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

1. Pre-sensitization of Cells

1. 24 h prior to the micro-irradiation experiment, seed 40,000 U2OS cells per well in 1x DMEM containing 10% FBS in 8-well culture slides with 170 µm-thick coverslip-like glass/polymer bottoms to ensure maximal transmittance of 405 nm laser light and excellent imaging with a laser-scanning inverted confocal microscope. If using an 8-well microslide, use 500 µL of media or solutions per well for all subsequent treatments and wash steps of the protocol.
   NOTE: Due to their flat morphology, good adherence, and large nuclei, U2OS cells facilitate the detection of protein recruitment at micro-irradiated sites but other adherent cell types can be used as needed. Cells should ideally be around 80% confluency at the time of micro-irradiation to increase the number of irradiated cells per experiment. Very high confluence may result in cell cycle alterations that can perturb specific repair pathways such as homologous recombination which takes place during the S and G2 phases of the cell cycle⁹.
2. Incubate the cells overnight at 37 °C with 5% CO₂, and then pre-sensitize the cells with (step 1.3) BrdU or (step 1.4) BBET (Hoechst 33342).
   NOTE: To observe live recruitment of a protein of interest (POI) to DNA lesions, transfect or transduce plasmid DNA carrying a fusion between a fluorescent protein and a POI 24 h after cell seeding. 24 h post-transfection/transduction, pre-sensitize and micro-irradiate the cells to monitor the recruitment of the POI at DNA damage sites in real time.
3. To increase the number of DSBs generated by exposition to the 405 nm laser, add 10 µM of BrdU to the media for 24 h prior to micro-irradiation. Prior to micro-irradiation, using a micropipette, rinse the cells twice with medium without phenol red to ensure maximal exposure of the cells to the 405 nm laser.
4. Alternatively, treat cells for 15 min with BBET at 10 µg/mL in the appropriate medium in a cell-culture incubator at 37 °C. After treatment, rinse twice with medium without phenol red prior to micro-irradiation.

2. Micro-irradiation of Cells

1. Select a suitable laser-scanning confocal microscope.
   NOTE: The experiments presented here were performed on a microscope system equipped with a 50 mW 405 nm laser line and a 63X 1.4 numerical aperture oil objective. To generate DSBs, cells were micro-irradiated at 1X scan zoom at 1,024 x 1,024 pixels/field (0.21 µm/pixel at 63X magnification) and in unidirectional mode at 8 µs/pixel. The laser power used was 25% and 80% for BBET- and BrdU-pre-sensitized cells, respectively. To help users configure their system, the laser power post-objective on the presented system was measured using a power meter according to manufacturer's instructions. The measurements correspond to 2.6 mW and 7.0 mW for BBET- and BrdU-pre-sensitized cells, respectively.
2. Turn on the microscope and the environmental chamber.
   1. Set the chamber to 37 °C with 5% CO₂ before samples are placed into the on-stage incubator to avoid unnecessary stress to the cells and to ensure homogeneous DDR kinetics across experiments.
3. Select field(s) with well-distributed cells, adjust the focus, and register their position to facilitate image acquisition after the IF protocol. Acquire at least one image that will serve as a pre-damage reference point to monitor the kinetics of protein recruitment after micro-irradiation.
   NOTE: Gridded culture vessels may also be used to facilitate the localization of micro-irradiated cells at subsequent steps.
   1. For IF experiments using BBET-labeled cells, adjust the focus on cells using the 405 nm laser at the lowest laser power sufficient to visualize the cells in order to avoid unwarranted DNA damage.
      1. Avoid using widefield fluorescence to adjust the focus on BBET-stained cells as the UV light emitted by the lamp will induce DNA damage at illuminated sites.
   2. If using BrdU-labeled cells, adjust the focus using differential interference contrast (DIC) or phase-contrast imaging by looking at sub-nuclear features such as nucleoli.
   3. If using cells expressing fluorescently-labeled POIs, select a focal plane with representative fluorescence intensity.
      NOTE: Avoid cells that express very high levels of the POI as strong overexpression may result in artifactual localization. Additionally, select cells that express the POI at comparable levels (selection of stable clones is strongly recommended to minimize variations in POI expression and recruitment levels).
4. Configure the microscope for the micro-irradiation step using the fluorescence-recovery after photobleaching (FRAP)_module of the system.
   NOTE: The most important parameters to consider when setting-up the microscope system for micro-irradiation experiments are: the output power of the 405 nm laser, number of iterations (i.e., number of times the laser will go over the same coordinates), number of pixels/field (at 63X magnification and 1,024 x 1,024 resolution, 1 pixel = 0.21 µm), and dwell time per pixel. All of these factors will impact the amount of energy that the cells experience and thus the amount and type of DNA damage generated (see Discussion for further dose optimization information).
5. Using the FRAP module of the microscope system, micro-irradiate the cells.
   NOTE: On most systems, micro-irradiation of multiple cells can be performed in a single field as spots or lines/rectangles (typically 1-2 µm wide). When performing live-imaging of fluorescently-labeled proteins immediately post-damage, limit the micro-irradiation to a small number of cells per field because the time taken to micro-irradiate each cell will result in a delayed induction of the DDR. This is particularly important when monitoring proteins that are recruited very rapidly to DNA lesions.
6. After micro-irradiation, put back the multi-well slides or plates in an incubator at 37 °C with 5% CO₂ for the required duration prior to processing samples for IF (Section 5) or proceed immediately with live-imaging (Section 3).
   1. As a first step, fix the cells within a few minutes and up to a few hours post-irradiation for the IF protocol. Depending on the POI, recruitment kinetics may vary. Typically, proteins that are chromatin-associated at DSBs and SSBs will localize very rapidly to the lesion and are sometimes removed within 5 min. Some factors such as those that assemble at ssDNA will be recruited at later time points once the resection machinery has been engaged and remain there for hours (See Figure 1 and reference⁴).

3. Live-imaging of Fluorescently-labeled Proteins

1. Perform time-lapse imaging of the micro-irradiated cells. For proteins that are recruited very rapidly to lesions, a few seconds per frame will be ideal, but for proteins that localize to the damaged area later (e.g., resection factors), acquisition may be performed every 1–2 min until a plateau is achieved.
   NOTE: Minimize the laser power for image acquisition to avoid bleaching as much as possible and to ensure that protein recruitment at damage sites does not reach saturation which would preclude signal quantification and subsequent analyses (see data analysis Section 4). On the presented system, the imaging was done with a 63X/1.4 oil objective using an 8 µs/pixel dwell time, 1,024 x 1,024 resolution, 1X zoom, and averaging of 2. Note that these parameters may need to be optimized for other systems and/or different experiments.
2. Micro-irradiate the cells in additional fields until kinetics measurements are obtained for 15-30 cells. Repeat the experiments at least 3 times in biological replicates to ensure the accuracy and reproducibility of the recruitment kinetics.
   NOTE : When adapting this protocol to a specific confocal system, it may be useful to start by monitoring the recruitment kinetics of a known fluorescently-labeled genome maintenance factor (e.g., 53BP1-GFP, RPA32-GFP, etc.). Using live-imaging to optimize micro-irradiation conditions will allow the user to test multiple parameters rapidly.

4. Recruitment Kinetics Analysis

1. Install the Microirradiation Analysis plugin¹⁰ in the Fiji image analysis software¹¹.
2. Open the acquired images in Fiji. Select "The Damage Analyzer" from the pull-down plugins menu in the Microirradiation Analysis suite. Follow the prompts to define a background region of interest (ROI) (typically 3 x 3 µm), and non-irradiated and irradiated ROIs in each micro-irradiated cell.
   1. Use ROIs of the same size to minimize potential biases in mean signal intensities. For acquired images, also include at least one pre-irradiation frame which will be used as a baseline to monitor recruitment at micro-irradiated sites.
   2. Once all ROI data have been collected over the full time-lapse series, save the file created by the plugin containing the results as a csv (comma delimited file) or txt file (tab delimited file). This file contains measurements of the mean intensity for each time point and each ROI (Mean Intensity Background, Mean Intensity Non-irradiated, and Mean Intensity Irradiated).
      NOTE: The plugin automatically subtracts the background intensity (I_b) from the intensity of the irradiated ROIs (I_s) and the non-irradiated ROIs (I_n). It also calculates a ratio between both values (see the formula below) to correct for photobleaching (Irradiated/Non-irradiated).
      
      The plugin also calculates a normalization between the first time point (value set to 1) and all the other time points for each nucleus (ratio relative to first time point). Additional information can be found on the plugin website¹⁰.
3. Perform the analysis using the plugin for all micro-irradiated cells (at least 15–30 cells) and graph the mean relative enrichment at micro-irradiated regions over time. Calculate the standard errors of the mean for each data point and plot them.
   1. Confirm differences in recruitment kinetics between two experimental conditions by performing Student's t-tests for each data point.

5. IF Protocol

1. Prepare IF solutions. Prepare pre/post-extraction solution (ice-cold 1x PBS containing 0.25% Triton X-100), washing solution (1x PBS containing 0.05% Tween-20), blocking solution (1x PBS containing 3% BSA and 0.05% Tween-20), and fixation solution (Paraformaldehyde 3% + sucrose 2% in PBS 1x) in conical polypropylene tubes.
   NOTE: For a single 8-well microslide, 10 mL of pre/post-extraction solution, 50 mL of washing solution, 10 mL of blocking solution, and 5 mL of fixation solution are required.
   1. Prepare the fixation solution.
      1. Under a chemical hood, mix 400 mL of double-distilled water (ddH₂O) with 15 g of paraformaldehyde and 30 µL of 10 N NaOH in a 1 L Erlenmeyer flask.
      2. Close the container and heat at 65 °C on a magnetic stirrer hot plate while stirring to solubilize the paraformaldehyde completely.
      3. Using a 25-mL pipette, add 50 mL of 10X PBS, stir, and filter using a 0.45 µm filter.
      4. Add 10 g of sucrose and complete to 500 mL with ddH₂O.
      5. Aliquot the solution in 15 mL conical tubes; store at -20 °C until use.
   2. Prepare the nuclei staining solution by diluting a stock solution of 1 mg/mL of 4,6-Diamidino-2-phenylindole (DAPI) 1:1,000 in 1x PBS.
2. Using a micropipette, carefully remove the media from the wells of the multi-well microscopy slides or plates and immediately wash with 500 µL of IF washing solution by changing the solution twice without waiting.
   NOTE: The IF data presented here were obtained using cells grown in 8-well microscopy slides that were washed and incubated in 500 µL of all solutions. Volumes should be adjusted depending on the cell culture containers used by experimenters. Additionally, perform the IF protocol with extreme care as cells can easily detach from the glass/polymer bottom. Use a micropipette for all solution changes to minimize cell loss.
3. Incubate on ice for 5 min with 500 µL of ice-cold IF pre/post-extraction solution and swirl gently by hand for 15–30 s to perform the pre-extraction step that removes most cytoplasmic and nucleoplasmic proteins and maximizes the signal from chromatin/DNA-associated factors.
4. Wash twice with 500 µL of IF washing solution.
5. Incubate for 15 min at room temperature in 500 µL of IF fixation solution. Alternatively, use a pre-made paraformaldehyde solution (3–4%) for the fixation step.
6. Wash twice with 500 µL of IF washing solution.
7. Incubate on ice for 5 min in 500 µL of ice-cold IF pre/post-extraction solution and swirl the slide gently by hand to perform the permeabilization step.
8. Wash twice with 500 µL of IF washing solution.
9. Incubate at least 30 min at room temperature with 500 µL of IF blocking solution.
10. Remove blocking solution using a micropipette and incubate overnight in a humidified chamber at 4 °C with 250 µL primary antibodies diluted in blocking solution.
    NOTE: It is good practice to perform the incubation with primary antibodies sequentially to avoid potential artifacts of co-localization. Once appropriate controls have been performed, simultaneously incubate primary antibodies to speed up the process.
11. Wash four times for 5 min in 500 µL of IF washing solution with gentle agitation (60 rpms on a rotator).
12. Incubate for 1 h at 37 °C with 250 µL of secondary antibodies diluted in blocking solution in a humidified chamber.
    NOTE: Protect the samples from light upon addition of fluorescently-labeled secondary antibodies.
13. Wash four times each for 5 min in 500 µL of IF washing solution with gentle agitation.
14. Incubate at room temperature for 5 min in 500 µL of 1x PBS containing 1 µg/mL DAPI to stain nuclei.
15. Wash twice with 500 µL of 1x PBS and leave cells in 500 µL of 1x PBS solution for imaging.
16. Using gridded slides or registered stage positions, find the micro-irradiated cells using a well-characterized DNA damage marker (e.g., γ-H2A.X, RPA32, etc.).
17. Image the multi-well culture slides or plates in all relevant channels using the appropriate settings on a laser-scanning confocal microscope.
    NOTE: On the presented system, the imaging was done with a 63X/1.4 oil objective using an 8 µs/pixel dwell time, 1,024 x 1,024 resolution, 1X zoom, and averaging of 2. Note that these parameters may need to be optimized for other systems and/or different experiments.