The overall goal of this protocol is to provide a detailed pipeline for the accurate purification and deposition of brain polyribosomes on mica and to obtain thousands of images at the nanoscale resolution without the need for heavy post-processing or 3D reconstruction analyses. This method can help answer key questions in the fields of translation and translational control, such as understanding the impact of ribosome organization within polysomes during the rewiring of gene expression. The main advantages of this technique are that no sample fixation or labeling are required, and measurements can be carried out in near physiological conditions to easily identify the ribosomes and naked RNA stands.
Although this method can provide insight into the organization of mammalian polyribosome, it can also be applied to other microorganisms, such as yeast, bacteria, and insect, as well as status involving translational controls of gene expression. Visual demonstration of this method is critical, as handling polysomal sample for absorption of mica and the washing steps are quite tricky and require handling skills and experience. Demonstrating the procedure will be Paola Bernabo, and Lorenzo Lunelli.
To begin, collect and freeze brain tissue as described in the accompanying text protocol. Then, pull the frozen tissue from the freezer and bring it to the bench. Place the frozen brain tissue and liquid nitrogen into a pre-chilled mortar and pulverize it using a pestle until it forms a powder.
Transfer about 25 milligrams of the brain powder to a cold microcentrifuge tube and immediately add 0.8 milliliters of lysis buffer. Pipette the mixture up and down 25 times quickly to disrupt the cells. Next, centrifuge the tube at 12, 000 x g for one minute at four degrees Celsius to pellet the cellular debris.
Transfer the supernatant to a new microcentrifuge tube and keep the tube on ice for 15 minutes. Then, centrifuge the tube for five minutes to pellet nuclei and mitochondria. Next, carefully remove one milliliter from the top of a previously prepared sucrose gradient.
Overlay the sucrose drop by drop with the supernatant from the cytosolic lysate. WIth the sample now loaded on top of the gradient, carefully lower the tube and a counterbalance tube into the buckets of a swinging bucket rotor. Centrifuge the gradient for 100 minutes.
After ultracentrifugation, leave the tubes in their buckets for 20 minutes at four degrees Celsius, to let the gradient stabilize. Then, carefully remove the sample tube and mount it on the collector device of a density gradient fractionation system. Collect one milliliter fractions while monitoring the absorbance of nucleic acids at 260 nanometers with a UV visible light detector and put them on ice.
Next, prepare 30-40 microliter aliquots of the fractions of interest, and keep them on ice. If the samples will not be used right away, store them at 80 degrees Celsius and take steps to limit the number of freeze-thaw cycles. To prepare samples for AFM imaging, first cover a washed mica sheet with 200 microliters of one millimolar nickel ii sulfate and incubate the sheet for three minutes at room temperature.
Then remove the solution, dry the surface with compressed air, and place the petri dish on ice. Next, gently add the entire aliquot from the fraction of interest drop by drop on the mica. Using a 100 or 200 microliter tip, spread the sample over the entire surface of the mica, and incubate the sample on ice for three minutes.
Then, cover the mica sheet drop by drop with 200 microliters of cold buffer AFM and incubate the sample on ice for one hour. Maintaining the sample at 40 degrees and using cold buffer that were prepared in RNase-free water are important to preserve polyribosome organization. Following incubation, carefully remove the buffer and wash the mica three to four times with 200 microliters of cold buffer AFM to remove any excess sucrose.
Then, wash the mica three times with cold washing solution and drain the excess water from the mica using paper. The complete removal of sucrose from the absorbant samples is crucial now that you have severed both ribosome and naked RNA in the polysome. Leave the sample to dry under the chemical hood with the top of the petri dish partially opened.
After two hours, close the petri dish. The sample can now be stored at room temperature. Attach the prepared sample to the sample holder of the AFM using double sided tape.
Then, insert the sample holder in the AFM stage in accordance with the manufacturer's instructions. Following calibration, approach the sample until the cantilever tip engages the surface. Select a scan area of two by two microns, a resolution of at least 512 x 512 pixels, choose live background subtraction mode, and select a Z-scale of 20-25 nanometers.
Then, begin image acquisition. Inspect the image, looking for the presence of round objects characterized by a height between 10 and 15 nanometers when acquiring images in the air. Next, adjust the set point and feedback parameters until sharp objects are visualized.
The background should appear relatively flat in good samples with some two to four nanometer high objects. Once the image looks good, acquire several two by two micron scans at different sample areas. After the deposition of sucrose aliquots on mica, AFM provides accurate size descriptions of single polysomes that appear as clusters of tightly packed ribosomes.
Taking a closer look at one of the ribosomal peaks using cross-section analysis reveals the height of ribosomal peaks are around 14 nanometers. This matches what was previously observed for human ribosomes and polysomes after air-drying. In the image on the right, a macro was used to identify ribosome location and mark each ribosome with a red circle.
Using this information, the frequency distribution of the number of ribosomes per polysome can be analyzed. This experimental distribution was fitted with a Gaussian curve that was centered at 5.8 ribosomes per polysome, with a standard deviation of 1.3. Once mastered, this technique from brain pulverization to polyribosome images acquisition can be done in less than eight hours if it is performed properly.
While attempting this procedure, it is important to remember to keep the sample on ice and use cold buffers for the sample deposition on the mica. Following this procedure, and using the ImageJ plugin of a developer to make available in your paper, you can precisely count the number of ribosome per polysome. After each development, this technique will pave the way to understand the kinetics of polysome formation and identifying the changes in polysome organization and their different cellular or tissue conditions.
After watching this video you should have a good understanding of how to obtain thousands of polysome images for systematically analyzing and studying their organization.
