Name: Proximity Ligand Assay to Localize Proteins in DNA Damage Sites (Video) | JoVE Journal
Uploaded: 2024-08-02T14:50:00.000+00:00
Duration: 9 min 39 s
Description: Learn proximity ligation assay to localize protein interactions at DNA damage sites using U2OS cells. This method combines Gamma-H2AX staining with PLA for spatial characterization.

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5 minutes

We thank Funda&#231;&#227;o de Amparo a Pesquisa do Estado de S&#227;o Paulo (FAPESP, through Grant Tem&#225;tico 2022/15126-9 to JK and fellowship 21/09439-1 to LARM) and the Conselho Nacional de Desenvolvimento Cientifico e Tecnol&#243;gico (CNPq) for funding this research.

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.
<ol>
	<li>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).
	<ol>
		<li>Culture U2OS cells in Dulbecco&#39;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 &#176;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.</li>
		<li>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 &#176;C in a humidified, 5% CO2 incubator.</li>
	</ol>
	</li>
</ol>
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.
<ol>
	<li>Prepare the stock solution of Etoposide at 50 mM concentration in DMSO. The stock solution can be kept at -20 &#176;C for several months. Dilute the etoposide solution for use in the complete culture medium at 25 &#181;M final concentration. Use DMSO at 0.05% diluted in the complete culture medium as vehicle control.</li>
	<li>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 &#176;C.</li>
</ol>
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.
<ol>
	<li>Remove the culture medium from the wells and add a culture medium containing etoposide or DMSO at 25 &#181;M or 0.05%, respectively. Incubate cells at 37 &#176;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.
	<ol>
		<li>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.</li>
	</ol>
	</li>
	<li>Remove the medium from the wells and wash the cells with PBS 3x.</li>
	<li>Fix the cells by adding ice-cold methanol with enough volume to cover the well surface. Incubate for 10 min at -20 &#176;C. Wash the wells with PBS 3x. 
	NOTE: At this point, the protocol can be paused. The plates can be stored at 4 &#176;C for 1 week.</li>
</ol>
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.
<ol>
	<li>Tap off the PBS from the samples.</li>
	<li>Add 30-40 &#181;L of permeabilization buffer (0.2% Triton X-100 in PBS) and incubate for 20 min at room temperature. Wash 3x with PBS</li>
	<li>Tap off the PBS from the samples and add 30-40 &#181;L 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.</li>
	<li>Incubate the plate in a pre-heated humidity chamber at 37 &#176;C for 1 h.</li>
</ol>
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 &#947;-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 &#947;-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.
<ol>
	<li>Dilute the primary antibodies (the mouse anti-Ku70 1:100 and goat anti-Nek4 1:50; rabbit anti-Nek5 1:25 and mouse anti-TOPII&#946; 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 &#181;L of antibody solution per condition if using coverslips or 20 &#181;L per well if using a 384-well plate.</li>
	<li>Tap off the blocking solution. Add the primary antibody solution to each well. Incubate at 4 &#176;C overnight in a humidity chamber.</li>
</ol>
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 &#176;C incubation, slow rotation can be used to improve the distribution of the antibody solution.
<ol>
	<li>Dilute the minus and plus probes 1:5 in the antibody diluent. Prepare 10 &#181;L per well if using 384-well plates or 20 &#181;L 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.</li>
	<li>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&#160;wash with TBS-T.</li>
	<li>Tap off and add the PLA probe solution with 10 &#181;L for the plates and 20 &#181;L for the coverslips. Incubate in the pre-heated humidity chamber at 37 &#176;C for 1 h.</li>
</ol>
7. Ligation
NOTE: Wait to add Ligase until immediately before adding it to the samples. Ligase must be kept in a freezer block (-20 &#176;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 &#176;C incubation, slow rotation can be used to improve the distribution of the antibody solution.
<ol>
	<li>Dilute the 5x ligation buffer in high-purity water. Prepare 10 &#181;L per well if using 384-well plates or 20 &#181;L if using coverslips.</li>
	<li>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.</li>
	<li>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 &#176;C for&#160;30 min to 1 h.</li>
</ol>
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 &#176;C). Avoid leaving the cells without a solution for a long period of time. When using 384-well plates for 37 &#176;C incubation, slow rotation can improve the distribution of the antibody solution.
<ol>
	<li>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 &#181;L per well if using 384-well plates or 20 &#181;L if using coverslips.</li>
	<li>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.</li>
	<li>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 &#176;C for 100 min. 
	NOTE: From this step on, avoid direct light exposition</li>
	<li>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.</li>
</ol>
9. &#947;-H2AX staining (Immunofluorescence - IF)
NOTE: The labeling of &#947;-H2AX is performed after the PLA is finished.
<ol>
	<li>Primary antibody incubation (IF)
	<ol>
		<li>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 &#181;L of antibody solution per well if using coverslips or 20 &#181;L if using a 384-well plate.</li>
		<li>Tap off the 0.01x SSC solution. Wash 2x with PBS.</li>
		<li>Add the primary antibody solution to each well. Incubate at room temperature for 1 h.</li>
	</ol>
	</li>
	<li>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 &#947;-H2AX detection is 488.
	<ol>
		<li>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 &#181;L of antibody solution per condition if using coverslips or 20 &#181;L per well if using a 384-well plate. Add Hoechst 33342 dye at 0.6 &#181;g/mL final concentration.</li>
		<li>Tap off the primary antibody solution. Wash 3x with TBS-T.</li>
		<li>Add the secondary antibody solution to each well. Incubate at room temperature for 20 min.</li>
		<li>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 &#176;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 &#176;C.</li>
	</ol>
	</li>
</ol>
10. Image acquisition
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
</ol>
11. Image analysis
NOTE: A&#160;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.
<ol>
	<li>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).</li>
	<li>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 &#62; Selection&#62; Add to Manager. Save the ROI by clicking More&#62; Save.</li>
	<li>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)
	<ol>
		<li>Background correction: To remove the background click Process&#62; Subtract Background. Test different rolling ball radius using the preview function). We have used rolling 10 for PLA dots and rolling 100 for nucleus.</li>
		<li>Remove noise by clicking Process &#62; Noise &#62; Despeckle. For nucleus, use the function smooth to improve the filling of the nucleus by clicking Process&#62;Smooth.</li>
		<li>Adjusting threshold: Convert to 8-bit image and adjust the threshold by clicking Image &#62; Type &#62; 8 bit and Image &#62; Adjust &#62;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.</li>
		<li>Convert to mask&#160;by clicking Process &#62; Binary &#62; Convert to mask. In the case of the nucleus, click Process &#62; Binary &#62; Fill holes followed by Process &#62; Binary &#62; Watershed. In the case of the PLA dots, click Process &#62; Binary &#62; Watershed.</li>
		<li>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 &#62; 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.</li>
		<li>For counting PLA dots, measure the area or the radius of the dots to estimate the average size.&#160;Select Apply Nucleus Mask (apply mask is a function on GDSC plugin, check if it is installed) followed by Analyze &#62; Analyze particles&#160;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.</li>
	</ol>
	</li>
	<li>The results show the number of dots per nucleus in each nucleus present in the image. Save this data.</li>
</ol>
12. Visualizing the merged images
<ol>
	<li>Open the channels corresponding to PLA, nucleus, and &#947;-H2AX. Subtract the background and remove noise, as mentioned in steps 11.4.1 and 11.4.2 for all images.</li>
	<li>Adjust brightness and contrast automatically or manually using the same value for all conditions from the same channel.&#160;Select Image &#62; Adjust &#62; Bright/contrast.</li>
	<li>Combine images (merging)by selecting Image &#62; Color &#62; Merge channels. Add scale bar by selecting Analyze &#62; Tools &#62; Scale bar. Save as.TIFF file.</li>
</ol>

Assessing Protein Interaction and Localization in DNA Damage

<ol><li>Xiao, W., Samson, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+vivo+evidence+for+endogenous+DNA+alkylation+damage+as+a+source+of+spontaneous+mutation+in+eukaryotic+cells%5BTitle%5D)+AND+%22Proc+Nat+Acad+Sci+U+S+A%22%5BJournal%5D)">In vivo evidence for endogenous DNA alkylation damage as a source of spontaneous mutation in eukaryotic cells.</a> Proc Nat Acad Sci U S A. 90 (6), 2117-2121 (1993).</li>
<li>Lindahl, T., Barnes, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Repair+of+endogenous+DNA+damage%5BTitle%5D)+AND+%22Cold+Spring+Harb+Symp+Quant+Biol%22%5BJournal%5D)">Repair of endogenous DNA damage.</a> Cold Spring Harb Symp Quant Biol. 65, 127-134 (2000).</li>
<li>DNA damage, aging, and cancer. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=%22N+Engl+J+Med%22">N Engl J Med</a>. Hoeijmakers Jan, H. J. 361 (15), 1475-1485 (2009).</li>
<li>Giglia-Mari, G., Zotter, A., Vermeulen, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((DNA+damage+response%5BTitle%5D)+AND+%22Cold+Spring+Harb+Perspect+Biol%22%5BJournal%5D)">DNA damage response.</a> Cold Spring Harb Perspect Biol. 3 (1), a000745(2011).</li>
<li>Ciccia, A., Elledge, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+DNA+damage+response%3A+Making+it+safe+to+play+with+knives%5BTitle%5D)+AND+%22Mol+Cell%22%5BJournal%5D)">The DNA damage response: Making it safe to play with knives.</a> Mol Cell. 40 (2), 179-204 (2010).</li>
<li>Chatterjee, N., Walker, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mechanisms+of+DNA+damage%2C+repair+and+mutagenesis%5BTitle%5D)+AND+%22EnvironMol+Mutagen%22%5BJournal%5D)">Mechanisms of DNA damage, repair and mutagenesis.</a> EnvironMol Mutagen. 58 (5), 235-263 (2017).</li>
<li>Mah, L. J., El-Osta, A., Karagiannis, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((GammaH2AX%3A+a+sensitive+molecular+marker+of+DNA+damage+and+repair%5BTitle%5D)+AND+%22Leukemia%22%5BJournal%5D)">GammaH2AX: a sensitive molecular marker of DNA damage and repair.</a> Leukemia. 24 (4), 679-686 (2010).</li>
<li>Paull, T. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+critical+role+for+histone+H2AX+in+recruitment+of+repair+factors+to+nuclear+foci+after+DNA+damage%5BTitle%5D)+AND+%22Curr+Biol%22%5BJournal%5D)">A critical role for histone H2AX in recruitment of repair factors to nuclear foci after DNA damage.</a> Curr Biol. 10 (15), 886-895 (2000).</li>
<li>Nakamura, A. J., Rao, V. A., Pommier, Y., Bonner, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+complexity+of+phosphorylated+H2AX+foci+formation+and+DNA+repair+assembly+at+DNA+double-strand+breaks%5BTitle%5D)+AND+%22Cell+cycle+%28Georgetown%2C+Tex%29%22%5BJournal%5D)">The complexity of phosphorylated H2AX foci formation and DNA repair assembly at DNA double-strand breaks.</a> Cell cycle (Georgetown, Tex). 9 (2), 389-397 (2010).</li>
<li>Karchugina, S., Chernoff, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Detection+of+heterodimerization+of+protein+isoforms+using+an+in+situ+proximity+ligation+assay%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">Detection of heterodimerization of protein isoforms using an in situ proximity ligation assay.</a> J Vis Exp. (140), (2018).</li>
<li>Tubbs, E., Rieusset, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Study+of+endoplasmic+reticulum+and+mitochondria+interactions+by+in+situ+proximity+ligation+assay+in+fixed+cells%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">Study of endoplasmic reticulum and mitochondria interactions by in situ proximity ligation assay in fixed cells.</a> J Vis Exp. (118), (2016).</li>
<li>Nguyen, C. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Nek4+regulates+entry+into+replicative+senescence+and+the+response+to+DNA+damage+in+human+fibroblasts%5BTitle%5D)+AND+%22Mol+CellBiol%22%5BJournal%5D)">Nek4 regulates entry into replicative senescence and the response to DNA damage in human fibroblasts.</a> Mol CellBiol. 32 (19), 3963-3977 (2012).</li>
<li>Basei, F. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((New+interaction+partners+for+Nek4.1+and+Nek4.2+isoforms%3A+from+the+DNA+damage+response+to+RNA+splicing%5BTitle%5D)+AND+%22Proteome+Sci%22%5BJournal%5D)">New interaction partners for Nek4.1 and Nek4.2 isoforms: from the DNA damage response to RNA splicing.</a> Proteome Sci. 13 (1), 11(2015).</li>
<li><a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=%22Animal+Cell+Culture+Guide%22">Animal Cell Culture Guide</a>. , https://www.atcc.org/resources/culture-guides/animal-cell-culture-guide (2024).</li>
<li>Soubeyrand, S., Pope, L., Haché, R. J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Topoisomerase+II%CE%B1-dependent+induction+of+a+persistent+DNA+damage+response+in+response+to+transient+etoposide+exposure%5BTitle%5D)+AND+%22MolOncol%22%5BJournal%5D)">Topoisomerase IIα-dependent induction of a persistent DNA damage response in response to transient etoposide exposure.</a> MolOncol. 4 (1), 38-51 (2010).</li>
<li>Wei, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Etoposide-induced+DNA+damage+affects+multiple+cellular+pathways+in+addition+to+DNA+damage+response%5BTitle%5D)+AND+%22Oncotarget%22%5BJournal%5D)">Etoposide-induced DNA damage affects multiple cellular pathways in addition to DNA damage response.</a> Oncotarget. 9 (35), 24122-24139 (2018).</li>
<li>Schindelin, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fiji%3A+an+open-source+platform+for+biological-image+analysis%5BTitle%5D)+AND+%22Nat+Methods%22%5BJournal%5D)">Fiji: an open-source platform for biological-image analysis.</a> Nat Methods. 9 (7), 676-682 (2012).</li>
<li>Melo-Hanchuk, T. D., Slepicka, P. F., Pelegrini, A. L., Menck, C. F. M., Kobarg, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((NEK5+interacts+with+topoisomerase+II%CE%B2+and+is+involved+in+the+DNA+damage+response+induced+by+etoposide%5BTitle%5D)+AND+%22J+Cell+Biochem%22%5BJournal%5D)">NEK5 interacts with topoisomerase IIβ and is involved in the DNA damage response induced by etoposide.</a> J Cell Biochem. 120 (10), 16853-16866 (2019).</li>
<li>George, J., Mittal, S., Kadamberi, I. P., Pradeep, S., Chaluvally-Raghavan, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Optimized+proximity+ligation+assay+%28PLA%29+for+detection+of+RNA-protein+complex+interactions+in+cell+lines%5BTitle%5D)+AND+%22STAR+Protoc%22%5BJournal%5D)">Optimized proximity ligation assay (PLA) for detection of RNA-protein complex interactions in cell lines.</a> STAR Protoc. 3 (2), 101340(2022).</li>
<li>Zhang, W., Xie, M., Shu, M. D., Steitz, J. A., DiMaio, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+proximity-dependent+assay+for+specific+RNA-protein+interactions+in+intact+cells%5BTitle%5D)+AND+%22RNA%22%5BJournal%5D)">A proximity-dependent assay for specific RNA-protein interactions in intact cells.</a> RNA. 22 (11), 1785-1792 (2016).</li>
<li>Hegazy, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Proximity+ligation+assay+for+detecting+protein-protein+interactions+and+protein+modifications+in+cells+and+tissues+in+situ%5BTitle%5D)+AND+%22Curr+Protoc+Cell+Biol%22%5BJournal%5D)">Proximity ligation assay for detecting protein-protein interactions and protein modifications in cells and tissues in situ.</a> Curr Protoc Cell Biol. 89 (1), e115(2020).</li>
</ol>

The authors declare that they have no competing financial interests.

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.&#160;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.

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 &#947;-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 &#947;-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 &#947;-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 &#947;-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.

<table><tbody><tr><td>Black 384-well plates</td><td>Perkin Elmer Cell carrier plates</td><td></td><td></td></tr><tr><td>Donkey&amp;nbsp; anti- Rabbit Alexa Fluor 488</td><td>Invitrogen</td><td>A21206</td><td>1:300</td></tr><tr><td>Duo link Donkey anti Mouse Minus</td><td>Sigma</td><td>DUO92004</td><td></td></tr><tr><td>Duolink antibody diluent</td><td>Sigma</td><td>DUO82008</td><td></td></tr><tr><td>Duolink blocking solution 1X</td><td>Sigma</td><td>DUO82007</td><td></td></tr><tr><td>Duolink Detection reagent Far red</td><td>Sigma</td><td>DUO92013</td><td></td></tr><tr><td>Duolink Donkey anti goat plus</td><td>Sigma</td><td>DUO92003</td><td></td></tr><tr><td>Duolink Donkey anti rabbit plus</td><td>Sigma</td><td>DUO92002</td><td></td></tr><tr><td>Etoposide</td><td>Sigma</td><td>E1383</td><td></td></tr><tr><td>Goat anti Nek4</td><td>Santa Cruz Biotechnology</td><td>SC-5517</td><td>goat anti Nek4 was used at 1:50 dilution</td></tr><tr><td>Hoechst 33342</td><td>Thermo</td><td>H1399</td><td>0.6 &amp;micro;g/mL</td></tr><tr><td>Leica DMI microscope</td><td>Leica</td><td></td><td></td></tr><tr><td>Mouse anti Ku70</td><td>Thermo</td><td>MA5-13110</td><td>mouse anti Ku70 was used at 1:100 dilution</td></tr><tr><td>Mouse anti TOPII&amp;beta;</td><td>Santa Cruz Biotechnology</td><td>SC-365071</td><td>1:25</td></tr><tr><td>Rabbit anti Nek5</td><td>Santa Cruz Biotechnology</td><td>SC-84527</td><td>1:25</td></tr><tr><td>Rabbit anti Y H2AX</td><td>Cell Signalling</td><td>9718S</td><td>1:100 dilution</td></tr><tr><td>U2OS cell line</td><td>ATCC</td><td>HTB-96&amp;nbsp;</td><td></td></tr></tbody></table>

proximity ligand assay to localize proteins in dna damage sites

Proximity Ligand Assay to Localize Proteins in 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.

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.
<img alt="Nek4-Ku70 and Nek5-TOPIIββ DNA damage foci; microscopy images and dose/nucleus bar graphs." class="xfigimg" src="/files/ftp_upload/67072/67072fig01v4.jpg" data-altupdatedat="1788304762000" data-alt="Figure 1"> 
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&lt;0.05, **p&lt;0,01, and *** p&lt;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. <a href="https://www.jove.com/files/ftp_upload/67072/67072fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
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). <a href="https://www.jove.com/files/ftp_upload/67072/Supplementary%20figure%201_RE.zip" target="_blank">Please click here to download this File.</a>
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. <a href="https://www.jove.com/files/ftp_upload/67072/Supplementary%20figure%202_RE.zip" target="_blank">Please click here to download this File.</a>
Supplementary Coding File 1: Macro File Created for ImageJ. <a href="https://www.jove.com/files/ftp_upload/67072/pla_dots_per_nucleus_basei_et_al_2024.zip" target="_blank">Please click here to download this File.</a>

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 ...

proximity-ligand-assay-to-localize-proteins-in-dna-damage-sites

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JoVE Journal

Biology

This video describes the proximity ligation assay to study in situ protein-protein interactions in nematode cells. The close proximity of the proteins of interest generates the circular DNA template with the help of a PLA probe attached to a secondary antibody. The probe is further amplified using the amplification solution, which produces a long single-stranded DNA followed by the hybridization of the detector probe, enabling the localization of protein-protein interactions in cells.

Proximity Ligation Assay to Study In Situ Protein-Protein Interactions

Lasers are frequently used in studies of the cellular response to DNA damage. However, they generate lesions whose spacing, frequency, and collisions with replication forks are rarely characterized. Here, we describe an approach that enables the determination of these parameters with laser localized interstrand crosslinks.

Single Molecule Analysis of Laser Localized Psoralen Adducts

Here, it is demonstrated how the in situ Proximity Ligation Assay (PLA) can be used to detect and visualize the direct protein-protein interactions between ATM and p53 in suspension cell cultures exposed to genotoxic stress.

Detection and Visualization of DNA Damage-induced Protein Complexes in Suspension Cell Cultures Using the Proximity Ligation Assay

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.

Advanced Confocal Microscopy Techniques to Study Protein-protein Interactions and Kinetics at DNA Lesions

Studying DNA damage repair kinetics requires a system to induce lesions at defined sub-nuclear regions. We describe a method to create localized double-stranded breaks using a laser-scanning confocal microscope equipped with a 405 nm laser and provide automated procedures to quantify the dynamics of repair factors at these lesions.

Investigation of Protein Recruitment to DNA Lesions Using 405 Nm Laser Micro-irradiation

The goal of this protocol is to quantify the repair of defined DNA-protein crosslinks on plasmid DNA. Lesioned plasmids are transfected into recipient mammalian cell lines and low-molecular weight harvested at multiple time points post-transfection. DNA repair kinetics are quantified using strand-specific primer extension followed by qPCR.

A Simple, Rapid, and Quantitative Assay to Measure Repair of DNA-protein Crosslinks on Plasmids Transfected into Mammalian Cells

Following DNA damage, human cells activate essential repair pathways to restore the integrity of their genome. Here, we describe the method of indirect immunofluorescence as a means to detect DNA repair proteins, analyze their spatial and temporal recruitment, and help interrogate protein-protein interaction at the sites of DNA damage.

Visualization of DNA Repair Proteins Interaction by Immunofluorescence

While replication fork collisions with DNA adducts can induce double strand breaks, less is known about the interaction between replisomes and blocking lesions. We have employed the proximity ligation assay to visualize these encounters and to characterize the consequences for replisome composition.

Visualization of Replisome Encounters with an Antigen Tagged Blocking Lesion

Here, a protocol for assaying nucleic acid metabolizing enzymes is presented, using examples of ligase, nuclease, and polymerase enzymes. The assay utilizes fluorescently labeled and unlabeled oligonucleotides that can be combined to form duplexes mimicking RNA and/or DNA damages or pathway intermediates, allowing for the characterization of enzyme behavior.

Using Modified Synthetic Oligonucleotides to Assay Nucleic Acid-Metabolizing Enzymes

We describe a simple protocol to identify brain proteins that bind to the full length C terminus of ATP-gated P2X2 receptors. The extension and systematic application of this approach to all P2X receptors is expected to lead to a better understanding of P2X receptor signaling.

Proteomics to Identify Proteins Interacting with P2X2 Ligand-Gated Cation Channels

Learn proximity ligation assay to localize protein interactions at DNA damage sites using U2OS cells. This method combines Gamma-H2AX staining with PLA for spatial characterization.

Proximity Ligand Assay to Localize Proteins in DNA Damage Sites

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