Proximity Ligand Assay to Localize Proteins in DNA Damage Sites

Summary

Here, we present a simple and rapid protocol for the detection of protein interaction at DNA damage sites.

Abstract

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.

Introduction

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 stress^1,2,3. 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 break^4,5.

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) repair^5,6. 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 structures^10,11and 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.

Protocol

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.

1. Plate cells considering a vehicle, technical controls, and biological controls. Use technical replicates at two or three wells per condition.
   NOTE: The use of technical control (both secondaries and one primary only) is mandatory. If possible, biological control should also be included (e.g., overexpression or knockdown of one of the target proteins or a condition that is known to change the interaction, for example).
   1. Culture U2OS cells in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% of Fetal Bovine Serum (FBS), 4.5 g/L Glucose, and 4 mM of L-Glutamine on tissue culture-treated plates at 37 °C in 5% CO₂ and a humidified atmosphere containing 90% air. Depending on the number of conditions to be tested and if using a 384 or coverslips at a 60 mm plate, start from a 60- or 100-mm plate, respectively.
   2. When the cells reach 60%-70% confluency, split cells using 0.25% trypsin-EDTA ¹⁴. Count cells using a Neubauer chamber or an automatic counter¹⁴. Plate 4 x 10⁵ cells in 60 mm plate containing multiple 13 mm round coverslips or 4 x 10⁵ cells per well in 384-well plate(s) and incubate for 12 - 24 hours at 37 °C in a humidified, 5% CO₂ incubator.

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 pathways^15,16.

1. Prepare the stock solution of Etoposide at 50 mM concentration in DMSO. The stock solution can be kept at -20 °C for several months. Dilute the etoposide solution for use in the complete culture medium at 25 µM final concentration. Use DMSO at 0.05% diluted in the complete culture medium as vehicle control.
2. Prepare 20x Saline Sodium Citrate (SSC; 3.0 M NaCl, 0.30 M trisodium citrate pH 7.0). This solution can be kept for several months at 4 °C.

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.

1. Remove the culture medium from the wells and add a culture medium containing etoposide or DMSO at 25 µM or 0.05%, respectively. Incubate cells at 37 °C in 5% CO₂ and a humidified atmosphere containing 90% air with etoposide or DMSO for 3 different periods of time: 20 min, 1 h, and 3 h. When using 384 plates, all conditions must be fixed simultaneously.
   1. For this, start by treating the condition with the longest time of treatment, such as 3 h. After 2 h, treat the 1 h treatment condition, and when there is 20 min left for fixation, treat the 20 min condition. Following this, all conditions can be fixed at the same time.
2. Remove the medium from the wells and wash the cells with PBS 3x.
3. Fix the cells by adding ice-cold methanol with enough volume to cover the well surface. Incubate for 10 min at -20 °C. Wash the wells with PBS 3x.
   NOTE: At this point, the protocol can be paused. The plates can be stored at 4 °C for 1 week.

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.

1. Tap off the PBS from the samples.
2. Add 30-40 µL of permeabilization buffer (0.2% Triton X-100 in PBS) and incubate for 20 min at room temperature. Wash 3x with PBS
3. Tap off the PBS from the samples and add 30-40 µL of 1x blocking solution provided in the PLA kit (alternatively, 3% of Bovine Serum Albumin and Triton X-100 0.1% in PBS solution can be used) to each well/coverslip.
4. Incubate the plate in a pre-heated humidity chamber at 37 °C for 1 h.

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.

1. Dilute the primary antibodies (the mouse anti-Ku70 1:100 and goat anti-Nek4 1:50; rabbit anti-Nek5 1:25 and mouse anti-TOPIIβ 1:25) in antibody diluent provided in the kit (alternatively, 3% of Bovine Serum Albumin and Triton X-100 0.1% in PBS solution can be used). Prepare 30 µL of antibody solution per condition if using coverslips or 20 µL per well if using a 384-well plate.
2. Tap off the blocking solution. Add the primary antibody solution to each well. Incubate at 4 °C overnight in a humidity chamber.

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.

1. Dilute the minus and plus probes 1:5 in the antibody diluent. Prepare 10 µL per well if using 384-well plates or 20 µL per sample if using coverslips. Utilize the following combinations: Donkey anti-mouse minus with Donkey anti-goat plus; Donkey anti-mouse minus with Donkey anti-rabbit plus.
2. Tap off the primary antibody solution. Wash 1x with TBS-T (50 mM Tris pH 7.5, 150 mM NaCl, 0.05% of Tween-20). Wash 3x for 5 min each with TBS-T at room temperature. Perform an additional wash with TBS-T.
3. Tap off and add the PLA probe solution with 10 µL for the plates and 20 µL for the coverslips. Incubate in the pre-heated humidity chamber at 37 °C for 1 h.

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.

1. Dilute the 5x ligation buffer in high-purity water. Prepare 10 µL per well if using 384-well plates or 20 µL if using coverslips.
2. Tap off the PLA probe solution. Wash 1x with TBS-T. Follow with three additional washes, each lasting 5 min, using TBS-T. Wash a final time and tap off the TBS-T before the next step.
3. Add Ligase to the 1x Ligation solution at 1:40, immediately before applying it to the samples. Incubate in the pre-heated humidity chamber at 37 °C for 30 min to 1 h.

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.

1. Remove completely all the wash solution before adding the polymerase solution. Avoid leaving the cells without a solution for a long period of time. Dilute the 5x amplification buffer in high-purity water. Prepare 10 µL per well if using 384-well plates or 20 µL if using coverslips.
2. Tap off the ligation solution. Wash 1x with TBS-T. Follow with three additional washes, each lasting 5 min, using TBS-T. Wash a final time and tap off the TBS-T before the next step.
3. Add the polymerase to the 1x amplification solution at 1:80 dilution, immediately before applying it to the samples. Incubate in the pre-heated humidity chamber at 37 °C for 100 min.
   NOTE: From this step on, avoid direct light exposition
4. Tap off the amplification buffer. Wash 2x with 1x SSC buffer for 10 min each. Wash 2x with 0.01x SSC buffer for 2 min each.

9. γ-H2AX staining (Immunofluorescence - IF)

NOTE: The labeling of γ-H2AX is performed after the PLA is finished.

1. Primary antibody incubation (IF)
   1. Dilute the primary antibodies (Rabbit anti yH2AX at 1:100) in blocking solution (alternatively, 3% of Bovine Serum Albumin and Triton X-100 0.1% in PBS solution can be used). Prepare 30 µL of antibody solution per well if using coverslips or 20 µL if using a 384-well plate.
   2. Tap off the 0.01x SSC solution. Wash 2x with PBS.
   3. Add the primary antibody solution to each well. Incubate at room temperature for 1 h.
2. Secondary antibody incubation (IF)
   NOTE: Make sure the chosen secondary antibodies do not recognize the PLA antibodies or probes. Additionally, the wavelength of the secondary antibody must be different from the PLA detection reagent wavelength. For example: our PLA detection reagent contains the fluorophore 647, and the secondary we used for γ-H2AX detection is 488.
   1. Dilute the secondary antibodies (Donkey anti-Rabbit Alexa Fluor 488) in blocking solution (alternatively, 3% of Bovine Serum Albumin and Triton X - 100 0.1% in PBS solution can be used) at 1:300 dilution. Prepare 30 µL of antibody solution per condition if using coverslips or 20 µL per well if using a 384-well plate. Add Hoechst 33342 dye at 0.6 µg/mL final concentration.
   2. Tap off the primary antibody solution. Wash 3x with TBS-T.
   3. Add the secondary antibody solution to each well. Incubate at room temperature for 20 min.
   4. Wash 5x with TBS-T. Wash 2x with 1x SSC buffer. Wash 2x with 0.01x SSC buffer.
      NOTE: At this point, the plates can be stored for several weeks at 4 °C (in SSC 0.01X) protected from light. If using coverslips, the slides can be mounted, and after 1 day, the slides can be stored at -20 °C.

10. Image acquisition

1. Use a conventional fluorescence microscope and the 63x objective. Determine the time of exposition by comparing negative and positive controls to avoid saturation and dots collapsing.
   NOTE: The use of confocal microscopes enhances the PLA dots detection. The 20x objective also can be used for increasing the number of nuclei detected.
2. Once the exposition time is adjusted, perform all acquisitions of PLA dots with the same parameters. Try to acquire images from different wells or coverslips regions to avoid artifacts due to non-homogeneous incubation.
3. Save all the channels with the same name pattern. If the microscope takes photos in the channels separately, save the corresponding images with the same name, adding only PLA or Nucleus at the beginning of the file name to differentiate the channel.

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.

1. The macro allows the selection of a specific region in the field. Use the option Select a ROI when a region in the image shows artifacts that can be detrimental to analysis. Choose ROI at the beginning of the analysis and use it for all the images.
   NOTE: This is an optional feature. If not selected, the entire image will be considered in the analysis (Supplementary Figure 2).
2. The ROI file should be created prior to the macro usage, as described below. For ROI selection, use a rectangle tool, select the region for analysis, and add it to the ROI manager by clicking Edit > Selection> Add to Manager. Save the ROI by clicking More> Save.
3. In the ImageJ ¹⁷, open images from different conditions to adjust the parameters for analysis. These parameters must be determined by the user and specified, along with the data, in the macro program (Supplementary Coding File 1)
   1. Background correction: To remove the background click Process> Subtract Background. Test different rolling ball radius using the preview function). We have used rolling 10 for PLA dots and rolling 100 for nucleus.
   2. Remove noise by clicking Process > Noise > Despeckle. For nucleus, use the function smooth to improve the filling of the nucleus by clicking Process>Smooth.
   3. Adjusting threshold: Convert to 8-bit image and adjust the threshold by clicking Image > Type > 8 bit and Image > Adjust >Threshold. Adjust the threshold accordingly to visualize all the nucleus or dots present in the image but avoiding over saturation and structures collapsing. Take notes of the values and test in different images to check if the threshold works for different images.
   4. Convert to mask by clicking Process > Binary > Convert to mask. In the case of the nucleus, click Process > Binary > Fill holes followed by Process > Binary > Watershed. In the case of the PLA dots, click Process > Binary > Watershed.
   5. For counting nuclei, measure the area or length of the different nuclei to estimate the average size. Use an approximate value to analyze particles. Click Analyze > Analyze particles, then select Set size: 15-infinity (these values depend on the size of the nuclei). Check the following options: Show: Mask; Display results; Clear results; Summarize; Add to Manager; Exclude on edges.
   6. For counting PLA dots, measure the area or the radius of the dots to estimate the average size. Select Apply Nucleus Mask (apply mask is a function on GDSC plugin, check if it is installed) followed by Analyze > Analyze particles and set. 0.02-3 (these values depend on the size of the nuclei). Check the following options: Show: Mask; Display results; Clear results; Summarize; Add to Manager; Exclude on edges.
4. The results show the number of dots per nucleus in each nucleus present in the image. Save this data.

12. Visualizing the merged images

1. Open the channels corresponding to PLA, nucleus, and γ-H2AX. Subtract the background and remove noise, as mentioned in steps 11.4.1 and 11.4.2 for all images.
2. Adjust brightness and contrast automatically or manually using the same value for all conditions from the same channel. Select Image > Adjust > Bright/contrast.
3. Combine images (merging)by selecting Image > Color > Merge channels. Add scale bar by selecting Analyze > Tools > Scale bar. Save as.TIFF file.

Results

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 etoposid...

Discussion

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 interaction^10,11,19,

Materials

Name	Company	Catalog Number	Comments
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