This research was supported by the AXonomIX research project financed by the Provincia Autonoma di Trento, Italy.

The practices used to obtain the mouse tissues were approved by the Body for the Protection of Animals (OPBA) of the University of Trento (Italy), protocol no. 04-2015, as per art.31 Legislative Decree no. 26/2014. All mice were maintained at the Model Organism Facility of the Centre for Integrative Biology (CIBIO), University of Trento, Italy.
Caution: To avoid any RNA degradation of the samples, prepare all buffers using DEPC-treated water for minimizing RNase contamination.
1. Preparation of Polysomes from...

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As the structure of DNA was proven to be of paramount importance to describe the process of transcription and as the organization of chromatin advanced our understanding of transcriptional control of gene expression, it is essential to analyze the organization and structure of polysomes to improve the truthful comprehension of translation and its regulation.
With the described protocol, an optimal density of polysomes gently immobilized on a flat surface, suitable for AFM imaging is obtained. ...

The synthesis of protein is the most energy consuming process in cells1,2. Hence, it is not surprising that protein abundances are mainly controlled at the translational rather than transcriptional level3,4,5. The polysome is the fundamental macromolecular component that converts the mRNA information into functional proteinaceous readouts. Polysomes are thus far recognized as macromolecular complexes where several translational controls converge6-13. Despite hundreds of studies on ribosome structure14- 16, the detailed molecular insights into the dynamics of translation and the topology of polysomes has encountered a limited interest. As a consequence, the organization of the native polysomal ribonucleoprotein complex and its potential effect on translation are still rather obscure issues. Polysomes may hide still unknown ordered and functional organizations, potentially mirroring what nucleosomes and epigenetic controls have represented for the transcription field. Indeed, the investigation of this intriguing hypothesis requires additional studies and new technical approaches. In this line, structural techniques and atomic force microscopy may fruitfully collaborate to unravel new mechanisms for controlling gene expression, similarly to what was achieved for the nucleosome17,18.
Since the discovery of the ribosome14-16, its structure has been extensively characterized in prokaryotes19,20, yeast21 and more recently in human22, providing the molecular description of the mechanisms at the base of protein synthesis. Polysomes were initially recognized on the membrane of the endoplasmic reticulum, forming typical 2D geometrical organizations14. As mentioned before, polysome assembly has not been the object of constant interest as the ribosome structure. In the past, polysomes have been studied essentially by transmission EM-based techniques. Only recently, cryo-EM techniques enabled the 3D reconstruction of purified polysomes from in vitro translation systems23-26, human cellular lysates27 or in cells28. These techniques offered more refined information about the ribosome-ribosome organization in polysomes23, 26-28 and a preliminary molecular description of the contact surfaces of adjacent ribosomes in wheat germ polysomes24. Thus, cryo-EM tomography allows the disclosure of ribosome-ribosome interactions with molecular detail, but it is burdened by extensive post-processing and reconstruction analyses that require heavy computational resources for data handling. Moreover, to obtain the molecular detail of ribosome-ribosome interactions, a highly resolved map of the ribosome is required, and this kind of reference ribosome is available only for a few species. Importantly, cryo-EM tomography is not able to detect free RNA. Therefore, new techniques are required to fully understand the organization of the translation machinery.
Beside cryo-EM, AFM has been also employed as a useful tool for direct imaging of polysomes in lower eukaryotes29-33 and humans27. Compared to EM, AFM requires no sample fixation or labeling. In addition, measurements can be carried out in near physiological conditions and with the unique possibility of clearly identifying both ribosomes and naked RNA strands27. Imaging of single polysomes can be performed relatively quickly, obtaining thousands of images at nano-resolution with little post-processing effort compared to the extensive and heavy post-processing and reconstruction analyses required by cryo-EM microscopy. Consequently, AFM data handling and analysis does not need expensive workstations and high computing power. As such, this technique collects information on polysomal shapes, morphological characteristics (such as height, length and width), ribosome densities, the presence of free RNA and the number of ribosomes per polysome27 with a higher throughput than cryo-EM. In such way, AFM represents a powerful and complementary approach to EM techniques to portray polysomes27.
Here we present a complete pipeline from purification to data analysis where AFM is applied to image and analyze mouse brain polysomes. The proposed protocol focuses on purification issues and on the accurate deposition of polysomes on mica substrates that are used for AFM imaging. In addition to conventional particle analysis that can be easily performed with common software used by the AFM community, an ImageJ34 plugin, called RiboPick, is presented for counting the number of ribosomes per polysome27, 35.

<table>
	<tbody>
		<tr>
			<td>Cycloheximide</td>
			<td>Sigma</td>
			<td>01810</td>
			<td>Prepararation of lysate</td>
		</tr>
		<tr>
			<td>DNAseI</td>
			<td>Thermo Scientific</td>
			<td>89836</td>
			<td>Prepararation of lysate</td>
		</tr>
		<tr>
			<td>RiboLock RNAse Inhibitor</td>
			<td>Life technologies</td>
			<td>EO-0381</td>
			<td>Prepararation of lysate</td>
		</tr>
		<tr>
			<td>DEPC</td>
			<td>Sigma</td>
			<td>40718</td>
			<td>Prepararation of lysate</td>
		</tr>
		<tr>
			<td>Triton X100</td>
			<td>Sigma</td>
			<td>T8532</td>
			<td>Prepararation of lysate</td>
		</tr>
		<tr>
			<td>DTT</td>
			<td>Sigma</td>
			<td>43815</td>
			<td>Prepararation of lysate</td>
		</tr>
		<tr>
			<td>Sodium Deoxycholate</td>
			<td>Sigma</td>
			<td>D6750</td>
			<td>Prepararation of lysate</td>
		</tr>
		<tr>
			<td>Microcentrifuge&#160;</td>
			<td>Eppendorf</td>
			<td>5417R</td>
			<td>Prepararation of lysate</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma</td>
			<td>S5016</td>
			<td>Sucrose gradient preparation</td>
		</tr>
		<tr>
			<td>Ultracentrifuge</td>
			<td>Beckman Coulter</td>
			<td>Optima LE-80K</td>
			<td>Sucrose gradient centrifugation</td>
		</tr>
		<tr>
			<td>Ultracentrifuge Rotor</td>
			<td>Beckman Coulter</td>
			<td>SW 41 Ti</td>
			<td>Sucrose gradient centrifugation</td>
		</tr>
		<tr>
			<td>Polyallomer tube</td>
			<td>Beckman Coulter</td>
			<td>331372</td>
			<td>Sucrose gradient centrifugation</td>
		</tr>
		<tr>
			<td>Density Gradient Fractionation System&#160;</td>
			<td>Teledyne Isco</td>
			<td>67-9000-176</td>
			<td>Sucrose gradient fractionation</td>
		</tr>
		<tr>
			<td>AFM</td>
			<td>Asylum Research</td>
			<td>Cypher</td>
			<td>Polysome visualization</td>
		</tr>
	</tbody>
</table>

peering at brain polysomes with atomic force microscopy

The translational machinery, i.e., the polysome or polyribosome, is one of the biggest and most complex cytoplasmic machineries in cells. Polysomes, formed by ribosomes, mRNAs, several proteins and non-coding RNAs, represent integrated platforms where translational controls take place. However, while the ribosome has been widely studied, the organization of polysomes is still lacking comprehensive understanding. Thus much effort is required in order to elucidate polysome organization and any novel mechanism of translational control that may be embedded. Atomic force microscopy (AFM) is a type of scanning probe microscopy that allows the acquisition of 3D images at nanoscale resolution. Compared to electron microscopy (EM) techniques, one of the main advantages of AFM is that it can acquire thousands of images both in air and in solution, enabling the sample to be maintained under near physiological conditions without any need for staining and fixing procedures. Here, a detailed protocol for the accurate purification of polysomes from mouse brain and their deposition on mica substrates is described. This protocol enables polysome imaging in air and liquid with AFM and their reconstruction as three-dimensional objects. Complementary to cryo-electron microscopy (cryo-EM), the proposed method can be conveniently used for systematically analyzing polysomes and studying their organization.

Sucrose gradient polysomal profiling of whole mouse brain 
It is possible to purify polysomes from a cellular lysate by polysomal profiling, which separates macromolecules in accordance to their weight and size. With polysomal profiling, cytoplasmic lysates obtained from cultured cells or tissues, such as in this example, are loaded onto a linear sucrose gradient and processed by ultracentrifugation to separate ...

Watch this Scientific Journal Video about Peering at Brain Polysomes with Atomic Force Microscopy at JoVE.com

Peering at Brain Polysomes with Atomic Force Microscopy

While ribosome structure has been extensively characterized, the organization of polysomes is still understudied. To overcome this lack of knowledge, we present here a detailed preparation protocol for accurate imaging of mammalian polysomes by atomic force microscopy (AFM) in air and liquid.

The translational machinery, i.e., the polysome or polyribosome, is one of the biggest and most complex cytoplasmic machineries in cells. Polysomes, ...

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.

peering-at-brain-polysomes-with-atomic-force-microscopy

Research

JoVE Journal

Neuroscience

8.2K Views.  Fondazione Bruno Kessler. 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 ...