The overall goal of this procedure is to directly visualize and quantify the activity of DNA binding proteins in live bacterial cells. This is accomplished by visualizing cells that express a photo activated fluorescent protein fused to A DNA binding protein of interest. The second step of the procedure is to acquire a movie of individual proteins getting photo activated while being imaged.
Then the data is analyzed to determine protein localizations and track proteins inside single cells. DNA binding events are identified by change in the diffusion coefficient of single proteins when they contact chromosomes. Ultimately, the results can provide a quantitative measure of protein DNA interactions at the single cell level by counting the number of bound and diffusing proteins per cell.
This method can help answering key questions in the DNA repair field, such as the in vivo repair rates and the spatial distribution of repair sites. Now we'll demonstrate the method by measuring the repair activity of DNA polymerase one in life e coli cells First make cover slips with no background fluorescent particles in a furnace at 500 degrees Celsius for an hour. Burn a stock supply of cover slips of thickness number 1.5, store them at room temperature in aluminum foil.
They're good for weeks to come. Next, concentrate a milliliter of early exponential phase E coli in a 1.5 milliliter micro centrifuge tube of the strain. AB 1157 poly a PA m cherry centrifuge the cells at 2, 300 G for five minutes.
Remove the supernatant and resuspend the cell pellet in 20 microliters of residual medium and vortex the cells into solution. Next, make a 1.5%low fluorescence aros solution in distilled water. Mix 500 microliters of the melted aeros with 500 microliters of two x minimal medium by gently pipetting up and down a few times for DNA damage experiments using methyl methane sulfonate or MMS taking precautions.
Add 8.3 microliters of MMS to the 500 microliters of minimal medium before mixing it with the aros before the mixture cools. Spread it on a burned cover slip. Spread it evenly and centered without making bubbles.
Flatten the pad with a second burned cover slip. Once cooled, remove the top cover from the pad and add a microliter of concentrated cell suspension. Immobilize the cells by covering the pad with a new burned cover slip and pressing down very gently.
The cells should be imaged within 45 minutes before they desiccate. To extend this time, the pad can be sealed inside a silicon gasket for DNA Damage experiments. Before imaging the cells incubate them on the pad for 20 minutes.
In a humidified container at room temperature, the microscope features a 405 nanometer photo activation laser and a 561 nanometer excitation laser. Single molecule sensitivity is reached by exciting only fluoro fours within a thin section above the cover slip surface using highly inclined illumination, the fluorescence emission is recorded on an electron multiplying CCD camera. Place the sample on the microscope stage and bring the cells into focus.
Under transmitted light illumination, define a cropped field of view to reduce data size and increase the camera readout speed. Cover the sample from ambient light and switch on the electron multiplying CCD camera gain for single fluoro four detection. Set the frame rate to 15.26 milliseconds per frame.
This includes the 0.26 millisecond camera readout Time Exposure times need to be sufficiently short to observe sharp fluorescent spots with little motion blaring. On the other hand, the tracks need to be sampled at sufficiently long time intervals to clearly distinguish the bound from the diffusing molecules. Now display the camera data to check the dark background signal.
Switch on the 561 nanometer laser and check the excitation background signal. Switch on the 405 nanometer laser for photo activation of the Paul one PA M cherry fusion proteins and increase the intensity until fluorescent spots of single molecules appear. Now adjust the angle of the excitation beam to illuminate only a thin section of the sample.
Close the cover slip surface. This method relies on the detection and precise localization of single fluorescent proteins, so the optimal alignment and sensitivity of the microscope will be crucial for the data quality. To this end, focus the laser beam into the back focal plane of a 100 x numerical aperture 1.4 objective.
Translating the focusing lens perpendicular to the beam moves the focus away from the center of the objective, causing the beam to exit the objective under an angle. Aim for the beam to maximize the fluorescence intensity and minimize the background. Find a new field of view of cells in transmitted light microscopy mode and focus the image.
Take a camera snapshot to record the cell outlines. Switch on the 561 nanometer laser and bleach the cellular autofluorescence and background spots on the cover. Slip for a few seconds.
Before starting data acquisition, start the acquisition of a palm movie under continuous 561 nanometer excitation. Switch on the 405 nanometer laser and gradually increase the intensity over the course of the movie reaching up to one watt per square centimeter. Avoid higher 405 nanometer intensities that cause cellular autofluorescence.
Pay attention to the density of fluorescent molecules. It is important to keep activation rates low such that the fluorescent spots are clearly isolated in each frame. Record about 10, 000 frames per movie, which with a cropped field of view typically takes up to three minutes and up to a gigabyte of hard disc space.
Note that once a field of view has been imaged, the pa m cherry fluorophores are irreversibly bleached and cannot be observed. Again, the following procedure uses custom software in matlab. Single fluorophores appear as point spread functions or psfs.
In the movie, psfs are first identified in a band pass filtered image using a Gaussian kernel with seven pixels diameter candidate positions correspond to P SFS with peak pixel intensities 4.5 times above the standard deviation of the background. The locally brightest pixel per candidate PSF serves as an initial guess for fitting an elliptical Gaussian function. The free fit parameters are X position y, position X width, Y width rotation, angle, amplitude, and background offset.
The elliptical Gaussian mask accounts for molecule movement during the exposure time, which blurs and deforms the PSF plot. The resulting XY localizations from all the frames of the palm movie onto the transmitted light microscopy image of the same field of view localizations of Paul one PAM Cherry should appear within the central area of e coli cells. Automated tracking measures the proteins movement, but successful choice of a tracking window is crucial.
First, run the tracking algorithm for a range of tracking window parameters. Plot the number of measured tracks per cell versus the tracking window to identify the smallest possible tracking window that does not split tracks display the resulting tracks on the transmitted light microscopy image of the same field of view. To visualize the spatial distribution of molecule movement within cells, P one tracks should display diffusion confined within single cells if a fraction of tracks appears to cross between cells, this suggests that separate molecules were erroneously linked because the tracking window was chosen too large and or the photo activation rate was too high.
Plot the cumulative distribution of the step lengths between consecutive localizations. The curve rises and saturates smoothly for sufficiently large tracking windows, but shows a cutoff edge if the window was chosen too small. Once a suitable tracking window has been chosen, the diffusion characteristics of Paul one can be analyzed.
Compute the mean square displacement or MSD between consecutive localizations for each track with a total of n steps only use tracks with at least five localizations to reduce the statistical uncertainty of the MSD. Next, calculate the apparent diffusion coefficient per track from the MSD. The second term corrects for the estimated localization error.
These are the values for the second term. In this example, now plot a histogram of the measured diffusion coefficient values from all tracks in the field of view for pol one in undamaged cells and for pol one in cells under DNA damage treatment with MMS, the red bars identify the population of individual pol one molecules that appear bound to the chromosome and diffuse at less than 0.15 square microns per second. Whereas freely diffusing molecules move around 0.9 square microns per second.
After the bound Paul one molecules have been identified. The positions of binding sites can be visualized in undamaged cells and in cells with mm MS damage. The fraction of bound tracks relative to the total number of observed tracks provides a direct quantitative measure of the DNA repair activity of pol.
One in vivo photo activation of pol one PA m cherry fusion proteins was carried out in live e coli cells. Photo activation could be limited to a single PA m cherry fluorophore in one cell, higher photo activation rates revealed more fluorescent molecules. Localization analysis was performed for each frame of a palm movie.
Precision was measured using immobile molecules in fixed cells or bound molecules in live cells, and found to be 40 nanometers in agreement with the theoretical prediction. Next, the localization threshold was set when it was too low. Random peaks in the background noise got erroneously selected as candidate positions, whereas if the threshold was too high, some spots were missed.
The resulting Paul one localizations occupied the central area of the cell, broadly recapitulating the spatial organization of the e coli nucleoid. The majority of Paul one tracks in undamaged cells display diffusion. A typical cell contains several hundred Paul one tracks consistent with the copy number of approximately 400 Paul one molecules per e coli cell.
Different types of molecular motion can be identified by calculating MSD values over a range of lag times directed motion gives a parabolic curve. Brownian motion is characterized by a straight line. A confined diffusion curve reaches a plateau and an offset of the MSD curve for immobile particles represents the localization uncertainty.
The resulting MSD curve for Paul one rose linearly for short lag times indicative of brownian motion and saturated at longer lag times due to cell confinement. Using these methods, DNA repair activity of Paul, one was measured in response to exogenous DNA alkylation damage in undamaged cells. The diffusion coefficient histogram of Paul one shows a dominant population of diffusing molecules.
The few bound molecules could be involved in DNA replication and repair of endogenous DNA damage under continuous 100 millimolar MMS damage. The frequency of tracks with near zero diffusion coefficients increases significantly. This supports the model that more Paul one molecules must be involved in DNA repair with mutagen present Following this procedure.
Other data analysis methods like localization, clustering can be performed in order to quantify the spatial distribution of proteins in the cell and to investigate the presence of larger protein complexes involved in DNA repair.
