting point of these methods is that the recruitment of exogenous proteins must be verified by appropriate antibodies on the endogenous level. Next, the critical step of such experiments is that over-transfected cells can provide false-positive results; thus, the best decision is to establish cell lines stably expressing the fluorescently tagged proteins of interest. Moreover, due to the phototoxic effects of the lasers used for image acquisition, either the conditions for scanning must be optimized or Trolox compound must be dissolved in the cell cultivation medium. Additionally elimination of the presensitization step before irradiation is recommended. After local microirradiation, DNA repair must proceed such that the cells undergo mitosis (Figure 3A-B and Video 3). Induction of apoptosis after laser irradiation is a less valuable process from the view of optimal DNA repair23. It is also evident that some DNA repair proteins recognize DNA damage only in specific cell cycle phases (e.g., Bártová et al.30). Thus, the use of the HeLa-Fucci cellular system, showing the cells in the individual phases of interphase, is a useful tool for studies of DNA damage response. In addition, cell cycle phases can be recognized according to the nuclear distribution pattern of the PCNA protein31.
It is well known that the FRAP and FRET methods represent very useful tools for investigating the characteristics of proteins that are recruited to DNA lesions7,8,32. Moreover, the FLIM technique32 can reveal information about the dynamic conformational changes of the proteins that accumulate at DNA lesions. The use of this method is important because FLIM measures dynamic cellular processes directly; thus, steady-state fluorescence intensity is measured over time. FLIM approaches increase image contrast by eliminating background fluorescence. This is an advantage of FRET-FLIM measurement in comparison with conventional acceptor-bleaching FRET. The fluorescence lifetimes measured by FLIM provide information about the fractions of fluorescently tagged proteins and show how heterogenic probes are due to environmental conditions. FLIM is also the best and most reliable biophysical approach to performing FRET for protein-protein interaction in living cells32,33.
Taken together, all of the described methods have the potential to reveal new mechanisms of the DNA damage response in human cells, which is important especially for radiotherapeutic approaches. For example, multiphoton FLIM has been applied to obtain high-resolution images in medicine, where it is called multiphoton tomography34. This highly sophisticated microscopic method can be applied in clinics for cancer diagnosis using histochemical samples and analyzed with high resolution. FLIM methods can additionally provide a precise analysis of tumor cell surfaces according to their autofluorescence. One disadvantage of the FLIM-FRET method is its highly sophisticated software background, which must be fully understood by the microscope operator. Further biological relevance of FLIM data, in general and in DNA repair processes, will certainly be revealed in the near future.
The authors have nothing to disclose.
This work was supported by the Grant Agency of the Czech Republic, project P302-12-G157. Experiments were also supported by the Czech-Norwegian Research Programme CZ09, which is supervised by Norwegian funds, and by the Ministry of Education, Youth and Sport of the Czech Republic (grant number: 7F14369).
Cell cultivation | |||
HeLa | ATCC | CCL-2TM | |
ES-D3 [D3] | ATCC | CRL-11632TM | |
HeLa -Fucci cells | http://ruo.mbl.co.jp/bio/e/product/flprotein/fucci.html | ||
DMEM | PAN-Biotech | P03-0710 | |
DMEM high glucose | Sigma-Aldrich | D6429-500ML | |
Fetal bovine serum (FBS) | HyClone | SV30180.03 | |
ES Cell FBS | Gibco | 16141-079 | |
Non-Essential Amino Acids (NEAA) | Gibco | 11140-035 | |
mLIF (mouse Leukemia Inhibitor Factor) | Merck Millipore | ESG1107 | |
MTG (1-Thioglycerol) | Sigma-Aldrich | M6145-25ML | |
Penicillin-Streptomycin Solution | Biosera | XC-A4122/100 | |
Trypsin – EDTA | Biosera | XC-T1717/100 | dilute with 1 × PBS in ratio 1:6 |
Nunclon cell culture dishes | Sigma-Aldrich | P7866 | cultivation of mESCs D3 cells |
µ-Dish 35m+A15:I35m Grid-500 | Ibidi GmbH | 81166 | microscopic dish |
0.2% Gelatine | Sigma-Aldrich | G1890-100G | dilute in destille water and autoclaved |
Name | Company | Catalog Number | Comments |
Cell transfection | |||
GFP-p53 plasmid | Addgene | 12091 | |
mCherry-PCNA plasmid | generous gift from Cristina Cardoso, Technische Universität Darmstadt | ||
mCherry-53BP1 plasmid | Addgene | 19835 | |
Metafecetene | Biontex Laboratories GmbH | T020–2.0 | |
10 × PBS | Thermo Fisher Scientific | AM9625 | for transfection use 1 × PBS diluted in nuclease-free water |
5-bromo-2’-deoxy-uridine | Sigma-Aldrich | 11296736001 | |
Name | Company | Catalog Number | Comments |
Confocal microscopy | |||
Microscope Leica TCS SP5 | Leica Microsystems | ||
Microscope Leica TCS SP8 | Leica Microsystems | ||
White-light laser | Leica Microsystems | ||
355-nm laser | Coherent Inc. | laser power 80 mW | |
405-nm laser | Leica Microsystems | laser power 50 mW | |
Name | Company | Catalog Number | Comments |
Immunofluorescence staining | |||
Coverslip | VWR International Ltd | 631-1580 | |
4% paraformaldehyde | Affymetrix | 19943 1 LT | |
Triton X100 | MP Biomedicals | 2194854 | |
Saponin from quillaja bark | Sigma-Aldrich | S4521 | |
BSA | Sigma-Aldrich | A2153 | |
53BP1 | Abcam | ab21083 | primary antibody |
AlexaFluore 647 | Thermo Fisher Scientific | A27040 | secondary antibody |
Vectashield | Vector Laboratories Ltd | H-1000 | mounting medium |