Visualization of miniSOG Tagged DNA Repair Proteins in Combination with Electron Spectroscopic Imaging (ESI)

The overall goal of the following experiment is to map the distribution of DNA repair proteins around double stranded DNA breaks at the nanoscale. In order to study the ultra structure of damaged chromatin undergoing repair using electron spectroscopic imaging. This is achieved by generating DNA expression constructs coding for fusion proteins of DNA damage response proteins using a fluorescent protein and the mini SOG tag.

As a second step constructs are introduced into cells by transfection, and then the cells are damaged by either laser or gamma irradiation. Next, the irradiated and fixed cells undergo electron microscopy preparation steps in order to make the distribution of the mini SOG tagged repair protein visible in the electron microscope. The resulting electron spectroscopic imaging provides detailed images that allow for the analysis of the chromatin ultra structure and the relationship of specific proteins with DNA repair foci.

The main advantage of this technology over other existing technologies like ImmunoGold labeling, is that it's not dependent on the accessibility of antibodies and that it labels the structures of interests quite densely. Generally, individuals that are new to this method will struggle because of the photo oxidation is very sensitive to pH light intensity and oxygen saturation. Visual demonstration of this method is critical because the photo oxidation, oxygen saturation and illumination steps are sensitive to very small changes, and so it's important to illustrate how the method is done in order to reproduce it.

Dr Hilmar Fadden, a postdoc in my lab, will demonstrate the method for you Culture. Human osteosarcoma cells that are stably expressing the mini SOG and M cherry tagged repair proteins as described in the accompanying text protocol. After splitting the cells from a 10 centimeter dish, grow the cells to 80%co fluency.

Next, place the cells under a fluorescence microscope and use a sterile diamond pen to outline a small area of approximately one square millimeter that contains cells expressing the ectopic proteins once marked, sensitize the cells by adding herx to the solution so that the final concentration is 0.5 micrograms per milliliter, and then place them in the incubator at 37 degrees Celsius for 20 minutes. Then laser micro irradiate the cells using the 405 nanometers solid state laser of a confocal microscope in order to induce DNA damage following DNA damage, fix the cells in the dark using one milliliter of 4%paraform aldehyde in 0.1 molar sodium. Cate buffer at room temperature after 30 minutes.

Wash the cells twice with four milliliters of 0.1 molar sodium Cate buffer at a pH of 7.4. Next, treat the fixed cells with two milliliters of a solution containing 50 millimolar glycine, five millimolar amino triazole, and 10 millimolar potassium cyanide at a pH of 7.4 for 30 minutes. Replace the buffer with two milliliters of photo oxidation buffer at a pH of 7.4, which was prepared as described in the accompanying text protocol.

Then incubate the sample in the dark on ice while bubbling oxygen in the solution to keep it oxygen saturated. Next, mount the dish with the fixed cells carefully onto an inverted fluorescence microscope equipped with a 40 x oil immersion objective lens and move it to the area of interest that was outlined earlier. Excite the mini SOG tag with blue light by using filter cubes for green fluorescent protein.

Continue with the illumination even after the green mini SOG fluorescence has completely disappeared. And until the brown photo oxidation product of the polymerized dia amino benadine emerges in the transmitted light channel. Then postfix the cells with one milliliter of 2%glutaraldehyde in 0.1 molar sodium Cate buffer at pH 7.4 for 30 minutes.

Next, fix the cellular membranes with 0.1 to 0.5%osmium tetroxide in 0.1 molar sodium Cate buffer for 20 minutes at pH 7.4. Once fixed, dehydrate the cells through a series of ethanol washes. Then incubate the cells in a one-to-one mixture of 100%ethanol and acrylic resin on a shaker for four hours.

Next, remove the mixture and add 100%acrylic resin to the samples for an additional four hours. In order to polymerize the resin, cut off the lid of a labeled two milliliter micro centrifuge tube with a razor blade and coat the rim with acrylic resin accelerator. Carefully fill the tube approximately two thirds full with LR white resin using a pasture pipe pad.

Take care that the resin does not get into contact with the accelerator on the rim. Remove the resin that infiltrated the cells in the dish and place the dish upside down onto the upright standing micro centrifuge tube so that the width of the tube almost completely fills up the glass covered observation window of the dish weight. One to two minutes for the accelerator to seal the tube and the cover slip.

Then invert the tube so that the resin in the tube now covers the cells. Place the dish with the tube into an oven at 60 degrees Celsius and cure it for 12 hours. When the block is cured, remove the dish with the attached resin filled micro centrifuge tube from the oven and separate the micro centrifuge tube from the dish.

Discard the glass bottom dish and carefully cut open the micro centrifuge tube with a razor blade in order to release the block. Using a razor blade, trim the block so that nothing but the one square millimeter region that contains the area previously marked with the diamond or tungsten pen remains. Next, mount the block in an ultra microtome and trim the block with a trimming knife until the cells are approached.

Then switch to a diamond knife and cut ultra thin sections of approximately 50 nanometers through the cells. Pick up the sections on high transmission 300 mesh grids. Then place the grids into a carbon coer and coat the sections with about 0.2 to 0.4 nanometers of carbon to help stabilize them under the electron beam of the transmission electron microscope.

Load the grids into a transmission electron microscope that is equipped with an energy filter. Inspect the cells on the sections using the low magnification normal transmission mode and compare them to fluorescence images taken earlier. Once a nucleus with a DNA damage track or DNA repair foci is found, switch to the energy filtering mode and record the sample with a thickness map.

Next, record the phosphorus map post edge images at 175 electron energy loss with a slit width of 20 electron volts. Also record preed images at 120 electron volts energy loss, also with a slit width of 20 electron volts for the nitrogen maps record. Post edge images at 447 electron volts with a slit width of 35 electron volts and preed images at 358 electron volts.

Also with a slit width of 35 electron volts. Open the elemental ratio maps in an image processing software such as this one. Copy the nitrogen map into the red channel and the phosphorus map into the green channel of an RGB image.

Then superpose and align the maps. Export the composite image as a tagged image file format file, and then open the image in an image processing software that can use layers. Next, adjust the dynamic range of each channel by rescaling the minimum and maximum values in the image to an eight bit data set.

Then subtract the phosphorus content from the nitrogen map within the layers window. Convert the image to an indexed color and create a yellow lookup table in the CMYK color space for the map showing the nucleic acid distribution and a cyan lookup table to show the non chromatin protein map. Then create a new image and import the maps showing the distributions of phosphorus and non chromatin protein as different layers.

Use the screen transparency mode in order to see both layers superimposed on each other. Shown here are electron spectroscopic ratio maps from inside a cell nucleus showing the locations of phosphorus and nitrogen when the two ratio maps are superimposed over each other in the RGB color space. An image like the one shown here is obtained in this image.

Yellow structures represent nuclear proteins reflecting the presence of both phosphorus and nitrogen. Here, electron spectroscopic imaging is used to view the changes in foci structure following gamma irradiation in U2 OS cells. Stably expressing M cherry tagged repair proteins in the top row are images of cells that were not irradiated.

The second row shows cells that were fixed three hours postradiation. The third row shows cells fixed six hours postradiation. The the electron spectroscopic imaging reveals that the structure of the foci appear to reorganize over time as the relative amount of 53 BP one protein in the focus indicated by the increase in nitrogen signal appears to increase while the chromatin indicated by the phosphorus signal takes a more peripheral position.

After watching this video, you should have a good understanding of how to prepare irradiated cells expressing mini so repair proteins to analyze the ultra structure of chromatin in DA repair foci using electron spectroscopic imaging. Don't forget that working with sodium ca correlate buffer potassium cyanide and osmium oxide can be extremely hazardous and precautions such as working in a fume hood and wearing gloves should always be taken while performing this procedure.