This protocol utilizes all major features of AFM, non-amenable to other single molecule techniques. As a result, we are able to resolve structure of nucleosome at nano scale. Importantly, direct visualization of nucleosomes was done at physiological conditions.
Using our AFM technique, we have recently revealed unique properties of the centromere nucleosome that can play a role on the function of the entire centromere. The method to be shown is currently applied in our lab to investigate self-assembly of proteins associated with formation of amyloid aggregates. These protein aggregates trigger the development of devastating diseases such as Alzheimer's and Parkinson's to name a few.
The simple preparation methods are also applicable to other protein-DNA complexes, including large systems such as DNA replication machinery. Although some modifications are needed. In order to characterize nucleosome particles at the single molecule level using static and time-lapse atomic force microscopy, begin by purifying, assembling, and validating the nucleosomes as described in the accompanying text protocol.
Next, prepare the mica surface for static AFM imaging. To do this, first prepare a fifty millimolar APS stock solution in deionized water. Store one milliliter aliquots of this solution at four degrees Celsius, until it is needed.
From the stock solution, prepare a working APS solution for mica modification by dissolving 50 microliters of the 50 micromolar APS stock in 15 milliliters of distilled deionized water. Mix the solution and then fill a cuvette with the solution. Next, cut one by three centimeter strips of mica from high quality mica sheets.
Check that the piece fits when placed diagonally in a cuvette. Then, cleave layers of the mica until both sides are freshly cleaved, and the piece is as thin as 0.1 millimeter. Immediately place the mica piece into the APS filled cuvette and incubate the mica for 30 minutes.
Transfer the mica piece to a cuvette filled with distilled deionized water and soak it for 30 seconds. Then, use argon to completely dry both sides of the APS mica strip. Apply double faced adhesive tape to several magnetic pucks and place them to the side.
Then, cut the APS mica substrate to one centimeter by one centimeter squares, and cover them in a clean Petri dish. Next, prepare three dilutions of the assembled nucleosomes using a 0.22 micron filtered buffer containing 10 millimolar hepes and four millimolar magnesium chloride at a PH of 7.5. To limit the loss of nucleosomes, at the low final concentration, the dilution should be done one at a time, immediately prior to deposition on the APS mica.
Deposit five to 10 microliters of each nucleosome sample at the center of an APS mica piece and let them incubate for two minutes. Then, gently rinse the sample with two to three milliliters of distilled deionized water to remove the buffer components. And dry the deposited sample under a light flow of argon.
To begin, mount a tip on the tip holder of the AFM setup. Then, mount the first sample on the AFM stage, being careful not to contact the sample surface. Position the laser over the cantilever until its sum is at the maximum and adjust the vertical and lateral deflection values to near zero.
Then, tune the AFM probe to find its resonant frequency, adjust the drive amplitude, and set the image size to 100 by 100 nanometers. Once set up, click the engage button to begin the approach. When the approach is complete, gradually optimize the amplitude set point until the surface of the sample is clearly seen.
Then, increase the scan size to one micron by one micron and the resolution to 512 by 512 pixels. Finally, click the capture button to begin image acquisition. Use glass rod scanner glue to attach a glass rod to the AFM scanner stage.
Allow this to dry for a minimum of 10 minutes. In the meantime, make 0.1 millimeter thick circular pieces of mica with a 1.5 millimeter diameter by punching them from a larger mica sheet. Use the high speed AFM mica glass rod glue to attach this mica piece to the glass rod on the high speed AFM, and allow it to remain untouched for a minimum of 10 minutes while it dries.
Then, cleave layers from the mica using a pressure sensitive tape until a well cleaved layer is seen on the tape. Next, dilute one microliter of 50 millimolar APS stock in 99 microliters of distilled deionized water to make a 500 micromolar APS solution. Deposit 2.5 microliters of the solution on the freshly cleaved mica surface and let the surface functionalize for 30 minutes.
Following the incubation, rinse the mica several times with distilled deionized water by applying three microliters rinses. Remove the water completely following each rinse by placing a non-woven wipe at the edge of the mica. After the final rinse, place three microliters of distilled deionized water on the surface, and let it sit for a minimum of five minutes to remove any non-specifically bound APS.
Next, place the probe in the high speed AFM holder, and position the holder on the AFM stage with the tip facing up. Rinse the holder using 100 microliters of distilled deionized water, followed by two 100 microliter rinses of 0.22 micron filtered nucleosome imaging buffer. With the rinses done, fill the chamber with 100 microliters of nucleosome imaging buffer, submerging the tip.
Adjust the cantilever position until it is hit with the laser. Then, rinse the APS mica five times with filtered nucleosome imaging buffer, using four microliters per rinse. Dilute one microliter of the nucleosome assembly stock into 250 microliters of filtered nucleosome imaging buffer for a final nucleosome concentration of one nanomolar.
Deposit 2.5 microliters of this dilution on the surface and let it sit for two minutes. Rinse the surface twice with four microliters of nucleosome imaging buffer to avoid over crowding. After the final rinse, leave the surface covered in imaging buffer.
Set the scanner and sample on top of the tip holder so that the sample is face down. To begin the approach, use the auto approach function with the set point amplitude close to the free oscillation amplitude. Adjust the set point until the surface is being well tracked.
Then, set the image area around 150 by 150 nanometers to 200 by 200 nanometers with the data acquisition rate of around 300 milliseconds per imaging frame. As a control for the assembly and deposition, H3 mononucleosomes were prepared and imaged using static AFM. This image provides a snapshot of the nucleosome population as it existed moments before deposition confirming that nucelosomes were successfully assembled.
With the H3 control assembly a success, the presented methods were next applied to the study of CENP-A nucleosomes. Static AFM imaging of this sample revealed its assembly was a success. From the static AFM images, the height and turn number of mononucleosomes could be characterized.
Both the angle between the free DNA arms and the length of the free DNA arms are used to determine the number of DNA turns in the individual nucleosome. In contrast, time lapse AFM imaging of the nucleosomes can be used to visualize the overall spontaneous unwrapping behavior of the nucleosomes. As the turn number of the nucleosome decreases, a corresponding decrease in the nucleosome core volume is also observed.
In addition to regular time lapse imaging, high speed time lapse AFM can be used to probe the more intricate nucleosome dynamics that is missed using standard time lapse imaging. The ability of this technique to capture the dynamics over a long period of time was essential to the visualization of a long distance translocation of a CENP-A nucleosome core, which was captured over the course of 1200 frames. This technique was also critical in capturing the rare transfer of a CENP-A nucleosome core from one DNA substrate to another.
The fast image capture rate made visualization of this dynamic event possible as it only took several frames to complete. In the broad chromatin field there are a set of important questions relating to the roles of liger histones and histone magnification of the chromatin dynamics. All these questions can be answered using our technique.
