Name: peering at brain polysomes with atomic force microscopy
Uploaded: 2016-03-16T15:00:00
Description: Watch this Scientific Journal Video about Peering at Brain Polysomes with Atomic Force Microscopy at JoVE.com

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...

<ol>
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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, ...

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Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow ...

The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology In Vivo

The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology In Vivo

The olfactory system has the unusual capacity to generate new neurons throughout the lifetime of an organism. Olfactory stem cells in the basal portion of ...

Experimental Demyelination and Remyelination of Murine Spinal Cord by Focal Injection of Lysolecithin

Multiple sclerosis is an inflammatory demyelinating disease of the central nervous system characterized by plaque formation containing lost ...

Characterizing Multiscale Mechanical Properties of Brain Tissue Using Atomic Force Microscopy, Impact Indentation, and Rheometry

To design and engineer materials inspired by the properties of the brain, whether for mechanical simulants or for tissue regeneration studies, the brain ...

In Vivo Single-Molecule Tracking at the Drosophila Presynaptic Motor Nerve Terminal

In Vivo Single-Molecule Tracking at the Drosophila Presynaptic Motor Nerve Terminal

An increasing number of super-resolution microscopy techniques are helping to uncover the mechanisms that govern the nanoscale cellular world. ...

A Novel In Vitro Model of Blast Traumatic Brain Injury

A Novel In Vitro Model of Blast Traumatic Brain Injury

Traumatic brain injury is a leading cause of death and disability in military and civilian populations. Blast traumatic brain injury results from the ...