This protocol helps to understand the spatial and temporal recruitment of DNA damage repair proteins, taking into consideration the cell cycle phase. For cell cycle discrimination, we use fluorescent protein-tagged PCNA as a marker of S phase, which circumvents artifacts introduced by other cell cycle synchronization methods. This combined with laser-based micro-irradiation gives unparalleled spatiotemporal resolution to DNA damage repair protein kinetics.
Successful in routine utilization of our protocol builds on basic knowledge of confocal imaging. 24 hours before the micro-irradiation, plate a total of eight by 10 to the fourth cells in 500 microliters to one milliliter of media on a four-well chambered cover glass. One hour before the micro-irradiation, exchange the regular growth medium with imaging medium containing either olaparib or a vehicle control such as DMSO.
At least four hours before imaging, turn on the environmental chamber and the microscope components. Switch on the heating, carbon dioxide supply, and the humidity regulator. Initialize light sources along with the laser lines at least one hour before transferring the cells to the microscope.
Oil the microscope objective and place a chambered cover glass under the microscope. To select S phase cells in an asynchronous population, look through the ocular for the unique localization pattern of the mPlum tagged PCNA in S phase and select a field of view that has enough S phase cells for micro-irradiation. Set the region of interest for micro-irradiation by using the associated software to insert binary lines.
To set the desired number of lines and spacing, click binary, then select insert line, circle, and ellipse for drawing the desired number of lines, then convert these binary lines into stimulation ROIs by clicking ROI, move binary to ROI, then right-click on any of the ROIs and select use as stimulation ROI S1.Place these lines in the field of view so that they pass through the nucleus of the cells. Before micro-irradiation, to identify PCNA foci for later analysis, take a higher resolution image of the field of view by setting the necessary parameters in the A1 LFOV compact GUI and the A1 LFOV scan area windows followed by hitting the capture button. To set up the micro-irradiation, open the ND stimulation tab, then access the time schedule window to acquire a series of pre-stimulation images and then a series of post-stimulation images using the Galvano scanners.
Set up three phases in the time schedule window. In the acquisition and stimulation column, select acquisition, stimulation, and acquisition for the three phases respectively. For the stimulation phase, set S1 as the ROI.
In the Galvano XY window, set 100%laser power output for the 405 nanometer laser line and set the dwell time for micro-irradiation. To set up timelapse imaging for the desired time window and intervals, use the time schedule A1 LFOV compact GUI and the A1 LFOV scan area windows. Optimize the laser power percentage, gain, and offset settings to reduce photo bleaching during imaging in the A1 LFOV compact GUI window.
Depending on the kinetics of the protein, extend or shorten the interval between images or the duration of the total timelapse by setting the desired interval and duration for the third phase acquisition row in the time schedule window. Press run now to execute the micro-irradiation and the subsequent timelapse imaging. At the end of the timelapse imaging, save the stimulation ROIs as separate images, which will be useful for identifying the coordinates of micro-irradiation in any downstream software used for analysis.
PCNA has a completely homogenous distribution in the nucleus in G1 and G2 phases. In S phase, PCNA localizes to sites of DNA replication, which can be visualized as bright spots in the nucleus. In early S phase cells, the spots are relatively small and equally distributed throughout the nucleus of the cell.
While progressing into mid S phase, the spots become blurred and localize more towards the perimeter of the nucleus and the nucleoli. In late S phase, the spots reduce in numbers, but become increasingly large as PCNA concentrates at late replication sites. Low doses of energy, such as 1, 000 microseconds of dwell time, do not induce recruitment of EGFP-FBXL10, a double stranded break responder, but are sufficient to induce recruitment of NTHL1-mCherry, a base excision repair pathway protein that is recruited to sites of oxidative DNA damage.
At 3, 000 microseconds dwell time, both EGFP-FBXL10 and NTHL1-mCherry are recruited, demonstrating a laser output that generates both oxidative lesions and double-stranded breaks. EXO1b reaches a maximum level of accumulation and micro-irradiation sites around one minute, and then slowly starts disengaging from the DNA lesions. In the presence of olaparib, accumulation of EXO1b at the laser stripe at one minute is significantly less compared to the vehicle control.
It is crucial that the microscope is given ample time to heat up and that culture and conditions are optimal for each experiment to ensure consistent results. Additionally, it is important to optimize your laser settings with different DNA damage reporters to test for specific DNA lesions. Following micro-irradiation, consider validating results with classical biochemical methods, such as fractionation, immunoprecipitation, or ChIP.
These approaches sample a larger cell population, thus providing statistical strength.
