Here, we present a simple and rapid protocol for the detection of protein interaction at DNA damage sites.
The DNA damage response is a genetic information safeguard that protects cells from perpetuating damaged DNA. The characterization of the proteins that cooperate in this process allows the identification of alternative targets for therapeutic intervention in several diseases, such as cancer, aging-related diseases, and chronic inflammation. The Proximity Ligand Assay (PLA) emerged as a tool for estimating interaction between proteins as well as spatial proximity among organelles or cellular structures and allows the temporal localization and co-localization analysis under stress conditions, for instance. The method is simple because it is similar to conventional immunofluorescence and allows the staining of an organelle, cellular structure, or a specific marker such as mitochondria, endoplasmic reticulum, PML bodies, or DNA double-strand marker, yH2AX simultaneously. The phosphorylation of the S139 at Histone 2A variant, H2AX, then referred to as yH2AX, is widely used as a very sensitive and specific marker of DNA double-strand breaks. Each focus of yH2AX staining corresponds to one break in DNA that occurs a few minutes after the damage. The analysis of changes in yH2AX foci is the most common assay for studying if the protein of interest is implicated in DNA damage response (DDR). Whether a direct role in the DNA damage site is expected, fluorescence microscopy is used to verify the colocalization of the protein of interest with yH2AX foci. However, except for the new super-resolution fluorescence methods, to conclude, the local interaction with DNA damage sites can be a little subjective. Here, we show an assay to evaluate the localization of proteins in the DDR pathway using yH2AX as a marker of the damage site. This assay can be used to characterize the temporal localization under different insults that cause DNA damage.
Cellular DNA damage occurs daily because of the spontaneous chemical reactions and is also increased by exogenous factors such as genotoxic agents (radiation and chemicals such as etoposide) and oxidative stress1,2,3. The cells have a complex machinery that corrects a myriad of different types of DNA damage, from removal of bases to replicative fork torsion or interruption to the most deleterious lesion: the DNA double-strand break4,5.
Several proteins that participate in the DDR have already been characterized, and excellent revisions explore the pathways of non-homologous end join (NHEJ) and homologous recombination (HR), the main pathways implied in double-strand breaks (DSB) repair5,6. The phosphorylation of the histone H2A variant, H2AX, at serine 139 (thereafter named y-H2AX) has been used for many years as a sensitive and reliable marker of the double-strand break. The phosphorylation of H2AX is observed in the first minutes following the damage and can persist for hours7.
The advantage of using the immunofluorescence detection of γ-H2AX foci is the characterization of the temporal and spatial distribution of the foci, as well as its co-staining with other proteins. Moreover, immunofluorescence does not require lysis, which preserves cellular context information and makes it more informative. Besides, as γ-H2AX is formed de novo, it is not abundantly present in the absence of DNA damage, making it a specific marker7.
H2AX is mostly phosphorylated by protein kinase ataxia-telangiectasia mutated (ATM) and DNA-dependent protein kinase (DNA-PK), the sensors of DSBs. Ku70/80 (XRCC6 X-ray repair cross-complementing 6/ XRCC5 X-ray repair cross-complementing 5) and DNA-protein kinase catalytic subunit (DNA-PKcs) are responsible for DSB identification and the protection of DNA ends, whereas Artemis carries out end-processing to facilitate ligation by the XRCC4-Ligase IV complex. The phosphorylation of H2AX at sites flanking DSBs acts as a signal for the recruitment of several repair proteins and signaling for cell cycle arrest, death, or survival8.
The analysis of changes in y-H2AX foci is the most common assay for studying if the protein of interest is implicated in DNA damage response. Whether a direct role in the DNA damage site is expected, fluorescence microscopy is used to verify the co-localization of the protein of interest with y-H2AX foci. However, except for the new super-resolution fluorescence methods, concluding the local interaction with DNA damage sites can be tricky. Furthermore, immunofluorescence detection usually depends on many protein molecules at the DNA damage site, making it difficult to identify scarce proteins9.
Here, we show an assay to evaluate the localization of proteins involved in the DDR pathway using y-H2AX as a marker of the damage site. This assay can be used to characterize temporal localization under different insults that cause DNA damage.
The Proximity Ligand Assay (PLA) emerged as a tool for estimating interaction between proteins as well as spatial proximity among organelles or cellular structures10,11and allows the temporal localization and co-localization analysis under stress conditions, for instance. The method is simple because it is like conventional immunofluorescence and allows the simultaneous staining of organelle or cellular structure-specific markers, such as mitochondria, endoplasmic reticulum, PML bodies, or DNA double-strand marker, yH2AX. The use of PLA allows the identification of protein interaction during DNA damage in a sensitive, specific, and reliable manner that can be used to monitor the interaction during the cell cycle, in response to different stimuli, and at different times after genotoxic stress.
We show here the use of PLA concomitantly to conventional γ-H2AX immunofluorescence to localize Nek4-Ku70 interaction after etoposide-induced DNA damage. Nek4, a Nek (NIMA-related kinases) family member, has no clear role in DNA Damage response. In 2012, Nguyen and colleagues showed that Nek4 interacts with Ku70/Ku80 proteins, and in the Nek4 absence, these proteins are not recruited to the DNA damage site, and H2AX phosphorylation is impaired12. The cellular localization of this interaction is unknown so far. We have observed a reduction in Ku70 phosphorylation in cells expressing Nek4 kinase-dead mutant13but whether Nek4 phosphorylates directly Ku70 remains to be elucidated. Considering that Nek4 interaction with Ku70 is increased after etoposide treatment according to immunoprecipitation studies12, we attempt to localize this interaction using PLA and the γ-H2AX marker.
Usually, the interaction studies are based on immunoprecipitation assays, but these are in vitro and do not provide information about the location of the interaction. When possible, an immunoprecipitation of fractioned cells (nucleus, mitochondrial, cellular membrane, or cytosol) can be performed, but performing a time course of the interaction is costly and laborious. Immunofluorescence can give information about the cellular localization of the proteins, but proving the interaction can be difficult and depends on the resolution of the microscopes. Here, we show the advantage of using the Proximity Ligand Assay to monitor the interaction of two proteins in a DNA damage context.
1. Cell plating
NOTE: Cells can be seeded in microscopic fluorescence slides, chambers, or plates. The use of small coverslips or 96/ 384 well plates is recommended for testing multiple conditions and spare reagents. The use of coverslips is advisable for cell lines that detach easily, like HEK293 cells. The coverslips can be previously coated with a poly-L-lysine solution to improve the attachment.
2. Preparation of solutions
NOTE: The concentration and times of the treatment were chosen based on literature results, which point out the activation of DNA damage repair pathways15,16.
3. Cell treatment and fixation
NOTE: Do not allow cells to dry at any moment; try to tap off the previous solutions as much as possible. The appropriate method for cell fixation (ice-cold methanol or 4% paraformaldehyde (PFA)) must be determined previously, as well as the antibody dilution, using a conventional immunofluorescence experiment.
CAUTION: Methanol must be disposed of properly.
4. Permeabilization and blocking
NOTE: If using the PFA fixation method, a step of blocking with glycine must be performed before permeabilization. The humidity chamber can be prepared by putting a water pan inside the incubator, resulting in a humidity of around 95%. The 384 plate or slides can be incubated in a closed plastic container above filter paper or moistened sponge and transparent film.
5. Primary antibody incubation (PLA)
NOTE: If using coverslips, it is important to ensure that the antibody solution covers all coverslip surfaces. Special attention must be paid to antibody selection. The antibodies must be from different animal species, and the secondary antibody used for γ-H2AX must not be against the species host of the PLA probes production. For example, the primary antibodies used for PLA were raised in mice and goat animals. The PLA probes, anti-mouse, and anti-goat were produced in the donkey. The antibody against γ-H2AX was produced in rabbits, and the secondary utilized is anti-rabbit produced in donkeys. In this way, the secondary will not recognize the PLA probes.
6. PLA probe incubation
NOTE: Choose the probes, taking into consideration not only the primary antibodies but also those intended for use in the immunofluorescence steps. When using 384-well plates for long 37 °C incubation, slow rotation can be used to improve the distribution of the antibody solution.
7. Ligation
NOTE: Wait to add Ligase until immediately before adding it to the samples. Ligase must be kept in a freezer block (-20 °C). Remove completely all the wash solution before adding the ligase solution. Avoid leaving the cells without a solution for long periods of time. When using 384-well plates for the 37 °C incubation, slow rotation can be used to improve the distribution of the antibody solution.
8. Amplification and washing
NOTE: Wait to add the polymerase until immediately prior to addition to the sample. The amplification buffer is light sensitive; protect all solutions containing the buffer from light. Polymerase must be kept in a freezer block (-20 °C). Avoid leaving the cells without a solution for a long period of time. When using 384-well plates for 37 °C incubation, slow rotation can improve the distribution of the antibody solution.
9. γ-H2AX staining (Immunofluorescence – IF)
NOTE: The labeling of γ-H2AX is performed after the PLA is finished.
10. Image acquisition
11. Image analysis
NOTE: A macro was created for analysis of dots per nucleus in Image J/FIJI17 and to be used with microscopes where the scale information must be added manually. The pixel size for the objective lens used must be known. Place the images for each channel in different folders (Supplementary Coding File 1, Supplementary Figure 1).
NOTE: The macro considers fluorescence channels obtained separately. In this case, it is important to save all the images with the same name.
12. Visualizing the merged images
We have observed Nek4-Ku70 interaction in the absence of etoposide treatment. However, this interaction can occur outside of the nucleus (Figure 1A). The Nek4-Ku70 interaction increases after DNA damage and is concentrated in the nucleus (Figure 1A). In the case of Nek5-Topoisomerase II β (TOPIIβ) interaction, used in this assay as a positive control based on literature results18, the interaction is greatly increased by etoposide treatment (Figure 1B).
The etoposide treatment promotes an increment of almost 40% (an average of 9 dots/nucleus in vehicle-treated cells versus 12 – 14 dots/nucleus in etoposide-treated cells). The interaction of Nek4 and Ku70 is not exclusive of DNA damage conditions. Indeed, Nguyen et al.12 showed that these proteins can interact in the absence of the cellular insult and are not dependent on DNA, meaning that the interaction is not always at DNA damage sites.
The γ-H2AX staining is absent in control conditions but increases after 20 min of etoposide treatment and is sustained until 3 h of treatment (Figure 1C). Some of the interaction dots are close to γ-H2AX staining (Figure 1C). After 1 h of treatment, most of the Nek4-Ku70 interaction dots are concentrated in DNA damage foci (Figure 1C).
The knockdown for Nek4 is around 70% (data not presented), meaning that the signal will not be completely eliminated. We believe that the difference in the number of dots/nuclei is not equivalent to protein depletion because the reminiscent protein will be dedicated to DNA damage response and then interact with Ku70. The average of dots/nucleus found in Nek4 knockdown cells treated for 3 h with etoposide is 10, meaning around a 30% reduction compared to control PLKO cells in the same condition. If we consider that etoposide increases Nek4-Ku interaction in 40% related to vehicle-treated cells, and we have 70% of Nek4 knockdown, knockdown cells will present around 10 dots/nucleus. The interaction of Nek4 and Ku70 is not exclusive of DNA damage response, and although it is increased after DNA damage, the major change is in the localization of the interaction.
The results shown here present an important tool to localize the interaction of proteins in the context of DNA damage response. Nek4 and Ku70 have already been implicated in DDR, and Nek4 seems to be important to Ku70/Ku80 recruitment to DNA damage foci12. However, the spatial and temporal characteristics of this interaction can be explored using PLA concomitantly with γ-H2AX staining.
Other techniques, such as co-immunoprecipitation and immunofluorescence, can provide similar information, but separately. Using the γ-H2AX marker as a reference allows temporal and spatial monitoring of the interaction.
Figure 1: Nek4 – Ku70 interaction in DNA damage conditions. (A) U2OS cells were incubated with etoposide (25 µM) for 20 min, 1 h and 3 h. DMSO (0.05% for 3 h) was used as a vehicle. After the treatment, cells were fixed with ice-cold methanol, and the PLA was performed using goat anti-Nek4 and mouse anti-Ku70 as primary antibodies. The nucleus was stained with Hoechst 33342 and is presented in blue; PLA dots are presented in red. Control cells were cells treated with etoposide for 1 h and incubated only with one primary antibody (goat anti-Nek4) and both secondary antibodies. Biological control was Nek4 knockdown cells (shRNA Nek4) treated for 3 h with etoposide. (B) Positive control of the assay based on literature results: U2OS cells were incubated with 25 µM of etoposide for 1 h, and the PLA was performed with mouse anti-Nek5 and rabbit anti-TOPIIβ. The graph is representative of one experiment and represents the average plus standard error mean (SEM) from all cells comprised in at least 4 different fields (at least 35 cells for control and 50 cells for all other conditions). The significance was obtained using the t-test and *: p<0.05, **p<0,01, and *** p<0,001 compared to the DMSO vehicle. (C) After PLA, cells presented in (A) were washed with PBS and incubated with rabbit anti- γ-H2AX antibody. After 1 h of incubation, the secondary antibody against rabbit was used for DNA damage foci detection and is presented in green. The regions containing co-staining of PLA and γ-H2AX are zoomed in. Please click here to view a larger version of this figure.
Supplementary Figure 1: Folder structure. The images from Nucleus and PLA should be placed in different folders, as shown here. The file names should start with a specific prefix (e.g., DAPI, PLA) and should have a corresponding image in each folder (e.g., DAPI_control1.tif and PLA_control1.tif). Please click here to download this File.
Supplementary Figure 2: Macro parameters window. After opening/running the macro in ImageJ/FIJI, the user will be prompted to choose data folders and enter the parameters. Please click here to download this File.
Supplementary Coding File 1: Macro File Created for ImageJ. Please click here to download this File.
The data show that the use of PLA concomitantly to a DNA-damaged marker can provide the most information in a DNA damage response profiling, showing the spatial and temporal behavior of the interaction after the insult. PLA is a versatile method that has been used for dimerization identification, organelles contact determination, protein-nucleic acid interaction, and mainly protein-protein interaction10,11,19,20. The critical steps in performing PLA are determining and validating primary antibodies, cell seeding density, washes, and temperature incubation. We believe that using this tool concomitantly with conventional immunofluorescence allows for specifying the region, time, and distribution of the interactions.
It is imperative to use the appropriate technical controls and it is strongly recommended to use biological control. Moreover, performing the analysis with the same parameters for all conditions allows the comparison among the samples.
Although the PLA is a simple assay that does not require special equipment, the commercial reagents are still a little expensive, and for this reason, this technique is indicated for confirmatory assays or when substantial information regarding the interaction and a role in DNA damage response is found in the literature. Another pitfall of the technique is the incompatibility of the reagents, which is also found in conventional fluorescence microscopy analysis. If the interaction is rare or very transient, PLA is a good choice because of its high sensitivity. However, when the number of interactions is small, it can be difficult to observe changes in different conditions, requiring an increase in the number of replicates. Also, if many molecules are participating in the interaction at the same site, the detection of PLA signals can be limited by the resolution of the objective lens, and the number of interactions can be underestimated. Particles that are closer than the resolution limit of the objective lens will coalesce, not allowing to distinguish individual protein-protein interactions. If the dots are dense and may comprise multiple spots, it is recommended to use the averaged intensity per area for quantification21.
The proximity ligand assay must be considered with caution because a positive signal can be generated from indirect interactions. The main limitations of the PLA are related to the cost of the reagents, with the requirement of specific antibodies that must be tested previously by immunofluorescence, and in the use of technical and biological controls. When performing simultaneously an assay with a marker to cellular specific localization, the antibody must be from a different species from all the other antibodies used in PLA assay. Moreover, the colocalization of the dots with the marker can only be determined using confocal microscopes.
The advantages are related to the smaller number of cells needed compared to immunoprecipitation studies, greater sensitivity since the signal from a few molecules is amplified, generating a strong signal, and concomitant information regarding the interaction and its cellular localization. We have provided here a simple protocol to localize protein interactions in the DNA damage context and a user-friendly macro for use in the analysis of the data. This protocol allows spatial and temporal localization of interactions after different stress genotoxic stimuli.
The authors have nothing to disclose.
We thank Fundação de Amparo a Pesquisa do Estado de São Paulo (FAPESP, through Grant Temático 2022/15126-9 to JK and fellowship 21/09439-1 to LARM) and the Conselho Nacional de Desenvolvimento Cientifico e Tecnológico (CNPq) for funding this research.
Black 384-well plates | Perkin Elmer Cell carrier plates | ||
Donkey anti- Rabbit Alexa Fluor 488 | Invitrogen | A21206 | 1:300 |
Duo link Donkey anti Mouse Minus | Sigma | DUO92004 | |
Duolink antibody diluent | Sigma | DUO82008 | |
Duolink blocking solution 1X | Sigma | DUO82007 | |
Duolink Detection reagent Far red | Sigma | DUO92013 | |
Duolink Donkey anti goat plus | Sigma | DUO92003 | |
Duolink Donkey anti rabbit plus | Sigma | DUO92002 | |
Etoposide | Sigma | E1383 | |
Goat anti Nek4 | Santa Cruz Biotechnology | SC-5517 | goat anti Nek4 was used at 1:50 dilution |
Hoechst 33342 | Thermo | H1399 | 0.6 µg/mL |
Leica DMI microscope | Leica | ||
Mouse anti Ku70 | Thermo | MA5-13110 | mouse anti Ku70 was used at 1:100 dilution |
Mouse anti TOPIIβ | Santa Cruz Biotechnology | SC-365071 | 1:25 |
Rabbit anti Nek5 | Santa Cruz Biotechnology | SC-84527 | 1:25 |
Rabbit anti Y H2AX | Cell Signalling | 9718S | 1:100 dilution |
U2OS cell line | ATCC | HTB-96 |
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